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BANTAO Journal 

Editors of this issue: Goce Spasovski 

Momir Polenakovic 

Official Publication of 

BANTAO Association 

Incorporating Procedings of the 

BANTAO Association 

Skopje 

Skopje 

Editor-in-Chief 

Associate Editors 

Ali Basci Izmir Dimitar Nenov Varna 


Skopje 

Ljubica Djukanovic 

Belgrade 

Deputy Editors Charalambos Stathakis Athens 

Adrian Covic 

Goce Spasovski 

Iasi 

Skopje 

Editorial Board 

Akcicek Fehmi Izmir John Boletis Athens 

Alketa Koroshi Tirana Kamil Serdengecti Istanbul 

Cengiz Utas Kayseri Katica Zafirovska Skopje 

Daniela Monova Sofia Ladislava Grcevska Skopje 

Dimitrios Goumenos Patra Liliana Garneata Bucharest 

Dimitris Tsakiris Veria Mirela Gherman-Caprioara Cluj-Napoka 

Dragan Ljutic Split Nada Dimkovic Belgrade 

Ekrem Erek Istanbul Petar Kes Zagreb 

Evgueniy Vazelov Sofija Rado Kveder Ljubljana 

Gabriel Mircescu Bucharest Sanja Simic-Ogrizovic Belgrade 

Georgios Vergoulas Thessaloniki Veselin Nenov Varna 

Halima Resic Sarajevo Vidosava Nesic Belgrade 

Huseyin Toz Izmir Visnja Lezaic Belgrade 

Igor Mitic Novi Sad Vladislav Stefanovic Nis 

Published by: Balkan Cities Association of Nephrology, 

Dialysis, Transplantation and Artificial Organs, 

Department of Nephrology, 

Vodnjanska 17 St, 

1000 Skopje, 

R. Macedonia 

Printing: Kiro Dandaro, Bitola

Congress report 

The dream is now a reality - The seventh Congress of the Balkan 

Cities Association of Nephrology, Dialysis, Transplantation and 

Artificial Organs (BANTAO) Ohrid, Republic of Macedonia, 8-11 

September 2005 

The 7 th BANTAO Congress was held from 8 th to 11 th September 

2005 in Ohrid, Republic of Macedonia. Ohrid is also the place where 

BANTAO was born in October 9 th , 1993. 

The main goal of BANTAO is to promote scientific and technical 

cooperation in the fields of renal disease and artificial organs among the 

Balkan cities, giving an opportunity for exchange of experience and 

knowledge among the experts in the area and to engage them in 

collaborative projects in order to demonstrate that cooperation is possible 

even on the turbulent Balkan Peninsula. 

Following the first six successful congresses: Varna (1995), 

Belgrade (1998), Izmir (1999), Thessaloniki (2001) and Varna (2003), 

the BANTAO Congress was established as the major and institutional 

forum for Balkan nephrologists, with its own journal including our will 

to communicate, to collaborate, to get to know each other and to share 

our difficulties. More than just a professional event, the BANTAO 

congress became a cultural phenomenon, through which we discovered 

that we have many more things in common than we previously thought, 

and that we must now take every advantage of being able to live and to 

communicate in a world without political boundaries. At present, the 

BANTAO Council has managed in a spirit of peace, friendship and 

collaboration to continuously strengthen this association and moreover, 

to make it a reputable part of the European and international ones. In this 

spirit the constitution of BANTAO has already been created. 

The Congress was attended by 570 physicians from the Balkan 

Peninsula cities as well as from Europe and USA. The Congress was 

held under the patronage of the President of the Republic of Macedonia, 

Mr. Branko Crvenkovski, Prof. M. Polenakovic was the Congress 

President and Doc. G. Spasovski the Secretary General. 

In the fields of the Epidemiology of Renal Disease, Basic Science, 

Clinical Nephrology, Dialysis, Transplantation and Artificial Organs we 

had 43 invited guest lectures, 42 oral and 223 poster presentations. 

The Congress was focused on several topics for example: how to 

improve the Balkan renal registry, prevention in nephrology, prevention 

of progression of renal diseases, understanding renal fibrosis and kidney 

transplantation on the Balkan Peninsula.

Companies invited speakers presented in six symposia, one 

workshop and three mini lectures their latest achievements in specific 

fields of nephrology, especially in dialysis. 

R. Vanholder, A. Perna, J. Jankowski and A. Argiles, on behalf of 

ESAO/EUTOX group, presented: An update on uremic toxicity- the role 

of EUTOX. D. Kerjaschki and H. Regele presented clinico-pathological 

correlation in renal transplant patients. 

For the first time at this 7 th BANTAO Congress we organized 

CME Course - Frontiers in nephrology, sponsored by ERA/EDTA and 

ISN/COMGAN. Six distinguished speakers: F. Locatelli, J. Mann, J. 

Floege, T. Risler, A. Wiecek, and M. Klinger presented the newest 

achievements in their fields. 

The abstracts were published in BANTAO Journal Vol. 3 (1), 

2005, pp 1-121, editor G. Spasovski as Abstract Book and 50 papers 

from the Congress in the BANTAO Journal Vol. 3 (2), 2005, pp 1-160, 

as Proceedings of the 7 th BANTAO Congress, editors G. Spasovski and 

M. Polenakovic. A. Basci (Izmir) was elected Editor in Chief. A. Covic 

(Iasi) and G. Spasovski (Skopje) were elected as deputy editors. 

The 7 th Congress of BANTAO, Ohrid, was a resounding success 

and confirmed our good will for mutual collaboration and friendship, so 

we can say our dream is now a reality. 

M. Polenakovic was elected President of BANTAO Association. 

The next - 8 th BANTAO Congress will be held in Belgrade in 2007. Prof. 

Ms. V. Nesic was elected as President of the Congress. 

M. Polenakovic 

G. Spasovski

Preface 

BANTAO Journal is an official publication of the BANTAO 

Association. The aim/intention of the Journal is to cover the issues of 

early diagnosis, prevention and treatment of kidney diseases, as well as 

other specific problems Balkan nephrologists are facing with. 

The Journal should improve the medical education and knowledge 

of nephrologists and other doctors through an integrative process for 

appropriate management of chronic kidney disease (CKD) as an 

increasing worldwide problem. 

After the successful 7 th BANTAO Congress in Ohrid, September 

2005, a need to improve Balkan renal registry, the early diagnosis of 

CKD, prevention and management of renal diseases in order to postpone 

the development of end stage renal failure (ESRF) was imposed. 

Papers published in this volume are dedicated to aforementioned 

problems, presented during our last BANTAO Congress. At the General 

Assembly of our BANTAO Association, A. Basci (Izmir) was elected 

Editor in Chief. A. Covic (Iasi) and G. Spasovski (Skopje) were elected 

as Deputy Editors. We congratulate them and wish a good and successful 

work in the Journal. 

Guest Editors of this issue: 


President of BANTAO Association 

Goce Spasovski 

Deputy Editor of the BANTAO Journal

Contents: 

Correlation between sera levels of sVCAM-1 and severity of kidney lesions in 

patients with lupus nephritis 

T. Ilic, I. Mitic, B. Milic and S. Curic......................................................................................... 1 

Continuous convective renal replacement (CCRR) system: A new modality of 

wearable artificial kidney 

M.N. Aboras and I.A. Kawalit…………………………………………………………………. 3 

Prevalence of hepatitis C virus infection in dialysis patients 

P. Dzekova, A. Slavkovska, L. Simjanovska, I. Nikolov, A. Sikole, R. Grozdanovski, 

A. Asani, G. Selim, S. Gelev, V. Amitov, G. Spasovski and M. Polenakovic............................ 6 

Influence of inflammation on nutritional status of dialysis patients 

P. Dzekova, I. Nikolov, G. Severova-Andreevska, A. Sikole, R. Grozdanovski, A. Asani, 

G. Selim, S. Gelev, V. Amitov, G. Spasovski and M. Polenakovic……………………………. 8 

Unfavorable prognostic factors in Immunoglobulin A Nephropathy 

L. Petrovic, S. Curic, I. Mitic, D. Bozic, S. Vodopivec, V. Sakac, T. Djurdjevic-Mirkovic 

and T. Ilic……………………………………………………………………………………...... 10 

Congestive heart failure and renal dysfunction 

D. Monova, E. Peneva and S. Monov………………………………………………………….. 13 

Sporadic Balkan Endemic Nephropathy (BEN) beyond the known regions of 

BEN 

J. Nikolic, T. Pejcic, D. Crnomarkovic, Z. Gavrilovic and C. Tulic…………………………… 15 

Amyloidosis in Turkish patients 

C. Ensari and A. Ensari………………………………………………………………………... 18 

Frequency of Alport syndrome at dialysis center “Vrsac”- Vrsac 

K. Djordjev, D. Tintor, G. Djurdjev, O. Vasilic-Kokotovic, I. Sokolovac and S. Bozic………. 21 

Eearly effects of the AT 2 receptor antagonist eprosartane mesylate (EM) in 

diabetic patients with and without chronic renal failure 

N. Nnchev, B. Deliyska, M. Bankova, P. Radulova, G. Kimenov and E. Marinova…………. 23 

Prealbumin and inflammatory markers in dialysis patients 

S. Chrysostomou, K. A. Poulia, Y. Jeanes, D. Perrea, M. Poulakou, B. Filiopoulos, 

D. Stamatiades and Ch. P. Stathakis…………………………………………………………… 26 

Liver and kidney damage in acute poisonings 

M. Mydlik and K. Derzsiova…………………………………………………………………… 30 

Vascular calcifications in patients on hemodialysis 

T. Damjanovic, N. Markovic, S. Djorovic, T. Djordjevic and N. Dimkovic………………… 34 

Is there a seasonal variation in mortality in hemodialysis patients? 

J. Popovic, N. Dimkovic, Z. Djuric, G. Popovic and N. Lazic………………………………… 36 

Propyl gallate-induced platelet aggregation in patients with end stage renal 

disease – The influence of the hemodialysis procedure 

T. Eleftheriadis, G. Antoniadi, V. Liakopoulos, A. Tsiandoulas, K. Barboutis 

and I. Stefanidis……………………………………………………………………….………... 40 

Serum cystatin C as an endogenous marker of kidney function in elderly with 

chronic kidney failure 

H. Resic and A. Mataradzija…………………………………………………………………… 46

Thyroid disfunction and ultrasonographic abnormalities in uremic patients 

undergoing conservative management and haemodialysis 

A. Mataradzija , H. Resic and E. Kucukalic-Selimovic…………………………………………. 50 

Prednisone/Cyclophosphamide treatment in adult-onset autosomal dominant 

familial focal segmental glomerulosclerosis (FSGS 1) 

L. Grcevska, S. Dzikova, V. Ristovska, M. Milovanceva Popovska, G. Petrusevska 

and M. Polenakovic…………………………………………………………………………….. 56 

Microalbuminuria – The new marker for Balkan Endemic Nephropathy? 

G. Imamovic, S. Trnacevic, M. Tabakovic, E. Mesic, M. Uzeirbegovic, M. Malohodzic, A. 

Halilbasic, V. Habul, D. Tulumovic, E. Hodzic, M. Atic, S. Lekic M. Dugonjic, 

A. Becirovic and E. Hasanovic..................................................................................................... 58 

Lymphatic neoangiogenesis in human neoplasia and transplantation as 

experiments of nature 

D. Kerjaschki…………………………………………………………………………………… 60 

Cytokines and acute renal failure 

K. Cakalaroski………………………………………………………………………………….. 62 

Amyloidosis 

M. Tunca……………………………………………………………………………………….. 66 

Oxidative stress evaluation in uraemic patients undergoing continuous 

ambulatory peritoneal dialysis 

G. Mircescu, C. Capusa, I. Stoian, D. Lixandru, E. Rus, L. Coltan and L. Gaman…….……… 69 

Hyperhomocysteinemia in ESRD 

A. F. Perna, C. Lombardi, R. Capasso, F. Acanfora, E. Satta, D. Ingrosso, 

M. G. Luciano and N. G. De Santo.............................................................................................. 74 

C.E.R.A. (Continuous Erythropoietin Receptor Activator): A new perspective 

in anaemia management 

W. Sulowicz……………………………………………………………………………………. 78 

Diagnosis and treatment of renal bone disease 

G. B. Spasovski………………………………………………………………………………… 82 

Efficiency of mycophenolate-mofetil in resistant nephrotic syndrome – case 

reports 

B. Milic, T. Ilic, I. Mitic, I. Budosan and S. Curic……………………………………………... 84 

Bacterial infections associated with double lumen central venous catheters 

M. S. Simin, M. Milosevic, S. Vodopivec, I. Mitic and S. Curic……………………………… 86 

Early renal protocol biopsies: beneficial effects of treatment of borderline 

changes and subclinical rejections on the histological changes for chronic 

allograft nephropathy 

J. Masin-Spasovska, N. Ivanovski, S. Dzikova, G. Petrusevska, B. Dimova, Lj. Lekovski, 

Z. Popov and G. Spasovski……………………………......................………………............... 88 

Factors associated with carotid and femoral atherosclerosis in non-diabetic 

hemodialysis patients 

S. Gelev, S. Dzikova, A. Sikole, Gj. Selim, P. Dzekova, V. Amitov and G. Spasovski............. 93 

Acid-base disorders in patients with hypoproteinemia 

P. Mavromatidou, N. Sotirakopoulos, T. Tsitsios, I. Skandalos, M. Peiou, 

and K. Mavromatidis.................................................................................................................... 99 

Instructions to authors…………………………………………………………………...… 104

BANTAO Journal 2006: 4 (1): 1 

Correlation between sera levels of sVCAM-1 and severity of kidney lesions 

in patients with lupus nephritis 

T. Ilic, I. Mitic, B. Milic and S. Curic 

Department of Nephrology, Clinical Center Novi Sad, Serbia and Montenegro 

Abstract 

We determined sera concentrations of soluble vascular 

adhesion molecule-1 (sVCAM-1) in group of 80 patients with 

SLE. Our aim was to investigate correlation between level of 

sVCAM-1 and degree of disease activity, also severity of 

lupus nephritis. Using ELISA procedure we determined sera 

levels of sVCAM-1 in 80 patients with SLE and in group of 

27 healthy volunteers. Patients with SLE had significantly 

higher sera levels of sVCAM-1 comparing healthy controls 

(p < 0,001). Patients with disease in active phase had higher 

sera levels of this adhesive molecule comparing patients with 

disease in phase of remission (p < 0,001). There was a high 

positive correlation between sera levels of sVCAM-1 and 

concentration of anti-ds DNK antibodies in sera of patients 

with SLE (r = 0,77, p < 0,001) and there was also negative 

correlation between sera levels of sVCAM-1 and sera 

concentrations of C 3 and C 4 component of complement (r = - 

0,64, r = - 0,58). In group of patients with lupus nephritis 

were detected significantly higher sera concentrations of 

sVCAM-1 comparing patients without nephritis. Using WHO 

classification, patients with lupus nephritis were systematized 

in three categories: class II (5 patients), classes III and IV (18 

patients) and class V (7 patients). Patients with class III and 

class IV of kidney changes had significantly higher levels of 

sVCAM-1 comparing patients with class II of kidney 

changes. At the same time, in group of patients with activity 

index (AI) of kidney changes over 4 sVCAM-1 sera levels 

were significantly higher comparing group with AI < 4. Sera 

level of sVCAM-1 is reliable parameter in evaluation of 

autoreactivity degree in SLE. At the same time, sVCAM-1 

sera level can be used as reliable marker in evaluation of 

renal lesion extensivity in SLE. 

Key words: SLE, sVCAM-1, lupus nephritis 

Introduction 

Systemic Lupus Erythematosus (SLE) is chronic, inflamatory 

connective tissue disease which contains loss of 

autotolerancy as main immunological disorder accompanied 

with breakdown of T lymphocytes, collapse of cytokine 

production and B lymphocite hiperactivity (1). Cytokines are 

essential regulating factors of immunology response which 

are included in migration process of inflammatory cells into 

the place of immunology caused inflammatory tissue 

reaction, together with adhesive molecules (2). The vascular 

cell adhesion molecule-1 (VCAM-1) is a member of the 

immunoglobulin gene superfamily. VCAM-1 supports the 

adhesion of lymphocytes, monocytes, natural killer cells, 

eosinophils and basophils through its interaction with 

leukocyte very late antigen-4 (VLA-4). VCAM-1/VLA-4 

interaction mediates firm adherence of circulating nonneutrophilic 

leukocyte to endothelium. VCAM-1 also 

participates in leukocyte adhesion outside of the vasculature, 

mediating presursor lymphocyte adhesion to bone marrow 

stromal cells and B cell binding to lymph node follicular 

dendritic cells (3). A soluble form of VCAM-1 (sVCAM-1) 

has been described. Soluble VCAM-1 levels have been found 

in the sera of healthy individuals and increased levels of 

sVCAM-1 can be detected in several diseases including 

connective tissue diseases (4). Studies have shown that 

elevated levels of sVCAM-1 are related to disease activity in 

patients with SLE (5). 

The aim of our study was to investigate changes of adhesive 

molecule sera levels in patients with SLE comparing different 

phases of disease activity and comparing presence/absence of 

lupus nephritis. At the same time relation of vascular cell 

adhesion molecule sera concentrations were analized with 

concentrations of other disease parameters (titre of ANA, 

SLED AI, titre of anti-ds DNA antibodies, concentrations of 

C3 and C4). 

Patients and methods 

The study was carried out in 80 patients included in 

crossection study and 10 patients included in longitudinaly 

study. We used ELISA procedure to determine sVCAM-1 

sera levels in 80 patients with SLE and in group of 27 healthy 

volunteers. An anti-sVCAM-1 monoclonal coating antibody 

is adsorbed onto microwells. sVCAM-1 present in the sample 

binds to antibodies adsorbed to the microwells. A mixtureof 

biotin–conjugated monoclonal anti-sVCAM-1 antibody and 

Streptavidin–HRP is added. Biotin conjugated anti-sVCAM- 

1 captured by the first antibody. Streptavidin-HRP binds to 

the biotin conjugated anti-VCAM-1. Following incubation 

unbound biotin conjugated anti-sVCAM-1 and Streptavidin – 

HRP is removed during a wash step and substrate solution 

reactive with HRP is added to the wells. Acoloured product is 

formed in proportion to the amount of soluble VCAM-1 

present in the sample. The reaction is terminated by addition 

of acid and absorbance is measured at 450 nm. A standard 

curve is prepared from six sVCAM-1 standard dilutions and 

sVCAM-1 sample concentration determined. 

______________________ 

Correspodence to: T. Ilic, Department of Nephrology, Clinical Center Novi Sad, Serbia and Montenegro


Results and discussion 

Patients with SLE had significantly higher sera levels of 

sVCAM-1 comparing healthy controls (p < 0,001). Patients 

with disease in active phase had higher sera levels of this 

adhesive molecule comparing patients with disease in phase 

of remission (p < 0,001). There was high positive correlation 

between sera levels of sVCAM-1 and concentration of anti-ds 

DNK antibodies in sera of patients with SLE (r = 0,77, p < 

0,001) and a negative correlation between sera levels of 

sVCAM-1 and sera concentrations of C 3 and C 4 component 

of complement (r = -0,64, r = - 0,58). In group of patients 

with lupus nephritis a significantly higher sera concentrations 

of sVCAM-1 were detected comparing patients without 

nephritis. Using WHO classification, patients with lupus 

nephritis were systematized in three categories: class II (5 

patients), classes III and IV (18 patients) and class V (7 

patients). Patients with class III and class IV of kidney 

changes had significantly higher levels of sVCAM-1 

comparing patients with class II of kidney changes. In the 

same time, in group of patients with activity index of kidney 

changes (AI) over 4 sVCAM-1 sera levels were significantly 

higher comparing group with AI < 4. 

Conclusions 

Sera level of sVCAM-1 is reliable parameter in evaluation of 

autoreactivity degree in SLE. Serial measurements of 

sVCAM-1 may be helpful for monitoring disease activity in 

patients with SLE and lupus nephritis. The serum level of 

sVCAM-1 was correlated with the clinical and histological 

activity score. Active lupus nephritis (proliferative lupus 

nephritis, WHO classes III and IV) was associated with 

significantly elevated sVCAM-1 levels. 

References 

1. Glandman DD, Urowitz MB. Systemic Lupus Erythematosus. 

In. Klippel JH, Dieppe PA. Rheumatology, Mosby, London 

1998; 7: 11-118 

2. Sfikakis PP, Mavrikakis M. Adhesion and lymphocyte 

costimulatory molecules in systemic rheumatic disease. Clin 

Rheumatol 1999; 18(4): 317-327 

3. Sharah SR, Winn R, Harlan J M. The adhesion cascade and 

anti-adhesion therapy: An overview. Springer Semin 

Immunopathol 1995; 16: 359 

4. Gearing AJ, Hemingway I, Pigott R et al. Circulating adhesion 

molecules in disease. Immunol Today 1993; 14: 506. 

5. Ikeda Y, Fujimoto T, Ameno M et al. Relationship between 

lupus nephritis activity and the serum level of soluble VCAM- 

1. Lupus 1998; 7: 347-354


Continuous convective renal replacement (CCRR) system: A new modality 

of wearable artificial kidney 

M.N. Aboras and I.A. Kawalit 

Saad Specialist Hospital, Al-Khobar, Kingdom of Saudi Arabia 

Abstract 

Wearable artificial kidney (WAK) has undergone clinical 

trials with results comparable to those of standard 

hemodialysis. Although they could have been excellent 

programs to rehabilitate End Stage Renal Disease (ESRD) 

patients and show them that despite their kidney problems, 

they could still enjoy the life. Unfortunately they were 

inconvenient due to different reasons, one of which includes 

the huge amount of hemofiltration and consequently the 

amount of replacement fluids. In this article will present our 

design of the WAK that theoretically intended to over come 

previous problems. 

Key words: hemodialysis, End Stage Renal Disease, 

wearable artificial kidney 

patients, the feasibility of using continuous hemofiltration in 

the regular design, as long-term dialysis program is very 

difficult. But the concept of continuous renal replacement 

therapy encouraged scientist to develop the wearable model 

of artificial kidney. 

Although “Wearable artificial kidneys” were intended to be 

excellent programs to rehabilitate renal patients and show 

them that despite their kidney problems, they could still enjoy 

the life. Unfortunately, they were inconvenient due to 

different reasons, such as the huge amount of hemofiltration 

and consequently the amount of replacement fluids, the 

volume of the design, the weight, the vascular access, and 

anticoagulation that made the realization of such a design not 

feasible. 

Diffusive versus Convective transport systems 

Introduction 

Dialysis as a practical treatment for kidney failure has 

evolved over centuries. Many have played a role in 

developing this medical technology, starting with Thomas 

Graham of Glasgow, who first presented the principles of 

solute transport across a semi permeable membrane in 1854 

(1). And since then continuous efforts were made to develop 

the ideal dialysis machine for patients with End Stage Renal 

Disease (ESRD) and while medical technology has made 

tremendous strides in treating kidney disease, quality of life 

issues and high mortality rates underscore the limitations of 

long-term dialysis. 

Intermittent Hemodialysis (HD) the most popular treatment 

for ESRD consumes about 12 to 15 hours weekly of the 

patient’s life during the dialysis sessions. Continuous 

Ambulatory Peritoneal Dialysis (CAPD) is a more 

convenient method but it needs special kind of intellectual 

and well-educated patients in order to reduce the frequency of 

peritonitis episodes. Haemofiltration has been developing 

during the last three decades as a possible alternative to the 

haemodialysis treatment. 

Recent clinical data comparing effective dose delivery by 

three acute dialysis therapies: continuous venovenous 

hemofiltration (CVVH), daily HD, and sustained lowefficiency 

dialysis (SLED), found that effective small solute 

clearance in CVVH is 8% and 60% higher than in SLED and 

daily HD, respectively. Differences were more pronounced 

for middle and large solute categories, the superior middle 

and large solute removal for CVVH is due to the powerful 

combination of convection and continuous operation (3, 4). 

Although it would seem an excellent program for ESRD 

______________________ 

Correspodence to: 

Before going over our design we will give a brief description 

on the differences between diffusive transport and convective 

transport systems to help understand our design of the 

Wearable artificial kidney (5-8). 

Diffusive transport 

Diffusion is the physical phenomenon upon which the 

process of hemodialysis depends. It is driven by the 

concentration differences between blood and dialysate 

compartments and the solutes pass through the 

semiperemeable membrane and it is most efficient for the 

non-protein bound, low-molecular weight solutes. 

Convective transport 

In contrast, in the convective therapies, the solvents are 

eliminated by solvent drag, secondary to the removal of 

plasma water from the blood stream. 

Convective transport is more efficient for transport of middle 

and high molecular weight non-protein bound molecules, 

which can pass through the greater pores in the applied 

membranes. 

Of course, also the elimination of potentially toxic middle 

and high molecular weight molecules could be improved. At 

least this has been shown for β 2 micro globulin which might 

be involved in arthralgia, carpal tunnel syndrome, neuropathy 

and bone disease. Also other molecules, like for instance the 

advanced glycation end products (AGEs) (9,10), which 

recently have been shown to be associated with 

cardiovascular disease, might be eliminated in a more 

M.N. Aboras, Saad Specialist Hospital, Al-Khobar, Kingdom of Saudi Arabia


efficient way, as well as inflammatory molecules like 

interleukins and endotoxins 

Continuous Convective Renal Replacement Therapy as a 

Wearable Artificial Kidney 

Haemofiltration, being superior to haemodialysis in its 

efficacy (5,10), could not be applied as an alternative for 

chronic renal failure treatment, due to the huge amounts of 

highly purified fluid and electrolytes replacement needed to 

be infused intravascularly. We present here our suggested 

innovation, which could overcome all the inconveniences of 

both haemodialysis and haemofiltration. 

Definition 

It is a continuous, portable, wearable and disposable renal 

replacement therapy system, depending on convective 

elimination of water and nitrogenous waist products, which 

can be used as a long-term treatment of end-stage renal 

failure. 

Description 

1. Vascular Access: 

Is a prerequisite for initiation of the therapy? The most 

convenient vascular access modality is Titanium 

subcutaneous catheters product of PakuMed® Medical 

products gmbh. Two ports are surgically inserted: one in the 

central venous system through the right internal jugular vein 

or the right subclavian vein, the other is installed in the 

arterial system through the right internal jugular or the right 

subclavian (innominate artery) Figure 1. (1) 

A third Titan Port catheter could optionally be implanted 

surgically opposite the suprapubic area, subcutaneously 

extending to inside of the urinary bladder. 

Fig 1. Graphic design illustrating the Continuous Convective Renal Replacement system 

Belt 

Arterial line 

Hollow fiber 

hemofilter 

Venous line 

Hemofiltrate 

collecting bag 

The Continuous Convective Renal Replacement system 

(Figure 1) is designed in the form of a tight jacket or belt to 

be worn by the patient. The material of the jacket can be any 

solid, light, leathery non-allergic substance or tissue water 

resistant. The jacket extends from the shoulders superiorly 

down to the hypogastrium inferiorly. The outside surface of 

the jacket is grooved for embedded bloodlines. Two sets of 

bloodlines are embedded. Another groove is prepared for the 

site of a small haemofilter. On the inside surface, there is two 

protruding needles gage 14 to 16, situated opposite the sites 

of the Titan Port. These needles are protected with plastic 

covers, to be removed at the time of wearing the jacket, and 

the needles will puncture the skin opposite the Titan Ports. 

In the blood line grooves, are embedded two sets of lines: a 

red coded set extending from the arterial needle to the arterial 

port of the haemofilter, while the blue coded set extends from 

the venous port of the haemofilter to the venous needle. A 

third wide bored needle (gage 14) protrudes from the lower 

end of the jacket opposite the vesicle Teten Port. This needle 

is connected to the ultrafiltration orifice of the haemofilter 

through a drainage line. 

The whole system of bloodlines, hemofilter and the needles 

are primed with heparinized sterile normal saline. 

2. The haemofilter: 

Is a hollow fiber, or parallel plate dialyser composed of a 

high flux, biocompatible membrane. The total surface area, 

the porosity, the membrane thickness and the length of the 

dialyser are to be calculated to give a rate of hemofiltration is 

about 250 ml/hour. Blood inlet and outlet are situated at both 

ends of the haemofilter, and a hemofiltrate drainage outlet is 

situated on the side of the haemofilter at the arterial end. It’s 

connected to either a urine bag or to the vesicle TITAN 

PORT. 

The whole system is completely sterile and packed in a sterile 

elegant package labeled with sufficient information and 

instructions about the system usage. A user manual is 

included inside the pack. 

The driving force of the extracorporeal circulation depends 

on the arterio-venous pressure gradient without the use of any 

sort of pump or any source of power. 

3. Replacement fluid: 

In this modality, the replacement fluid is to be given orally to 

the patient. Sachets containing salts, replacement materials,


vitamins and sugar are to be prepared. Different formulae of 

these sachets are to be prepared according to the nature of the 

most common clinical presentations: e.g. those patients who 

are salt loosing or salt retaining, those who are usually 

acidotic should be given high alkali (sodium bicarbonate) 

content and so on. 

The patient is to be instructed to dissolve the contents of one 

or two sachets in an amount of water to be calculated by his 

physician according to the degree of water retention, the 

degree of dehydration or over hydration, and the degree of 

residual renal function etc… 

4. Anticoagulation: 

Could be achieved either by an oral anticoagulant 

(Coumadine) with adjustment of PT and INR; or low 

molecular weight heparin (Clexan) in a prophylactic 12 

hourly dose. 

Modified design 

This model is designed for patient who can’t tolerate large 

volume of replacement fluid. It has the same idea of a 

continuous, portable, disposable renal replacement therapy 

system, it depends on hemofiltration properties (convection) 

but, in addition, it has hemodialysis properties (diffusion) and 

better control of ultrafiltrate volume and consequently fluid 

replacement. This design is techniquely sophisticated. 

Description: 

The modified design (Figure 2) is divided into three filters; 

the first one is a high flux filter will refer to as dialyzer-1, 

second one is very low flux filter will refer to as V-filter and 

a third one is a low flux-high efficiency filter with dialysate 

compartment will refer to as dialyzer-2. 

Fig 2. 

Dialyzer-1 intended for the medium and large molecular 

weight solutes clearance and the V-Filter is used to 

ultrafiltrate the drainage from dialyzer-1 to decrease volume 

loss (fluid loss), and by doing so the filtrate blood from 

dialyzer-1 and the ultafiltrate from the V-Filter will pass to 

dialyzer-2 which has a high surface area and small to medium 

size pores. Dialyzer-2 has fluid, which act as dialysate fluid 

contains removable adsorbent cartilages around its casing bag 

to keep the fluid at continuous lower concentration of 

diffusible solutes. 

The filtrate drainage from the V-Filter, which contains a 

concentrated large molecular weight waist solutes with low 

volume, will be drained with the drained filtered of the 

Dilayzer-2 into a one-way valve urinal bag attached to the 

patient’s leg. 

In addition, the dialyzer-2 dialysate fluid casing will have a 

sensor for the most important diffusible solutes that will 

alarm or change in colour to notify the patients to change the 

adsorbent cartilage and/or exchange dialysate fluid. Our 

intention is to be once daily. 

References 

1. Graham T. The Bakeries lecture on osmotic force. 

Philosophical Transactions of the Royal Society in London. 

1854; 144: 177–228 

2. Ludger A, Graf H, Prager R et al. Continuous arteriovenous 

hemofiltration in the therapy of acute renal insufficiency. Wien 

Klin Wochenschr 1984; 6: 96(1): 1-4 

3. Liao Z, Zhang W, Hardy PA et al. Kinetic comparison of 

different acute dialysis therapies. Artif Organs 2003; 27 (9): 

802-7 

4. Graham T. The Bakerian lecture—on osmotic force. Philos 

Trans R Soc Lond 1854; 144:177–228 

5. Bland LA, Favero MS. Microbiologic aspects of hemodialysis 

systems. In AAMI Standards and Recommended Practices, vol. 

3. Arlington, VA: Association for the Advancement of Medical 

Instrumentation 1993; 257–265 

6. Daniels F, Alberty RA. Physical Chemistry. New York : John 

Wiley& Sons 1955. 

7. Gottschalk CW, Fellner SK. History of the science of dialysis. 

Am J Nephrol 1997; 17(3-4): 289-98 

8. Gerdemann A. Wagner Z, Solf A, et al. Plasma levels of 

advanced glycation end products during haemodialysis, 

haemodiafiltration and haemofiltration: potential importance of 

dialysate quality. Nephrol Dial Transplant 2002; 17(6): 1045-9 

9. Lin CL, Huang CC, Yu CC et al. Reduction of advanced 

glycation end product levels by on-line hemodiafiltration in 

long-term hemodialysis patients. Am J Kidney Dis 2003; 42(3): 

524 

10. Subramanian, Sanjay; Kellum, John A. Convection of diffusion 

in continuous renal replacement therapy for sepsis. Current 

Opinion in Critical Care 2000; 6(6): 426-430


Prevalence of hepatitis C virus infection in dialysis patients 

P. Dzekova 1 , A. Slavkovska 2 , L. Simjanovska 2 , I. Nikolov 1 , A. Sikole 1 , R. Grozdanovski 1 , A. Asani 1 , G. 

Selim 1 , S. Gelev 1 , V. Amitov 1 , G. Spasovski 1 and M. Polenakovic 1 

1 Department of Nephrology, Clinical Center, Skopje, Republic of Macedonia 

2 Research Center for genetic engineering and biotechnology, Macedonian Academy of Sciences and Arts, Skopje, 

Republic of Macedonia 

Abstract 

Hemodialysis patients are at particularly high risk for 

hepatitis C virus infection because of the exposure to blood 

products and contaminated equipment. The high prevalence 

of HCV infection in dialysis patients is of great concern 

because these patients have a higher mortality than HCV 

negative patients. 

Our study aimed to evaluate the prevalence of HCV infection 

among patients at our dialysis unit. 

A cohort of 178 patients on maintenance hemodialysis at the 

Department of Nephrology, Clinical Center Skopje, was 

retrospectively evaluated for the levels of transaminases and 

the number of transfused blood products. The presence of 

HCV antibodies was determined by second-generation assay 

and the presence or absence of HCV RNA in the serum of the 

patients was determined by reverse-transcriptase PCR. 

The results from this study showed that from 178 patients, 

114 (64%) were anti-HCV positive and 64 (36%) were anti- 

HCV negative. The duration of dialysis, transaminase levels 

and number of transfusions was significantly higher in anti- 

HCV positive group of patients when compared with the anti- 

HCV negative group. 

The high prevalence of anti-HCV positive patients in our 

dialysis unit was associated with greater dialysis duration, 

blood products transfusions and liver enzyme levels. 

Measures to prevent the spread of HCV in dialysis units 

should include isolation of HCV RNA positive patients 

equipment and possibly their treatment with interferon 

therapy. 

Key words: hepatitis C virus, prevalence, dialysis patients 

Introduction 

Hemodialysis patients are at particularly high risk for 

hepatitis C (HCV) virus infection because of the exposure to 

blood products and contaminated equipment. There is 

considerable variation in the prevalence of anti-HCV in 

dialysis units worldwide, ranging from as low as 1% to as 

high as 63%. Risk factors for spread include a history of 

transfusion, number of blood products transfused, and 

number of years on hemodialysis therapy (1). The high 

prevalence of HCV infection in dialysis patients is of great 

concern because these patients have a higher mortality than 

HCV negative patients (2). Although HCV transmission 

through blood products transfusion previously was a 

significant source of infection, current cases are more likely 

related to nosocomial exposure (3). Hepatitis C virus is 

characterized by spectrum of outcomes. Asymptomatic in the 

vast majority of patients, transition from acute to chronic 

infection is usually without notice (4,5). In addition to viral 

and environmental factors, host genetic and immunological 

factors are believed to exert an impact on outcome of HCV 

infection (6). The mechanisms involved in viral clearance are 

not yet fully understood, although increasing evidence 

suggests that cellular immune responses to HCV play a 

central role (7). The aim of this study was to evaluate the 

prevalence of HCV infection among patients at our dialysis 

unit. 

Patients and Methods 

The study was carried on 178 (113 men) patients on 

maintenance hemodialysis at the Department of Nephrology, 

Clinical Center, Skopje. The mean age of patients was 54.8 

years and mean time on dialysis 86 months. The levels of 

liver aminotransferase enzymes: alanine aminotransferase 

(ALT) and aspartate aminotransferase (AST), and the number 

of transfused blood products, were recorded from the 

histories of the patients. The presence of HCV antibodies was 

determined by second-generation assay. The presence or 

absence of HCV RNA in the serum of the patients was 

determined by reverse-transcriptase PCR (RT/PCR) at 

Research Center for genetic engineering and biotechnology, 

Macedonian Academy of Sciences and Arts, Skopje. 

Student’s t-test was used for group mean comparison 

between anti-HCV positive and anti-HCV negative patients. 

Results 

Fig 1. Distribution of anti-HCV positive and anti-HCV 

negative patients in the study 

anti-HCV - 

64(36%) 

anti-HCV + anti-HCV - 

anti-HCV + 

114 (64%) 

The results from this study showed that from 178 patients, 

114 (64%) were anti-HCV positive and 64 (36%) were anti- 

______________________ 

Correspodence to: P. Dzekova, Department of Nephrology, Clinical Center Skopje, Medical Faculty, 

University "Sts. Cyril and Methodius", Republic of Macedonia


HCV negative (Figure.1). 

The mean values of age, duration on dialysis, levels of AST 

and ALT, and number of transfusions in anti-HCV positive 

and anti-HCV negative patients, and comparison between 

them are given in Table 1. 

Table 1. Mean values of age, duration on dialysis, levels of AST and ALT, and number of transfusions, and 

comparison between anti-HCV positive and anti-HCV negative patients 

anti-HCV positive anti-HCV negative p value 

age (years) 53.81 ± 13.61 56.62 ± 12.06 NS 

duration (months) 103.7 ± 71.6 53.41 ±33.7 0.00 

AST (U/L) 37.87 ± 21.96 23.05 ±11.63 0.00 

ALT (U/L) 53.74 ± 34.71 25.06 ± 14.05 0.00 

No. of transfusions 8.91 ± 13.53 4.84 ± 5.27 0.03 

The duration of dialysis in months was significantly longer in 

anti-HCV positive than in anti-HCV negative patients (103 

vs. 53, p


Influence of inflammation on nutritional status of dialysis patients 

P. Dzekova, I. Nikolov, G. Severova-Andreevska, A. Sikole, R. Grozdanovski, A. Asani, G. Selim, 

S. Gelev, V. Amitov, G. Spasovski and M. Polenakovic 

Department of Nephrology, Clinical Center Skopje, Medical Faculty, University Sts Cyril and Methodius, Republic of Macedonia 

Abstract 

Malnutrition and inflammation are frequently observed in 

maintenance hemodialysis patients as risk factors for poor 

quality of life and increased morbidity and mortality. The 

causes and consequences of both are anorexia, 

hypoalbuminemia, muscle wasting, refractory anemia, and, 

possibly accelerated arteriosclerosis. 

The aim of our study was to evaluate the influence of 

inflammation on parameters of nutritional status dialysis 

patients at the Department of Nephrology, Clinical Center 

Skopje. 

We crossectionally analysed 154 dialysis patients (93 men) 

which were then followed in a period of six consecutive 

months. A number of biochemical and anthropometrical 

parameters were evaluated as measures of nutritional status. 

The mean (±SD) age of all patients was 54.8 ± 12.7 years and 

vintage (duration of dialysis therapy) from 7 to 288 months. 

CRP and serum albumin levels were significantly greater in 

men than in women (12.9 vs. 7.97, p < 0.04 and 40.2 vs. 38.8, 

p < 0.02, respectively). Triceps skin fold, mid arm 

circumference and body mass index measurements were 

statistically greater in women than in men (p < 0.01). CRP 

level strongly correlated only with serum concentration of 

cholesterol (r =0.49, p

BANTAO Journal 2006: 4 (1): p 9 

Results 

Our results showed that CRP level was significantly greater 

in men than in women (12.9 vs. 7.97, p < 0.04). Serum 

albumin level was significantly greater in men than in women 

(40.2 vs. 38.8, p < 0.02). Serum cholesterol level tended to be 

greater in men than in women (p=NS). Triceps skin fold 

measurements were statistically greater in women than in 

men (1.37 vs. 0.96, p < 0.001). Mid arm circumference and 

body mass index were greater in women, whereas mid arm 

muscle circumference was greater in men, but not statistically 

(Table 1). 

Table 1. Laboratory and anthropometrical data, and a comparison 

between men and women 

men women p< 

No. of patients 93 61 

CRP (mg/l) 12.9 ±16.9 7.97 ± 10.9 0.04 

albumin (g/l) 40.2 ±4.0 38.8 ± 3.7 0.02 

cholesterol 

(mmol/L) 3.7 ± 1.4 3.2 ± 1.5 NS 

TSF (cm) 0.96 ±0.3 1.37 ± 0.6 0.0001 

MAC (cm) 26.3 ± 2.7 27.3 ± 4.4 NS 

MAMC (cm) 23.27 ± 3.1 23.03 ± 3.5 NS 

BMI (kg/m2) 23.03 ± 3.2 23.7 ± 4.7 NS 

CRP level showed strong correlation only with serum 

concentration of cholesterol (r =0.49, p


Unfavorable prognostic factors in Immunoglobulin A Nephropathy 

L. Petrovic, S. Curic, I. Mitic, D. Bozic, S. Vodopivec, V. Sakac, T. Djurdjevic-Mirkovic and T. Ilic 

Clinical Centar Novi Sad, Clinic for Nephrology and Clinical Immunology, Novi Sad, Serbia and Montenegro 

Abstract 

Immunoglobulin A nephropathy (IgAN) is a 

clinicopathological entity characterized by diffuse glomerular 

mesangial deposition of IgA as the predominant 

immunoglobulin. Renal biopsy reveals a spectrum of changes 

in the glomerul, tubulointerstitium and blood vessels. 20-50% 

of all patients develope end-stage renal failure 20 years after 

onset of the disease. 

The aim of this stady was to analized influence of 

clinicopathological and laboratory changes to prognosis of 

IgAN. The study included 60 patients with biopsy-proven 

IgAN without some other systemic diseases or purpura 

Henoch-Schnlein. We analized influence of clinical features 

of the disease, laboratory and immunofluorescence/light 

microscopy findings on the prognosis of IgAN. The study 

was partly retrospective and partly prospective. At the 

moment of renal biopsy 63,16% of patients had normal renal 

function, 31,58% had stage I and 5,25% had stage II chronic 

renal failure. At the end of study 21,05% of investigated 

patients progressed to a worse stage of renal failure with 

regard to the initial stage. 

In this study we found severe histopathological changes in 

the group with already impaired renal function and these 

changes correlated with laboratory findings, clinical features 

and prognosis of the disease. Normal renal function at the 

moment of renal biopsy provides smaller risk of further 

damage. Changes in the tubulointerstitium, mesangium, 

heavy proteinuria and hypertension influence to worse 

prognosis of disease. Crossing to the higher stage of renal 

failure was 1,24% per year and this requires long-term 

follow-up of patients with IgAN. 

Key words: IgA nephropathy, renal biopsy, outcome 

Introduction 

Immunoglobulin A nephropathy (IgAN), immunoglobulin A 

glomerulonephritis (IgAGN) or Berger’s disease is a special 

clinical/pathological entity which is characterized by the 

finding of immunofluorescent microscopy with predominant 

IgA deposits in mesangium of the glomerul. 

Morphological characteristics of the nephrotic damages in 

IgAN, similarly to clinical manifestation of the disease, are of 

a different form and degree of attack on glomerul, tubule, 

interstitium and blood vessels. Clinical progress of the 

disease is in correlation with histopathological changes with 

existence of significant clinical/pathological correlation with 

disease prognosis (1,2). Clinical and histopathological 

indicators (present at the moment of diagnosis) of the 

unfavorable disease prognosis are: 1) older age, 2) male sex, 

3) absence of episodes of macrohematuria, 4) lower intensity 

value of glomerular filtration (IGF), 5) heavier proteinuria 

(>1000mg/24h) or proteinuria of nephrotic rank, 6) arterial 

hypertension, 7) more pronounced histopathological changes, 

8) intensive IgA and C3 immunofluorescent deposits in 

glomerul and capillary wall, commonly associated with IgG 

deposits and fibrinogen are also considered to be the markers 

of unfavorable prognosis, 9) as the independent factors of the 

unfavorable disease prognosis, one can account for diabetes 

mellitus, hypertriglyceridemia, hyperuricemia, period of 

disease duration, positive family history (3-8). 

IgA nephropathy was firstly called benign recurrent 

hematuria, however, a long-term follow-up of the renal 

function has shown that IgAN represents a slow, progressive 

illness of a different result which includes a high incidence of 

progressive damage of renal function with development of 

terminal nephritic insufficiency in 20-50% of the diseased, 20 

years after the beginning of the disease. The research has 

shown that the damage of the renal function is developed 

faster in patients with an initial impairment of renal function, 

in contrast to patients with relatively normal renal function at 

diagnosis (4,6). The most significant prognostic factors in 

IgA nephropathy are histopathological changes, so that the 

expressed mesangial proliferation, glomerulosclerosis, 

presence of crescent formations, arteriosclerosis, interstitial 

fibrosis represent histological markers of unfavorable 

prognosis. Glomerulosclerosis, focal-segmented and 

especially diffuse, influence renal function substantially more 

intensively than the proliferative changes. Tubulointerstitial 

infiltrate, especially T cellular (CD4+, CD8+) is in 

correlation with faster progression of the disease. Interstitial 

fibrosis, regardless of simultaneous presence or absence of 

glomerulosclerosis, is also a significant prognostic factor. 

This explains different rate of renal function damaging in 

patients with the same level of glomerular damage, but 

different considering the tubulointerstitial inflammatory 

infiltrate or sclerosis (1,3,6,9,10). 

The aim of this study was to analyze the influence of clinicalpathological 

and laboratory changes on prognosis of IgAN. 

Considering the possibility of development and progression 

of chronic renal insufficiency, the justification of longer 

follow-up of the diseased was reviewed. 


Testing covered 60 patients, who had percutaneous renal 

biopsy at the Clinic of Nephrology and Clinical Immunology 

of Clinical Center Novi Sad. The tested patients had 

documentation which consisted of at least, the first control 

______________________ 

Correspodence to: L. Petrovic, Clinical Centar Novi Sad, Clinic for Nephrology and Clinical Immunology, 

Novi Sad, Serbia and Montenegro

BANTAO Journal2006: 4 (1): p 11 

examination, i.e. they were followed-up for at least one 

month up to maximum 17 years. Criterion for inclusion into 

the study was the finding of IgA deposits, as a dominant 

immunoglobulin in the mesangium of glomerul, which was 

deduced by immunofluorescent microscopy, while the 

presence of Henoch-Schonlein purpura and some other 

systematic illness were excluded. 

Immunohistological technique included the application of 

standard antiserums: anti-IgA, -IgG, -IgM, -C3, -C4, -C1q, - 

fibrin. The intensity of positive immunofluorescent finding 

was estimated semi-quantitatively with a precise localization 

of the deposits. 

Results 

Patients’ age ranged from 14-56 years, with an average of 

34.19 years. From the cohort of 60 patients, 70% were of 

male sex, while 30% of them were female. 

At the moment of renal biopsy, i.e. the diagnostics of the 

disease, 36 patients (63.16%) had a normal renal function 

(creatinine < 107 mol/l, clearance creatinine > 90 

ml/min/m 2 ), 18 patients (31.58%) were in stadium I of renal 

insufficiency (creatinine < 133 mol/l, clearance of creatinine 

60-90 ml/min/m 2 ) and 3 patients (5.26%) were in stadium II 

of renal insufficiency (creatinine 133-440 mol/L, clearance 

of creatinine 10-60 ml/min/m 2 ). Out of 36 patients, who at 

the moment of diagnostics had normal renal function, 31 

patient (86,11%) preserved the renal function until the end of 

the follow-up, 5 patients (13,89%) moved to stadium I of 

renal insufficiency. 12 patients (66.67%) from the group of 

18 patients with initial stadium I of renal insufficiency 

remained in this stadium, while 6 patients (33.33%) moved to 

stadium II. 2 patients (66.67%) from the group of 3 patients 

with initial stadium II remained in this stadium, while 1 

patient (33.37%) moved to stadium III. 

If we observe the total number of patients who moved from 

one stadium of renal insufficiency to another (0I, III, 

IIIII), their total number was 12 (21.05%) for the follow-up 

period of 17 years, i.e. 1.24% of patients annually moved to a 

higher stadium in relation to the initial renal function (Table 

1). 

Table 1. Relationship between beginning and end stage renal failure 

Beginning 

stage 

Normal 

function 

Number 

Percent 

36 63,16 % 

Stage of renal function at the 

end of the study 

Number 

Percent 

Normal function 31 86,11% 

I stage of renal failure 5 13,89% 

I stage of 

renal 

failure 

II stage of 

renal 

failure 

18 31,58 % 

3 5,26 % 

I stage of renal failure 12 16,67% 

II stage of renal failure 6 33,33% 

II stage of renal failure 2 66,67% 

III stage of renal failure 1 33,37% 

57 100 % 57 100% 

Within the group of patients with normal initial renal 

function, the existence of positive correlation was identified 

among: 1) changes in mesangium and increase of creatinine 

concentration, 2) changes in mesangium and appearance of 

microhematuria in clinical manifestation of the disease, 3) 

glomerulosclerosis and higher values of systolic blood 

pressure, 4) crescent formations and severity of proteinuria 

(Table 2). 

Table 2. Positive correlation between pathological findings with 

clinical features and laboratory parameters in the group of patients 

with normal renal function at the beginning 

Pathological 

Degree of 

Laboratory findings 

findings 

correlation 

Creatinine concentraction 

Mesangium increase 

r=0,47 

Microhematuria 

r=0,35 

Glomerulosclerosis Systolic blood presure r=0,21 

Crescent Proteinuria r=0,24 

The conclusions of the testing of patients with the initial 

stadium I renal insufficiency are the following: 1) there is an 

easy connection of changes in mesangium with the increase 

of creatinine concentration, higher values of systolic and 

diastolic blood pressure, 2) there is a significant correlation 

of glomerulosclerosis with microhematuria and heavier 

proteinuria, 3) there is an easy correlation between the 

presence of crescent formations and heavier proteinuria, 4) 

the changes in interstitium are in correlation with systolic, 

diastolic blood pressure and heavier proteinuria, 5) the 

changes in tubule are in easy correlation with a heavier 

proteinuria and the appearance of microhematuria (Table 3). 

Table 3. Positive correlation between pathological findings with 

clinical features and laboratory parameters in the group of patients 

with Ist stage of renal failure 

Pathological 

findings 

Laboratory findings 

Degree of 

correlation 

Creatinine concentration 

increase 

r=0,30 

Mesangium Systolic blood pressure r=0,37 

Diastolic blood pressure r=0,31 

Glomerulosclerosis Proteinuria r=0,35 


r=0,66 

Crescent Proteinuria r=0,35 

Proteinuria 

r=0,71 

Intestitium Systolic blood pressure r=0,39 

Diastolic blood pressure r=0,35 

Tubulus 

Proteinuria 

r=0,33 


r=0,25


Conclusions 

In this study, we found severe histopathological changes in 

the group with already impaired renal function and these 

changes correlated with laboratory findings, clinical features 

and prognosis of disease. Normal renal function at the 

moment of renal biopsy provides smaller risk of further 

damage. Changes in the tubulointerstitium, mesangium, 

heavy proteinuria and hypertension influence the worsening 

of prognosis of the disease. Crossing to the higher stage of 

renal failure was 1.24% per year and this requires a long-term 

follow-up of patients with IgAN. 

References 

1. Amico GD. Natural history of idiopathic IgA nephropathy: role 

of clinical and histological prognostic factors. Am J Kidney Dis 

2000; 36 (2): 227-37 

2. Droz D. IgA nephropathy: clinicopathologic correlations. In: 

Clarkson A. IgA Nephropathy. Martinus Nijhoff Publishing, 

Boston 1987; 97-101 

3. Wyatt RJ, Kritchevsky SB, Woodford SY et al. IgA 

nephropathy: long-term prognosis for pediatric patients. J 

Pediatr 1995; 127 (6): 913-919 

4. Ibels LS, Gyory AZ. IgAN: analysis of the natural history, 

important factors in the progression of of renal disease, and 

review of the literature. Medicine-Baltimore 1994; 73(2): 79- 

102 

5. Droz D. IgA nephropathy: clinicopathologic correlations. In: 

Clarkson A. IgA Nephropathy. Martinus Nijhoff Publishing, 

Boston, 1987; 101-102 

6. Donadio JV, Bergstra EJ, KP. Offord et al. Clinical and 

histopathologic associations with impaired renal function in 

IgAN. Clin Nephrol 1994; 41(2): 65-71 

7. Kenji W, Shigene N, Seicli M et al. Risk Factors for IgA 

Nephropathy: A Case-Control Study with Incident Cases in 

Japan. Nephron 2002; 90(1): 16-23 

8. Mustonen J, Syrjanen J, Rantala I et al. A. Clinical course and 

treatment of IgA nephropathy. J Nephrol 2001; 14: 440-446 

9. Alexopoulos E, Papaghianni A, Papadimitriou M. The 

pathogenetic significance of C5b-9 in IgA nephropathy. 

Nephrol Dial Transplant 1995; 10(7): 1166-72 

10. Daniel L, Saingra Y, Giorgi R et al. Tubular lesions determine 

prognosis of IgA nephropathy. Am J Kidney Dis 2000; 35(1): 

13-20


Congestive heart failure and renal dysfunction 

D. Monova 1 , E. Peneva 1 and S. Monov 2 

Department of Internal Diseases, Medical Institute 1 – MIA, Medical University 2 , Sofia, Bulgaria 

Abstract 

The kidney plays a key role in the homeostatic maintenance 

of fluids and electrolytes in the context of chronic congestive 

heart failure (CHF). CHF carries a spectrum of 

pathophysiological aberrations, which constitute a stress on 

the respective renal regulatory mechanisms. 

We defined bedside clinical, laboratory and 

electrocardiographic parameters characterizing CHF patients 

with and without concomitant renal failure (RF), and 

analyzed their impact on mortality. We studied 146 

symptomatic unselected consecutive furosemide-treated CHF 

patients hospitalized for various acute conditions. On 

admission, history taking, physical examination, chest x-ray, 

ECG and routine laboratory tests were performed. 

Subsequently, patients were divided into 2 subgroups, those 

with serum creatinine > 140 µmol/l (58) and those with 

lower values (88). Prevailing in RF subgroup were older age, 

male gender, admission pulmonary edema, cardiac 

arrhythmias, cardiac condition disturbances, lower ejection 

fraction, anemia, higher furosemide maintenance dosages, 

insulin treatment and receiving less ACE inhibitors. RF being 

the parameter most significantly associated with low survival. 

Using multivariate analysis in the RF subgroup, older age, 

female gender and diabetes mellitus proved most 

significantly associated with poorer survival. In the non-RF 

subgroup, only older age and diabetes mellitus were 

significantly associated with low survival. 

Renal failure is a marker of severity in CHF. Its full-blown 

deleterious prognostic effect is already manifested at serum 

creatinine 140 µmol/l. Older age, diabetes mellitus and 

female gender significantly heralded a shorter survival. Such 

patients require special care. 

Key words: congestive heart failure, renal failure, diabetes 

mellitus, gender, age 

Introduction 

The kidney plays a key role in the homeostatic maintenance 

of fluids and electrolytes in the context of chronic congestive 

heart failure (CHF)(1). CHF carries a spectrum of 

pathophysiological aberrations, which constitute a stress on 

the respective renal regulatory mechanisms. It has been 

shown that the presence of renal failure in patients with 

congestive heart failure is associated with shorter survival (2- 

4). Moreover, renal failure, even moderate, represents not 

only a marker for poor prognosis, but also an independent 

risk factor for increased mortality (3). 

We defined bedside clinical, laboratory and 

electrocardiographic parameters, characterizing CHF patients 

with and without concomitant renal failure (RF), and 

analyzed their impact on mortality. 


We studied 146 symptomatic unselected furosemide-treated 

patients with congestive heart failure, hospitalized for various 

acute conditions. Congestive heart failure was of various 

etiologies and had been present for at least 6 months prior to 

admission. All patients had been on chronic furosemide 

treatments of at least 40 mg/day for more than 3 months. 

On admission, blood was drawn for serum biochemical 

determinations, including glucose, creatinine, chloride, 

potassium, sodium, bicarbonate, blood pH, calcium, 

phosphorus, cholesterol, triglycerides and blood count. All 

determinations were performed using conventional methods. 

The history taking, physical examination, chest x-ray and 

ECG were performed. Subsequently, patients were divided 

into 2 subgroups, those with serum creatinine > 140 µmol/l 

(58 patients) and those with lower values (88 patients). 

Results and discussion 

The study group included 146 patients (84 females and 62 

males, mean (±SD) age 71,4±19,6 years). Renal failure was 

present in 58 (39,73%) patients. Table 1 depicts the mean 

values of quantitative variables in the patients with renal 

failure as compared to those with normal creatinine values. 

Table 1. Mean values of quantitative variables in congestive heart 

failure patients with versus without renal failure 

Characteristic 

Patients with Patients without 

renal failure renal failure 

Weight (kg) 68,7±12,4 70,4±13,6 

Age (years) 65,4±6,4 60,3±8,3 

Serum potassium 4,71±0,82 4,5±0,52 

(mmol/l) 

Serum bicarbonate 24,4±4,22 26,24±3,96 

(mmol/l) 

Serum chloride 

104,0±5,4 102,88±4,6 

(mmol/l) 

Serum phosphorous 3,8±0,8 3,5±0,5 

(mmol/l) 

Blood pH 7,37±0,06 7,39±0,05 

It can be seen that patients with renal failure were 

significantly older (P


Table 2. Qualitative variables prevailing in congestive heart failure 

patients with versus without renal failure 

Characteristic 

Patients with 

renal failure 

- N(%) 

Patients 

without renal 

failure – N(%) 

Male gender 37 (63,79%) 25 (28,41%) 

Pulmonary edema on 

admission 

36 (62,07%) 29 (32,95%) 

Cardiac arrhythmias on 

34 (58,62%) 28 (31,82%) 

admission 

NYHA class III-IV 35 (60,34%) 31 (35,23%) 

Ejection fraction < 30% 25 (43,10%) 15 (17,04%) 

Presence of anaemia 30 (51,72%) 20 (22,73%) 

Furosemide dosage>80mg 20 (34,48%) 10 (11,36%) 

Treatment with ACE 

inhibitors 

28 (48,28%) 59 (67,04%) 

Male gender, admission pulmonary edema, cardiac 

arrhythmias (atrial or ventricular premature beats >6 min, 

couplets, atrial fibrillation, supraventricular or ventricular 

tachycardia), severe CHF, lower ejection fraction, anemia, 

higher furosemide maintenance dosages and receiving less 

ACE inhibitors prevailed in the subgroup with renal failure. 

Duration of furosemide treatment, presence of hypertension, 

periferal vascular disease, ischemic heart disease, chronic 

obstructive pulmonary disease, hypokalemia or hyponatremia 

on admission, treatment with calcium channel blockers, 

nitrates or digoxin, were not significantly different between 

the two subgroups. 

The follow-up period extended up to 40 months, median 

34±12 months. During this period 24 (16,44%) patients died. 

17 patients (70,83%) were from subgroup with renal failure. 

Renal failure was the parameter most significantly associated 

with low survival. Using multivariate analysis in the 

subgroup with renal failure, older age, severity of CHF, 

female gender proved to be significantly associated with 

poorer survival. In the subgroup without renal failure, only 

older age was significantly associated with low survival. 

Parameters which did not influence survival in the subgroup 

with renal failure, included duration of furosemide treatment, 

hypertension, hyperlipidemia, anaemia, various cardiac 

arrhythmias, use of vasodilators or digoxin and hypokalemia 

or hyponatremia on admission. 

The pathophysiological interrelationship between CHF and 

renal insufficiency is bidirectional, involving a variety of 

factors (1). Activation of the neurohormonal and reninangiotensin 

systems, mainly via reduction of effective blood 

volume in CHF leads to stimulation of aldosterone and ADH 

secretion. These may produce a rise in cardiac filling 

pressure, increased extracelular volume, edema and organ 

dysfunction, including deterioration of CHF. Within the 

context of the kidney, intravascular volume depletion and the 

ensuing GFR reduction, eventually limit natriuresis and 

diuretic capacity despite enhanced nitric oxide production 

and activation of natriuretic substances. Superposition of 

renal failure on CHF implies an additional burden of poor 

prognostic factors. These may include various electrolite and 

acid-base balance aberrations, decreased immunological 

competence, osteoporosis, enhanced bleeding tendency and 

other factors, which may shorten survival from cardiac and 

non-cardiac death (5). 

Conclusions 

Renal failure is a marker of severity in CHF. Its full-blown 

deleterious prognostic effect is already manifested at serum 

creatinine of 140 µmol/l. Older age and a female gender 

significantly heralded a shorter survival. We were unable to 

find such association in the literature (3). Such patients 

require special care. Following discharge, patients were 

managed exclusively by their primary physicians. Therefore 

our results are not biased by other factors such as those 

introduced by pharmaceutically oriented studies or those 

involving intervention by specialized heart failure clinics. 

References 

1. Zanchetti, A and Stella A. Cardiovascular disease and the 

kidney: an epidemiologic overview. – J Cardiovasc. Pharmacol 

1999; 33, 1-6 

2. Al-Ahmad, Rand WM, Manjundth G et al. Reduced kidney 

function and anemia as risk factors for mortality in patients 

with left ventricular dysfunction. – J Am Coll Cardiol 2001; 

38: 955-962 

3. Dries DL, Exner DV, Domanski MJ et al. The prognostic 

implications of renal insufficiency in asymptomatic and 

symptomatic patients with left ventricular systolic dysfunction. 

– J Am Coll Cardiol 2000; 35: 681-689 

4. Mosterd A, Cast B, Hoes AW et al. The prognosis of heart 

failure in the general population: The Rotterdam Study - Eur 

Heart J 2001; 22: 1318-1327 

5. Muntner P, Hamm L, Lorid C et al. Renal insufficiency and 

subsequent death resulting from cardiovascular disease in the 

United States. – J Am Soc Nephrol 2002; 13: 745-75


Sporadic Balkan Endemic Nephropathy (BEN) beyond the known regions of 

BEN 

J. Nikolic¹, T. Pejcic¹, D. Crnomarkovic¹, Z. Gavrilovic² and C. Tulic¹ 

¹Urologic Clinic-Faculty of Medicine Belgrade, ² Institute for developement of water resources, Jaroslav Cerni, 

Belgrade, Serbia and Montenegro 

Abstract 

There is an very old opinion that we diagnose only most 

prominent BEN cases and miss latent and mild ones, that 

unrecognized BEN is possible in endemic villages and the 

number of such cases, their characteristics, and origin are 

unknown. 

From 1955 to 1998 year we evaluated 1235 patients with 

histologicaly proven upper urothelial tumors, who underwent 

surgery. The diagnosis of sporadic BEN, was made after 

exclusion of other known nephropaties. 

In the analyzed region, BEN was present in 170 of 808 

settlements, and in 104 of them, 534 cases of UUT were 

reported. In 66 possible BEN settlements, there were no UUT 

cases. In the next 457 of 808 settlements (56.6%) neither 

BEN, or UUT were registered. 

Sporadic BEN is an atypical, mild clinical variety of BEN, 

with simultaneous presence of UUT. Both sexes were equally 

affected, with the majority of patients in sixth and seventh 

decade of. The presence of sporadic BEN cases gives a 

possibility to speculate that the existence of BEN without 

familiar agreggations is possible elsevere, not only in 

Balkans. 

Key words: Balkan Endemic Nephropathy, familiar 

agreggations, distribution 

Introduction 

The idea of »ice berg phenomenon«, (1,2) suggesting that we 

diagnose only most prominent BEN cases and miss latent and 

mild ones, is very old. There is a similar opinion, that 

unrecognized BEN is possible in endemic villages (in 

»healthy« famillies), and in the neighborhood of BEN areas, 

(3) but the number of such cases, their characteristics, and 

origin are unknown. Territorial distribution of cases with 

upper urothelial tumors (UUT) in Serbia is much wider than 

distribution of BEN (4, 5), suggesting the similar distribution 

of BEN. 

The diagnosis of sporadic BEN , presented as non-famillar, 

non-endemic disease (6), is hardly possible with standard 

criteria, because two main criteria for the diagnosis are 

missing. 


urologists and patients from Serbia came from the region of 

31 communities with 808 settlements, with total of 1.162.225 

inhabitants (5). 

The diagnosis of sporadic BEN, was made in patients that 

underwent surgery because of UUT, after exclusion of other 

known nephropaties. The presence of UUT in all such cases, 

enables the diagnosis, knowing that other nephropaties 

followed by UUT (CHN, AN etc.) are extremely rare in 

Serbia. The data source of territorial distribution of BEN, was 

standard one (7). 

Non-endemic villages with sporadic BEN cases were divided 

into three groups, according to the number of cases (more 

than 3, 2, or 1 case) in settlement, during four decades of 

observation. The first group of settlements with sporadic 

BEN (≥ 3 cases) from non-endemic settlements is shown on 

the map, compared to 104 villages from BEN register, with 

simultaneous UUT presence. 

Results 

In the analyzed region, BEN was present in 170 of 808 

settlements (7), and in 104 of them, 534 cases of UUT were 

reported. In 66 possible BEN settlements, there were no UUT 

cases. In the next 457 of 808 settlements (56.6% ) neither 

BEN, or UUT were registered (Table 1). 

Table 1. The presence of BEN and the number of UUT in the 

analyzed region villages, according to BEN register (1955- 1998 

year) 

BEN Villages UUT cases 

From 1955 to 1998 year, 1235 patients with histologicaly 

proven UUT, underwent surgery. Data collected from 

______________________ 

Correspodence to: J. Nikolic, Urologic Clinic-Faculty of Medicine Belgrade, Jaroslav Cerni, 

Belgrade, Serbia and Montenegro 

Tumors per 

village 

BEN present 40 301 7,5 

BEN possible 64 233 3,6 

No BEN 33 145 4,39 

No BEN 36 72 2 

No BEN 95 95 1 

No BEN 457 0 0 

BEN possible 66 0 0 

The UUT was registered in 285 of 808 settlements. The UUT 

cases from 16 cities (182 pts.) and Belgrade (207 pts. ), with 

no autochtonous BEN cases, were excluded from the study. 

We found 312 sporadic BEN cases, with concomitant UUT, 

in 164 non-endemic settlements. The both sexes were equally 

affected: 140 males with average age of 61.06 years, and 172 

females with an average age of 64.99 years.


Table 2. The number and sex distribution of sporadic BEN cases in settlements 

Settlement Community 

Sporadic 

BEN 

Males Females Inhabitants 

1 Orljevo Petrovac 3 3 - 546 

2 Srpce Kuevo 3 2 1 293 

3 Rakinac Vel.Plana 3 - 3 1404 

4 Kula M.Crnie 3 - 3 908 

5 Klievac Požarevac 3 2 1 2084 

6 Nemenikue Sopot 3 2 1 1933 

7 Brzohode Žabari 3 - 3 1225 

8 Bošnjak Petrovac 3 2 1 696 

9 V.Crnie M.Crnie 3 - 3 926 

10 irikovac Požarevac 3 1 2 1645 

11 Kuevo Kuevo 3 1 2 4846 

12 Živica Požarevac 3 - 3 914 

13 Sopot Sopot 3 - 3 1720 

14 M.Ivana Sopot 3 1 2 1857 

15 Vrani Barajevo 3 1 2 3288 

16 Golubac Golubac 3 1 2 1995 

17 Viteževo Žabari 4 2 2 1402 

18 Voluja Kuevo 4 1 3 1318 

19 Vel.Selo M.Crnie 4 2 2 694 

20 Brgule Ub 4 2 2 1356 

21 Selevac Sm.Palanka 4 2 2 4618 

22 Dubravica Požarevac 4 1 3 1521 

23 Boževac M.Crnie 5 1 4 2480 

24 Toponica M.Crnie 5 3 2 1374 

25 Vel.Laole Petrovac 5 1 4 2694 

26 Mala Krsna Smederevo 5 2 3 1829 

27 Carevac V.Gradište 5 2 3 1102 

28 Rabrovo Kuevo 5 2 3 1441 

29 Ranovac Petrovac 6 1 5 2787 

30 Vrin Grocka 8 6 2 8034 

31 Dragovac Požarevac 8 2 6 1166 

32 Barajevo Barajevo 10 6 4 6243 

33 Porodin Žabari 11 6 5 3454 

145 59 86 69793 

Discussion 

The diagnosis of BEN is not absolutely accurate, just very 

possible (8). The similar situation is with sporadic BEN 

cases, where simultaneous occurrence of UUT, after 

exclusion of all other nephropathies, makes a diagnosis more 

accurate. Both sexes were affected equally, the patients were 

in sixth or seventh decade of life, with mild to moderate 

degree of renal failure in 39,4%. In the most endemic BEN 

areas, the patients with BEN and UUT, had renal failure in 

60% (9). The cases with BEN and UUT, presented without 

the signs of renal damage at the time of surgery for UUT, 

developed renal failure later on. 

The distribution of 312 sporadic BEN cases in 164 nonendemic 

settlements was: 33 villages with ≥ 3 cases (4.39 per 

settlement), 36 villages with two, and 95 with only one case. 

Like in our previous report (5), the territorial distribution of 

villages with various UUT incidence had a concentric, rather 

than mosaic pattern. The endemic villages with highest 

incidence, were situated in central and the lowest parts of the 

river valleys, and less endemic, in their surroundings and 

higher altitudes. These 33 villages with sporadic BEN, were 

located like a ring, around groups of BEN villages. The same 

pattern existed in the valleys of rivers Kolubara, Morava and 

Mlava.The villages with two and one sporadic BEN cases, 

were located over wider territory. 

The basic differencies between BEN and sporadic BEN, are 

that there are no young sporadic BEN cases with very small 

kidneys and severe renal failure, and there is no familiar 

occurence of the disease. The incidence of sporadic cases in 

the settlements, during 40 years of observation, was one case 

every 10, 20 or 40 years. 

One can speculate that sporadic BEN is the result of low 

concentration of ethiological agent in nature and therefore the 

incidence is minimal, the age of onset is high, and the renal 

failure is not severe, with normal or slightly smaller kidneys. 

Here documented territory, endangered with BEN, including 

sporadic BEN cases, is actually much larger, which means 

that the distribution of ethiologic agent in the nature is not 

limited only in known endemic or hypoendemic areas. 

Conclusions 

Sporadic BEN is an atypical, mild clinical variety of BEN, 

with simultaneous presence of UUT, characterized with very 

low incidence in the non-endemic areas and without familiar 

aglommeration, which are the crucial caracteristics of BEN. 

The number of sporadic BEN cases without UUT, remains 

undetected, because of rigidity in diagnostic criteria and the 

abscence of specific findings. 

We found 312 sporadic BEN cases in 164 non-endemic 

villages.


Both sexes were equally affected, with the majority of 

patients in sixth and seventh decade of life (male average age 

was 61.06, and female average age was 64.99 years). 

Moderate or mild renal failure was found in 39.4%. 

The 33 villages with ≥ 3 sporadic BEN, were located in the 

circle arround the known BEN zones, certifying the diagnosis 

of BEN. The rest of villages were spread over wider territory. 

The presence of sporadic BEN cases gives a possibility to 

speculate that the existence of BEN without familar 

agreggations, with low incidence in the settlement (one case 

per few decades) is possible elsevere, not only in Balkans, 

but it is still impossible to detect sporadic BEN using the 

standard diagnostic procedures. 

Map 1. Territorial distribution of 33 settlements with sporadic BEN cases associated with UUT cases, 

localized out of known BEN regions. 

References 

1. Zacharia C. Uber endemische Nephropatien (Nierenatrophien) 

eine epidemioologische untersuchung. In: Int Symp on 

Endemic Nephropathy. Sofia 1963. Bulg. Acad Sc Press, Sofia 

1965; 73-76 

2. Zacharia C. A modern approach to the concept of familiar 

endemic nephropathy. Rev Roum Med 1978; Med Int 16:2 

3. Polenakovi M, Stefanovi V. Balkan Nephropathy. In: Oxford 

Textbook of Clinical Nephrology. Oxford medical Publications. 

Sec ed. Eds. Davidson A., Cameron JS, Grunfeld JP, Kerr 

DNS, Ritz E and Winearls Ch. Oxford, Oxford University 

Press, 1998; 1202-1210 

4. Nikoli J, Djoki M, Todorovi D et al. 1998; Characteristic of 

patients suffering from ureteral or renal pelvic tumors in lower 

flow of the Morava and Mlava rivers: Third Congr.of the 

Balkan Cities Associastion of Nephrology, Dialysis, 

Transplantation and Arteficial organs, Belgrade, 1998; abstract 

book p42 

5. Nikoli J, Djoki M, Crnomarkovi D et al. Upper urothelial 

tumors and Balkan nephropathy-dose responsible diseases in 

Facta Universitatis univ of Nis, vol 9, No1, 2002; 114-118 

6. Danilovi V. Endemic nephropathy in Yugoslavia. In: Endemic 

(Balkan) nephropathy, Proc. 4th Symp., Niš 1979. Eds. S. 

Strahinji V. Stefanovi Instr Nephr Haemod Niš 1981; 1-5 

7. Radovanovi Z. Topographical distribution of the Balkan 

endemic nephropathy in Serbia (Yugoslavia). Trop geogr Med 

1979; 31: 185-189 

8. Radoni M, eovi S, Radoševi Z. Clinical picture of 

Endemic nephropathy 3rd simp on Endemic Nephropathy, 

Beograd 1982 ed.S. Petkovi SANU, book XXlll Beograd 

1985; 87-196 (in serbo-croat) 

9. Djoki M, Hadžidjoki J, Nikoli J et al. Comparison des 

tumeurs de haut appareil urinaire dans la region de la 

nepphropatie balkanique avec celles d,autres regions de 

Yugoslavie. Prog Urol 1999; 61-68 

10. Cancer incidence in five continents, data for Vojvodina. 2005; 

htpp//www.janc.fr


Amyloidosis in Turkish patients 

C. Ensari 1 and A. Ensari 2 

1 Ankara Atatürk Research Hospital Dialysis Unit, 2 University of Ankara, Medical School - Turkey 

Abstract 

Amyloidosis is characterized by the systemic or localized 

extra cellular deposition of amyloid, a proteinaceous fibrillar 

material in various tissues and organs, mainly classified into 

primary (AL) or secondary (AA) groups according to the 

biochemical nature of the fibril forming protein. 

The histopathological diagnosis was made on Congo red 

stained biopsy specimens using polarized light and the 

subtyping of amyloidosis was made by immunohistochemical 

analysis. One hundred twenty eight amyloid positive biopsies 

obtained from 111 patients whose paraffin blocks were 

available for immunohistochemical analysis were included in 

the study. 111 patients (70 male) with an average age of 34.2 

years (range 6-65 years), were included in the study. 

Amyloid deposits were subtyped immunohistochemically in 

128 biopsies. The vast majority (90.9 %) of patients in our 

study presented with AA amyloidosis. 

This retrospective study demonstrates that the patient 

population with amyloidosis in Turkey is significantly 

different from the western countries. Our results indicate the 

predominance of AA amyloidosis and also suggest that 

routine immunohistochemical analysis of amyloidosis cases 

with certain ethnic background is sufficient to classify the 

subtype of amyloid fibril protein and the related disease. 

Key words: amyloidosis, histopathological diagnosis, 

immunohistochemistry 

Introduction 

Amyloidosis is characterized by the systemic or localized 

extra cellular deposition of amyloid, a proteinaceous fibrillar 

material in various tissues and organs. Although many types 

of fibril proteins have been defined, amyloidosis is mainly 

classified into primary (AL) or secondary (AA) groups 

according to the biochemical nature of the fibril forming 

protein. The results of previously published reports on 

amyloidosis including autopsy series strongly suggested that 

the frequency and type of amyloidosis and related disease 

may vary significantly in different patient populations (1-4). 

There is not much information in the literature concerning the 

accuracy of immunohistochemical subtyping in relation to 

clinical diagnosis (5-7), though classifying primary 

amyloidosis by immunohistochemistry has been reported to 

be more difficult (8). 

The aim of the present study was, therefore, to classify 

amyloidosis by means of immunohistochemical subtyping of 

amyloid fibril proteins and assessing the correlation of 

immunohistochemistry with clinical diagnosis in order to 

retrieve useful epidemiological data from these patients. 


The histopathological diagnosis was made on Congo red 

stained biopsy specimens using polarized light and the 

subtyping of amyloidosis was made by immunohistochemical 

analysis. One hundred twenty eight amyloid positive biopsies 

obtained from 111 patients whose paraffin blocks were 

available for immunohistochemical analysis were included in 

the study. Mean patient age was 34.2 years with a range of 6- 

65 years. There were 70 male, 41 female patients in the 

study. 

Paraffin sections, 4 microns thick, were taken from 128 

biopsy specimens comprising 69 renal, 36 rectal, 15 

testicular, 5 liver, 2 small intestinal biopsies and 1 bladder 

biopsy. An antibody panel including monoclonal antibodies 

to Amyloid A (1:100), lambda (1:50) and kappa (1:50) light 

chains, transthyretin (1:100), and beta-2-microglobulin (1:50) 

(DAKO), leaving out the extremely rare forms of amyloid 

fibrils, was selected for the study. Patient files were reviewed 

and clinical histories were recorded. 

Table 1: The results of immunohistochemical analysis 

Biopsy site # of 

biopsies 

AA AL AK ATTR β2MG Mixed 

Kidney 69 65 4 (2 AA+AL) 

(2 AA+ATTR) 

Rectum 36 35 1 (AA+ATTR) 

Testicle 15 10 4 1 

(AA+AL+ATTR) 

Liver 5 0 2 1 2 (1 AA+AL) 

(1 AA+β2MG) 

Small Int. 2 2 

Bladder 1 1 

Total 128 113 6 0 0 1 8 

______________________ 

Correspodence to: C. Ensari, Ankara Atatürk Research Hospital Dialysis Unit, Ankara, Turkey


Results 

Amyloid deposits were subtyped immunohistochemically in 

128 biopsies taken from 111 cases. In 17 cases, multiple 

organ biopsies were taken; 10 cases had renal + rectal 

biopsies, whereas 7 cases had renal + testicular biopsies. 

The results of the immunohistochemical analysis are 

presented in Table 1. Amyloid deposits were stained by a 

single antibody in 120 (93.8 %) whereas 8 biopsies (6.2 %) 

stained positively by more than one antibody. 

Table 2: The relation of clinical diagnosis to biopsy site 

Disease 

# of # of 

Small 

Kidney Rectum Testicle Liver 

cases biopsy 

Intestine 

Bladder 

FMF 81 98 48 36 10 2 1 1 

Tuberculosis 8 8 8 

Bronchiectasis 4 4 4 

RA 7 7 7 

Crohn’s Disease 1 1 1 

Plasma CD 7 7 1 4 2 

Hemodialysis 1 1 1 

Unknown 2 2 1 1 

Total 111 128 69 36 15 5 2 1 

FMF: Familial Mediterranean Fever, RA: Rheumatoid Arthritis, Plasma CD: Plasma cell dyscrasias 

The relation between clinical diagnosis and biopsy site is 

presented in Table 2. The correlation of 

immunohistochemical subtyping and clinical diagnosis is 

summarized in Table 3. 

Table 3: The correlation of immunohistochemical sub typing and clinical diagnosis 

Disease 

# of 

cases 

# of 

biopsy 

AA AL AK ATTR β2MG Mixed 

3 (1 AA+AL) 

FMF 81 98 95 (1 AA+ATTR) 

(1 AA+β2MG) 

Tuberculosis 8 8 8 

Bronchiectasis 4 4 4 

RA 7 7 5 2 (2 AA+ATTR) 

Plasma CD 7 7 6 1 (1 AL+AA) 

Crohn’s Disease 1 1 1 

Hemodialysis 1 1 1 

Unknown 2 2 

2 (1 AA+AL) 

(1AA+AL+ATTR) 

Total 111 128 113 6 0 0 1 8 

FMF: Familial Mediterranean Fever, RA: Rheumatoid Arthritis, Plasma CD: Plasma cell dyscrasias 

Discussion 

Lachmann et al (8), reported that immunohistochemistry is 

usually definitive in identifying reactive (AA) amyloidosis, 

though, it is frequently not diagnostic with respect to AL 

amyloidosis either due to abnormal fibril conformation of 

light chain fragments or to the background staining caused by 

normal immunoglobulins in the tissues. In a recent study, 

immunofluorescence technique yielded negative staining in 

35.3 % of cases with proven AL amyloidosis in renal 

biopsies (9). Since the vast majority (90.9 %) of patients in 

our study presented with AA amyloidosis, the technical 

limitations of immunohistochemistry are not evident in our 

patients except for two cases (1.8 %) in which no clinical 

condition related to amyloidosis could be documented. In 

contrast to the reports from western countries where the 

majority of cases are AL amyloidosis (8, 9), the few cases 

with AL amyloidosis in the present study were also 

accurately diagnosed by means of immunohistochemistry. 

Amyloidosis can be diagnosed and classified in any affected 

tissue sample. Nevertheless, it is still important to define the 

most suitable diagnostic site to be biopsied and examined for 

the early detection of amyloid deposition. For systemic 

amyloidosis, however, no such favorite site has been 

documented in any of the studies performed so far (2, 3) 

although rectal biopsy seems to be more commonly used 

mainly because of its easy access by the clinician (3, 10). 

Another easy access, abdominal fat biopsy has been reported 

as insensitive for AA amyloidosis while renal biopsy, 

probably due to the increase in the number of well-trained 

nephrologists, is becoming more popular in the diagnosis of 

amyloidosis (4). Similarly, it was the most commonly used 

intervention to diagnose amyloidosis in our study. In practice, 

however, clinical presentation seems to be the main 

determinant of the biopsy site; i.e. a patient with proteinuria 

would undergo a renal rather than a rectal biopsy. Renal or 

rectal biopsies were performed commonly in our cases 

depending on the presenting clinical symptoms. Though a 

variety of anatomical sites have been used for the detection of 

amyloid (3,4), we found only few reports mentioning 

testicular biopsy despite the high incidence observed in our 

study (11,12). In one of these reports, testis biopsy was found 

valuable and more sensitive than rectal biopsy in the 

diagnosis of systemic amyloidosis (12). 

In many developed countries primary amyloidosis is the more 

common form of systemic amyloidosis (1-3). With the virtual


abolition of chronic infectious diseases, the incidence of 

secondary amyloidosis has been reduced in such countries. 

However, it still occurs mainly in patients suffering from RA 

followed by chronic infectious diseases and Crohn’s disease 

while TBC and FMF were found to be very rare as amyloid 

related diseases (5,13). On the other hand, secondary 

amyloidosis is still the more common type of systemic 

amyloidosis in developing countries (1,3) where TBC 

together with other chronic infectious diseases are the leading 

causes of systemic amyloidosis followed by RA which is 

observed only in a small percentage of patients with 

amyloidosis (14,15). In the Middle East, however, among 

patients with different ethnic origins, FMF is the leading 

etiologic condition in the development of amyloidosis. This 

ethnically restricted genetic disorder mainly affects 

populations originated from the Mediterranean-Middle 

Eastern populations such as Jews, Arabs, Armenians and 

Turks (16). In a recent report, a lower incidence of 

amyloidosis was observed in a group of Italian patients with 

FMF (17). Although the true Figures are not well 

documented, Turkey is known to have a high prevalence of 

FMF and FMF-related amyloidosis (18,19). 

Not surprisingly, secondary amyloidosis is the main form of 

systemic amyloidosis in our cases, many with a clinical 

diagnosis of FMF followed by TBC, RA and bronchiectasis 

in similar frequencies. This finding seems to be in 

concordance with previous reports from Turkey (19,20). 

Conclusions 

This retrospective study demonstrates that the patient 

population with amyloidosis in Turkey is significantly 

different from the western countries. Our results indicate the 

predominance of AA amyloidosis associated with FMF and 

also suggest that routine immunohistochemical analysis of 

amyloidosis cases with certain ethnic background is sufficient 

to classify the subtype of amyloid fibril protein and the 

related disease. However, detailed clinical information is 

mandatory before making a definite diagnosis based solely on 

immunohistochemical observations. Moreover, due to 

increased rate of immigration affecting the ethnic structure of 

the developed countries, physicians’ awareness of the 

prevalence of certain diseases related to ethnic origin is 

essential to prevent long term serious complications such as 

amyloidosis and improve the quality of patients’ lives. 

References 

1. Simms RW, Prout MN, Cohen AS. The epidemiology of AL 

and AA amyloidosis. Baillieres Clin Rheumatol 1994; 8: 627- 

634 

2. Falk RH, Comenzo RL, Skinner M. The systemic amyloidoses. 

N Eng J Med 1997; 337: 898-909 

3. Sipe JD, Cohen AS. Amyloidosis. In Braunwald A, Fauci AS, 

Kasper DL, Hauser SL, Longo DL, Jameson JL, Eds. 

Harrison’s Principles of Internal Medicine (15 th edn). New- 

York: McGraw-Hill 2001; 1974-1979 

4. Röcken C, Sletten K. Amyloid in surgical pathology. Virchows 

Arch 2003; 443: 3-16 

5. Hoshii Y, Takahashi M, Ishihara T, Uchino F. 

Immunohistochemical classification of 140 autopsy cases with 

systemic amyloidosis. Am J Pathol 1994; 4: 352-358 

6. Röcken C, Schwotzer EB, Linke RP, Saeger W. The 

classification of amyloid deposits in clinicopathological 

practice. Histopathol 1996; 29: 325-335 

7. Strege RJ, Saeger W, Linke RP. Diagnosis and 

immunohistochemical classification of systemic amyloidoses. 

Virchows Arch 1998; 433: 19-27 

8. Lachmann HS, Booth DR, Booth SE, Bybee A, Gilbertson JA, 

Gilmore JD, Pepys MB, Hawkins PN: Misdiagnosis of 

hereditary amyloidosis as AL (primasy) amyloidosis. N Eng J 

Med 2002; 346: 1786-1791 

9. Novak L, Cook WJ, Herrera GA, Sanders PW. AL-amyloidosis 

is underdiagnosed in renal biopsies. Nephrol Dial Transplant 

2004; 19: 3050-3053 

10. Lee JG, Wilson JA, Gottfried MR. Gastrointestinal 

manifestations of amyloidosis. South Med J 1994; 87: 243-247 

11. Haimov-Kochman R, Prus D, Ben-Chetrit E. Azoospermia due 

to testicular amyloidosis in a patient with familial 

Mediterranean fever. Hum Reprod 2001; 16: 1218-1220 

12. Ozdemir BH, Ozdemir OG, Ozdemir FN, Ozdemir AI. Value 

of testis biopsy in the diagnosis of systemic amyloidosis. 

Urology 2002; 59: 201-205 

13. Joss N, McLaughlin K, Simpson K, Boulton-Jones JM. 

Presentation, survival and prognostic markers in AA 

amyloidosis. QJM 2000; 93: 535-542 

14. Shah VB, Phatak AM, Shah BS, Kandalkar BM, Haldankar 

AR, Ranganathan S. Renal amyloidosis – a clinicopathologic 

study. Indian J Pathol Microbiol 1996; 39: 179-185 

15. McAdam KP, Raynes JG, Alpers MP, Westermark GT, 

Westermark P. Amyloidosis: a global problem common in 

Papua New Guinea. P N G Med J 1996; 39: 284-296 

16. Orbach H, Ben-Chetrit E. Familial Mediterranean fever – a 

review and update. Minerva Med 2001; 92: 421-430 

17. Regina ML, Nucera G, Diaco M, Procopio A, Gasbarrini G, 

Notarnicola C, Kone-Paut I, Touitou I, Manna R. Familial 

Mediterranean fever is no longer a rare disease in Italy. Eur J 

Hum Genet 2003; 11: 50-56 

18. Yazici H, Özdogan H. Familial Mediterranean Fever in Turkey. 

In: Sohar E, Gafni J, Pras M Eds. Familial Mediterranean 

Fever. London and Tel Aviv: Freund Publishing House Ltd 

1997; 66-71 

19. Tuglular S, Yalcinkaya F, Paydas S, Oner A, Utas C, 

Bozfakioglu S, Ataman R, Akpolat T, Ok E, Sen S, Dusunsel 

R, Evrenkaya R, Akoglu E. A retrospective analysis for 

etiology and clinical findings of 287 secondary amyloidosis 

cases in Turkey. Nephrol Dial Transplant 2002; 17: 2003-2005 

20. Paydas S. Report on 59 patients with renal amyloidosis. Int 

Urol Nephrol 1999; 31: 619-631


Frequency of Alport syndrome at dialysis center “Vrsac”- Vrsac 

K. Djordjev¹, D. Tintor¹, G. Djurdjev¹, O. Vasilic-Kokotovic¹, I. Sokolovac² and S. Bozic³ 

¹Department of Nephrology and Haemodialysis, Health Center “Vrsac”, Vrsac; ²Clinic of ORL,University of Medicine, 

Novi Sad; ³Department of Ophtalmology, Health Center “Vrsac”, Vrsac, Serbia and Montenegro 

Abstract 


Alport syndrome (AS) is a hereditary nephropathy 

characterized by persistent haematuria that may evolve to end 

stage renal disease, associated with sensorineural hearing loss, 

ocular abnormalities. Th aim of the study was to establish 

correct diagnosis and to estimate the frequency of disease. 

We evauated 44 members in four generations from one Gipsy 

family in Vrsac county. Through the family inquiry and a 

review of medical records, the genealogic tree was made. The 

frequency of Alport syndrome in Dialysis Center Vrsac is 

12.5% (5 out of 40 patients). 

Considerably great frequency of AS in our dialysis center is 

confirmed in comparison to other centers in our country, 

which is probably caused by consanguinity or mutation that 

is not connected with X chromosome, or due to other factors 

that are not of genetic nature like lifestyle, diet, infections or 

something else. 

Key words: Alport syndrome, disease frequency 

Introduction 

Alport syndrome (AS) is a hereditary nephropathy 

characterized by persistent haematuria that may evolve to end 

stage renal disease (ESRD). This syndrome is associated with 

sensorineural hearing loss, ocular abnormalities (anterior 

lenticonus, macular or peripheral flecks), and ultrastructural 

abnormalities of the glomerular basement membrane (GBM) 

(1). Mutation in collagen type IV genes are responsible for 

disorders of glomerular basement membrane, that produce 

broad spectrum of disease phenotypes ranging from benign 

haematuria to most severe forms of AS. This leads us to 

agglutinate different entities under the name of “collagen 

type IV” nephropathy (2). AS displays considerable 

phenotypic and genetic heterogeneity (3), and this syndrome 

is relatively rare. There are three genetic forms of AS exist: 

X-linked form (accounts for 85% cases), autosomal-recessive 

(responsible for approximately 10-15% of cases; and 

autosomal-dominant form (less than 1%), in at least some 

families. The estimated gene frequency ratio of AS is 1:5000 

(5). 

This study is implemented with the aim to establish correct 

diagnosis, to estimate frequency of disease, to make a 

genealogical tree, to collect data from epidemiological, 

ophthalmic, audiometric tests and to compare them with the 

other experiences from literature, and to confirm the type of 

heredity through a review the genealogic tree. 

A great frequency of Alport syndrome is presented in Vrsac 

county. There is a large percentage of this phenomenon 

causing end stage renal disease (ESRD) at our center and it is 

12.5% (5 out of 40); and this percentage is high and is very 

likely to increase. All the patients are from one Gipsy family, 

at this moment with 44 members in four generations, and all 

of them live in the same village 65 km away from Belgrade. 

The oldest alive member is 40, and the youngest one is onemonth-old 

baby. 

Results 

Through the family inquiry, as well as the review of medical 

records, the genealogic tree was made (Figure 1). The 

frequency of Alport syndrome in Dialysis Center Vrsac is 

12.5% (5 out of 40 patients). AS is considered the be a cause 

of approximately 0.6 to 2.3% of ESRD in Europe and the 

United States (6,7), and from the registry 2004 in Serbia and 

Montenegro it is less than 1%. This proportion is probably 

underestimated because of the diagnostic difficulties. 

Average ages when started with dialysis is 21.5 years for 

males and for females is 35 years (with the deceased family 

members). The male patients III:5, III:6, III:12 and III:13 

started the dialysis at the age of 22, 21, 23 and 24 

respectively, and the female patient II:2 started at the age of 

34. At present, the male patients spent average 4 years and 

female spent on dialysis 6 years. Thus the deceased members 

(two female and one male patient) from the same family, 

started the dialysis at the age of 41 and 31(females), and 17 

(male) and spent less than one month, the first, and less than 

two years, the other female; and less than a year for the male. 

All patients have had macroscopic haematuria before they 

started with dialysis. They should probably have started 

sooner, given the fact that they had come in a very bad 

condition, with extremely high values of serum creatinine and 

blood urea concentrations and had urgently started with 

haemodialysis. They have not consulted a doctor before, 

considering that they had painful haematuria, nausea, 

frequent vomiting, and a peripheral edema. Clinical 

examinations have shown a great percentage of extrarenal 

abnormalities with variability of signs, which is rare. All the 

patients who were on dialysis have progressive sensorineural 

deaffnes (5 out of 5), both for high and low frequencies with 

similar findings in the study of autosomal dominant 

nephropathy (8). Ocular abnormalities had 4 out of 5 patients, 

except a female patient. They had lenticonus anterior and 

retinitis pigmentosa. Two biopsies at light microscopy 

indicated disorders of GBM. Three out of five patients on 

______________________ 

Correspodence to: K. Djordjev, Department of Nephrology and Haemodialysis, Health Center “Vrsac”, 

Vrsac, Serbia and Montenegro


dialysis had hypercholesterolaemia (total cholesterol more 

than 8.3, 7.1 and 6.7 mm/l) (9), but unlike those who had 

Fetchner syndrome, they didn’t have trombopenia (10). 

Complications occured in a form of arterial hypertension in 4 

out of 5 patients. All of them are anemic (average value of 

haemaglobin was 11.7 g/dl) and they were all on Epoetin 

Beta (Recormon®). Two out of five patients were hepatitis 

“B” seropositive. They had mild intellectual deficit. In 

addition to this, the patients were very undisciplined, had a 

great increase in weight interdialysis and had a poor social 

background. 

Conclusions 

This data obviously leads to a confirmation of the diagnosis 

of Alport syndrome, as a severe (juvenile) form (


Eearly effects of the AT 2 receptor antagonist eprosartane mesylate (EM) in 

diabetic patients with and without chronic renal failure 

N. Nnchev, B. Deliyska, M. Bankova, P. Radulova, G. Kimenov and E. Marinova 

Medical University, Sofia, Bulgaria 

Abstract 

ATII receptor antagonists are effective in the control of the 

arterial hypertension (AH) in patients with CRF. Different 

factors take part in the pathogenesis of the AH in the diabetic 

mellitus (DM) patients: volume overload, sodium retention, 

renin-angiotensin system (RAAS), activity of the sympatic 

system (SNS) and endothelin system. 

The aim of the study is assessment of the effectiveness of the 

EM on the blood pressure (BP) and pulse pressure (PP) 

which are incriminated factors for the cardiovascular 

morbidity and mortality. 

22 patients (14 male and 8 female) with DM and AH, mean 

age 52±8 years, divided in 2 groups were followed for 3- 

month period. On 0, 1 st , 4 th , 12 th week the following 

parameters were followed: systolic BP, diastolic BP, mean 

arterial pressure (MAP), PP, heart rate, weight, height, basic 

laboratory tests. All the patients received 600 mg EM 

(Teveten ® ) once a day in the morning (10 of them as a mono 

therapy and the rest in a combination with other 

antihypertensive therapy, which was not changed during the 

study). 

There were significant changes in the mean blood pressure 

and PP in both groups at 4 and 12 week. There were not 

significant changes in HF between the groups. At the end of 

the study mean BP decreased in group A for 20,4% and in 

group B for 26,9%. PP decreased in group A for 35,7% and 

in group B for 38,9%. Serum creatinine did not changed 

significantly neither in group A nor in group B (group A 

from 98+/-11,1 to 95,9+/-8,4 mol/l; group B from 321+/-76 

to 349+/-21,3 mol/l). We did not observed also a significant 

changes in creatinine clearance in the two groups (group A 

from 111+/-15,1 to102+/-11 mmol/min; group B from 18+/- 

2, 2 to 20,6+/-4,8 mmol/min). 

ATII receptor blocker EM leads to significant control over 

BP and PP after 4 th week in DM patients with and without 

CRF. This effect is probably due to a dual mechanism of 

action over AH – control over RAAS and SNS. The drug is 

well tolerated with no side effects. 

Key words: AT 2 receptor antagonist, diabetes mellitus, renal 

failure 

Introduction 

There is a relationship between cardiovascular and kidney 

diseases. Treatment of an early nephropathy has also 

cardioprotective effect in the hypertensive patients. 

Renoprotective drugs improve the renal and endothelial 

dysfunction that is connected with overactivation of the 

______________________ 

Correspodence to: N. Nnchev, Medical University, Sofia, Bulgaria 

renin-angiotensin-aldosterone system (RAAS). RAASdirected 

antihypertensive agents are both angiotensin 

converting enzyme (ACE) inhibitors and angiotensin II 

receptor blockers (ARBs) that have renoprotective effects. 

They decrease both high blood pressure and proteinuria (8). 

Metabolic syndrome is a cluster of common cardiovascular 

risk factors that includes hypertension and insulin resistance. 

Hypertension and diabetes mellitus are frequent 

comorbidities and like metabolic syndrome, increase the risk 

of cardiovascular events. Eprosartane, an antihypertensive 

agent with evidence of partial peroxisome proliferatoractivated 

receptor activity-gamma (PPARgamma) activity, 

may improve insulin sensitivity and lipid profile in patients 

with metabolic syndrome (1). 

The aim of the study was to evaluate early efficacy of 

eprosartane mesilate (EM) (Teveten ® -Solvei) on the blood 

pressure (BP) and pulse pressure (PP) in diabetic patients 

with and without chronic renal failure (CRF). 


The study was conducted in 22 patients with diabetic 

nephropathy. Fourteen from them were males with a mean 

age of 52±8 years. The patients were divided into 2 groups. 

Group A, 8 patients without CRF and group B, 14 CRF 

patients. The follow-up period was 12 weeks. At 0, 4 and 12 

week the followed parameters were: mean blood pressure 

(BP), PP, hearth frequency (HF), body weight, high, serum 

creatinine and creatinine clearance. All patients were treated 

with EM, 600 mg once daily. Ten patients were with 

monotheray by EM, all others received before EM another 

antihypertensive treatment and EM were additional included. 

Results 

The results of PB, PP, HF of the patients from two groups are 

shown in Table 1. 

Group A 

1 st 

Parameter Baseline 

week 

4 th week 12 th week 

MAP 113 ± 6 110 ± 5 96 ± 7 * 90 ± 5 ** 

(mm Hg) 

PP 

70 ± 8 62 ± 6 51 ± 5 * 45 ± 4 ** 

(mm Hg) 

Heart rate 75 ± 9 75 ± 4 70 ± 4 72 ± 6 

There were significant changes in the mean blood pressure 

and PP in both groups at 4 and 12 week. There were not 

significant changes in HF between the groups. At the end of


the study mean BP decreased in group A for 20,4% and in 

group B for 26,9%. PP decreased in group A for 35,7% and 

in group B for 38,9%. Serum creatinine did not changed 

significantly neither in group A nor in group B (group A 

from 98+/-11,1 to 95,9+/-8,4 mol/l; group B from 321+/-76 

to 349+/-21,3 mol/l). We did not observe also a significant 

changes in creatinine clearance in the two groups (group A 

from 111+/-15,1 to102+/-11 mmol/min; group B from 18+/- 

2, 2 to 20,6+/-4,8 mmol/min). 

Group B 

Parameter 

Baseline 

1 st 

week 

4 th 

week 

12 th week 

MAP 

(mm Hg) 

130 ± 9 127 ± 4 105 ± 7 * 95 ± 8 

PP 

(mm Hg) 

90 ± 8 100 ± 2 80 ± 5 * 55 ± 9 ** 

Heart rate 88 ± 9 86 ± 3 82 ± 4 75 ± 4 

* p < 0,01 ; ** p< 0,001 

Discussion 

Eprosartane is a well tolerated ARB drug. There were 

evidences for its reno- and cardioprotection especially in 

diabetic patients. Therapy with ARB is suitable also for 

patients with primary and secondary glomerulonephritis. The 

effectiveness of a combined therapy with ARB and ACE 

inhibitors may have been at least partly due to the greater 

inhibition of the action of angiotensin II in patients with IgA 

nephropathy. This strategy apparently reduced mild-tomoderate 

proteinuria in patients with normotensive IgA 

nephropathy (9). The angiotensin II changes glomerular 

permeselective function via the opening of large pores after 

elevations in transmembrane pressure and by acting on the 

glomerular pressure, too. There is evidence that angiotensinconverting 

enzyme inhibitors (ACEI) alone or with the 

ARB’s may improve the glomerular size-selective function 

and the hemodynamic intrarenal accounted output of plasma 

proteins that result in decreasing of proteinuria. It was not 

only by the drug related reduction in systemic blood pressure 

(4). The combination of ARB and ACEI seems to be 

beneficial and may offer an additional renoprotective effect 

(10). Hemodialysis patients have uremic dyslipidemia, 

represented by elevated serum intermediate-density 

lipoprotein cholesterol (IDL-C) levels, and an increased 

cardiovascular mortality rate. The effect may be in 

connection with changes on pulse wave velocity (PWV), 

which predicts cardiovascular morbidity and mortality. Longterm 

blockade of the RAAS may have a beneficial effect on 

the acceleration of atherosclerosis and uremic dyslipidemia in 

hemodialysis patients (5). Several recent morbidity and 

mortality trials with ARB’s provide an evidence-based 

rationale for the use of the drugs in patients with 

hypertension. Studies with comparison to conventional 

antihypertensive agents showed improved morbidity and 

mortality outcomes in patients with hypertension and left 

ventricular hypertrophy (Losartan ® ) and diabetes mellitus 

(Losartan ® and Irbesartan ® ) (13). Systemic and renal 

hemodynamic responses to acute AT1R blockade are, at least 

in part, genetically determined (12). The Seventh Report of 

the Joint National Committee on Prevention, Detection, 

Evaluation, and Treatment of High Blood Pressure 

emphasizes the urgent need to lower blood pressure to a goal 

of


converting enzyme inhibitors alone or in combination for 

treatment of primary glomerulonephritis. Scand J Urol Nephrol 

2004; 38(5): 427-33 

10. Scheen AJ. Renin-angiotensin system inhibition prevents type 

2 diabetes mellitus. Part 1. A meta-analysis of randomised 

clinical trials. Diabetes Metab 2004; 30(6): 487-96 

11. Makris TK, Stavroulakis G, Papadopoulos DP. Eprosartan 

effect on fibrinolytic/hemostatic variables in arterial 

hypertension: a comparative study to losartan. Drugs Exp Clin 

Res 2004; 30(3): 125-32 

12. Spiering W, Kroon AA, Fuss-Lejeune MJ et al. Genetic 

contribution to the acute effects of angiotensin II type 1 

receptor blockade. J Hypertens 2005; 23(4): 753-8 

13. Vitale C, Mercuro G, Castiglioni C et al. Metabolic effect of 

telmisartan and losartan in hypertensive patients with metabolic 

syndrome. Cardiovasc Diabetol 2005; 15: 4(1): 6-9


Prealbumin and inflammatory markers in dialysis patients 

S. Chrysostomou, 1 K. A. Poulia, 1 Y. Jeanes, 2 D. Perrea, 3 M. Poulakou, 3 B. Filiopoulos, 4 

D. Stamatiades 4 and Ch. P. Stathakis 4 

1 Department of Nutrition and Dietetics, Laikon General Hospital of Athens, 2 School of Human and Life Sciences, Roehampton 

University, UK, 3 Laboratory for Experimental Surgery and Surgical Research "Christeas Hall", University of Athens, 4 Division of 

Nephrology, General Hospital of Athens, Greece 

Abstract 

Inflammation is one of the main causes of high mortality and 

morbidity rates in patients with chronic renal failure treated 

with dialysis. In these cases, the use of anthropometric or 

biochemical markers alone, is not enough, but it is important 

to use the combination of them. 

The aim of this study is to evaluate serum prealbumin as an 

inflammatory and nutritional marker and determine whether 

there is an association between prealbumin and other 

anthropometric and biochemical markers. 

A total of 30, clinically stable dialysis patients (15 HD and 15 

PD) participated in the study. The investigated 

anthropometric and biochemical measurements of the study 

are body weight (BW), Body Mass Index (BMI), serum 

prealbumin, albumin, transferrin, C - reactive protein (CRP), 

creatinine, ferritin, Interleukin-6 (IL-6), cholesterol, Iron (Fe) 

and Prognostic Inflammatory and Nutritional index (PINI). 

There are no significant differences in age, duration of 

dialysis, dry weight and BMI between patients on HD or PD. 

Albumin is significantly lower in the PD group, whereas, 

transferrin and total cholesterol are significantly greater in the 

HD group. A higher percentage of the HD patients have 

inflammation with CRP>5g/l and IL-6>8.8ng/ml compared 

with the PD patients. 

According to the present study, prealbumin levels are lower 

in the PD group compared with the HD group but it is not 

significantly correlated with any of the investigated 

parameters, apart from ferritin, in the HD group. Moreover, 

further analysis showed that patients are malnourished 

independently whether they have inflammation or not. 

Key words: inflammation, dialysis, prealbumin, morbidity, 

mortality 

Introduction 

Inflammation is one of the main causes of high mortality and 

morbidity rates in patients with chronic renal failure treated 

with dialysis methods (1). In these cases, the use of 

anthropometric or biochemical markers alone, is not enough. 

It is very important to use the combination of them, in order 

to have more reliable assessment (1). Further studies in this 

area, are required to examine the association between 

markers of visceral protein stores, inflammatory markers and 

clinical outcomes, in dialysis patients (2). 

Prealbumin is a visceral protein with short half-life (2-3 

days), a rapid turnover and a simple method exists to measure 

serum levels (3). It is an earlier nutritional marker compared 

with serum albumin and more responsive to immediate 

changes in nutritional intake and clinical status than albumin. 

Although prealbumin is considered as a more sensitive index 

compared with albumin (4), it is not commonly used and its 

associations with other inflammatory and nutritional indices, 

during End Stage Renal Disease (ESRD), have not been well 

documented (4). 

Inflammation is a key determinant of serum prealbumin (an 

acute phase protein) (4). However, a few studies support that 

prealbumin is a reliable marker of inflammation but still more 

studies are required to confirm this hypothesis (2). 

The aim of this study is to evaluate serum prealbumin as an 

inflammatory and nutritional marker and determine whether 

there is an association between prealbumin and other 

anthropometric and biochemical markers. Furthermore, this 

study will compare the nutritional and inflammatory status 

between HD and PD patients, through prealbumin and other 

indices. 


The study is performed at the Outpatient Dialysis Unit at 

“Laikon” General Hospital (Athens, Greece). A total of 30 

dialysis patients (15 HD and 15 PD) participated in the study. 

Moreover, only clinically stable patients are included in this 

study. 

An Ethical approval is granted from University of Surrey 

Roehampton ethics committee and approval from the director 

of Laikon General Hospital of Athens. 

The investigated anthropometric and biochemical 

measurements of the study are body weight (BW), Body 

Mass Index (BMI), percentage body fat derived from skinfold 

thickness measurements (%BF) and serum prealbumin, 

albumin, transferrin, C - reactive protein (CRP), creatinine, 

ferritin, Interleukin-6 (IL-6), cholesterol, Iron (Fe) and 

Prognostic Inflammatory and Nutritional index (PINI). The 

investigated variables of this study are compared with the 

outcome goals for dialysis patients based on Kidney Disease 

Outcome Quality Initiative (K/DOQI). Inflammatory markers 

are compared with the laboratory reference ranges. 

BMI (in kilograms per meter²) is calculated from patient 

height and dry body weight. This study considers 

undernourished patients as those with BMI< 18.5 kg/m². 

The same observer took the anthropometric measurements, 

immediately before the dialysis session (weight empty of 

dialysis fluid). Skinfold measurement was taken at 4 sites in 

triplicate (triceps, biceps, superilliac and subscapular 

______________________ 

Correspodence to: S. Chrysostomou, “Laikon” General Hospital of Athens, Department of Nephrology, 

Agiou Thoma 17, 11527, Athens-Greece


skinfolds) using Harpenden callipers (Baty, British Indicators 

CE 0120). Percentage body fat was calculated using Durnin 

and Womersley equation. 

Non-fasting blood samples were taken before dialysis 

treatment for serum prealbumin, albumin, creatinine, ferritin, 

transferrin, IL-6, CRP, cholesterol and iron. All these 

parameters, apart from prealbumin and IL-6 immunoassays, 

were analysed on the day of sampling at the Department of 

Clinical Chemistry Laboratory at “Laikon” General Hospital. 

Prealbumin and IL-6 mmunoassays were performed at the 

laboratory for Experimental Surgery and Surgical Research 

“Christeas Hall”. Serum prealbumin was measured by a 

photometric enzyme-linked assay (ELISA, Immune 

Diagnostic, Cat. No: K 6331) and serum albumin was 

determined with a photometric method (bromcresol method, 

BCG-Olympus). Cholesterol was analysed by a photometric 

enzyme-linked assay with Olympus analysers (RA-XT 1000). 

Serum transferrin, iron and ferritin were analysed by 

Tholosimetry (Hitachi 912). CRP was analysed by 

nephelometry (SEAC, RADIM GROUP, DELTA) and High- 

Sensitive IL-6 was analysed by a photometric enzyme-linked 

assay (ELISA, Quantikine HS, Cat. No: HS 600B). 

The PINI value was calculated using markers from the 

biochemical tests, in the following equation: (C-reactive 

protein X alpha (1)-acid glycoprotein): (albumin x 

transthyretin) (16). 

The statistical programme SPSS for Windows (version 11.5) 

is used for data analysis. Results are presented as mean ± 

standard deviation (Mean ± SD). A P-value of 8.8ng/ml 

compared with the PD patients. Results are shown in Table 3. 

Table 3. Prevalence of inflammation based on CRP, IL-6 and 

ferritin cut off points, in our study-group 

Haemodialysis 

Group 

Peritoneal 

dialysis Group 

Marker of inflammation % % 

CRP (>5g/l) 46.7 40 

IL-6 (>8.8ng/ml) 53.3 30.8 

Ferritin Males 

(>400ng/ml) 

37.5 50 

Females 

(>150ng/ml) 

85.7 88.9 

The prevalence of malnutrition based only on prealbumin 

levels shows that a much higher percentage of PD patients 

are severely malnourished with prealbumin


prevalence on malnutrition based on the other investigated 

variables is presented. 

Table 4. Prevalence of malnutrition based on prealbumin 

Haemodialysis 

Group 

Peritoneal dialysis 

Group 

Prealbumin 

(mg/ml) 

Percent 

Percent 

0-200 13 46 

200-300 0 23 

300+ 87 31 

Malnutrition and inflammation: Malnutrition indices are 

analysed against inflammation indices. In the current study, a 

patient is characterised as malnourished if he/she is found 

malnourished for at least one nutritional marker. Moreover, a 

patient is considered to have inflammation if he/she is found 

to have values above the cut-off points for of the 

inflammatory markers. A higher percentage of PD patients 

have both malnutrition and inflammation compared with the 

HD patients as shown in Table 6. 

Table 5. Prevalence of malnutrition based on Albumin, BMI, 

Creatinine, Cholesterol, Transferrin and % Body fat cut off point in 

our study-group 

Haemodialysis 

group 

Peritoneal 

dialysis 

group 

Marker of malnutrition Percent (%) Percent (%) 

Albumin (


with any of the investigated parameters, apart from ferritin, in 

the HD group. Moreover, further analysis showed that 

patients are malnourished independently whether they have 

inflammation or not. None of the patients have inflammation 

without being malnourished at the same time. 

References 

1. Kaysen G. Malnutrition and the acute-phase reaction in dialysis 

patients-how to measure and how to distinguish. Nephrol Dial 

Transplant 2000; 15: 1521-1524 

2. Mehrotra R, Kopple J. Nutritional management of maintenance 

dialysis patients: why aren’t we doing better? Annual Review of 

Nutrition 2001; 21: 243-379 

3. Holland D, Meers C, Lawlor M et al. Serial prealbumin levels 

as predictors of outcomes in a retrospective cohort of peritoneal 

and hemodialysis patients. Journal of Renal Nutrition, 2002; 

11(3): 129-138 

4. Sreedhara R, Avram M, Blanco M et al. Prealbumin is the best 

nutritional predictor of survival in hemodialysis and peritoneal 

dialysis. Am J Kidney Dis 1996; 28: 937-942 

5. Qureshi A, Alvestrand A, Danielsson A et al. Factors 

predicting malnutrition in hemodialysis patients: A crosssectional 

study. Kidney Int, 1998; 53: 773-782 

6. Herzig K, Purdie D, Chang W et al. Is C-reactive protein a 

usefull predictor of outcome in peritoneal dialysis patients. J 

Am Soc Nephrol, 2001; 12: 814-821 

7. Owen W, Lowrie E. C-reactive protein as an outcome predictor 

for maintenance hemodialysis patients. Kidney Int 1998; 54: 

627-636 

8. Goldwasser P, Feldman J, Barth R. Serum prealbumin is higher 

in peritoneal dialysis than in hemodialysis: A meta-analysis. 

Kidney International 2002; 62: 276-281 

9. Chumlea C, Dwyer J, Bergen C et al. Nutritional status 

assessed from anthropometric measures in the HEMO study. 

Journal of Renal Nutrition, 2003; 13(1): 31-38 

10. Kooistra M, Niemantsverdriet E, Van Es A et al. Iron 

absorption in erythropoietin-treated hemodialysis patients: 

effects of iron availability, inflammation and aluminium. 

Nephrol Dial Transplant 1998; 13: 82-88


Liver and kidney damage in acute poisonings 

M. Mydlik and K. Derzsiova 

Nephrological Clinic, Faculty Hospital of L. Pasteur, Kosice, Slovak Republic 

Abstract 

In the paper the retrospective analysis of simultaneous 

damage of liver and kidneys after acute carbon tetrachloride 

poisoning in 60 patients, after mushroom poisoning (Amanita 

phalloides) in 81 patients and after ethylene glycol poisoning 

in 20 patients from the view of differential diagnosis, 

conservative, intra- and extracorporeal elimination therapy is 

described. Acute toxic hepatitis with the different degree of 

severity was present in all patients and even acute renal 

failure was present in some patients. After acute carbon 

tetrachloride poisoning acute hepatitis developed 

approximately simultaneously with the development of acute 

renal failure. After acute Amanita phalloides poisoning acute 

toxic hepatitis, which was the cause of death in liver coma in 

16 patients, quickly developed in the foreground of the 

clinical picture. Renal damage was less frequent and it was 

not the cause of death even in one patient. After acute 

ethylene glycol poisoning acute renal failure dominated with 

severe metabolic acidosis, oxaluria and leucocytosis, acute 

toxic hepatitis was less severe. After ethylene glycol 

poisoning 3 patients died in the period before the use of the 

bicarbonate haemodialysis was possible. During 30 years the 

intra- and extracorporeal elimination therapy qualitatively 

developed (bicarbonate haemodialysis, haemoperfusion 

through active charcoal and other sorbents, plasmapheresis, 

haemofiltration), which participates to the significant degree 

on the recovery and improvement of the prognosis of patients 

after acute poisonings with simultaneous damage of liver and 

kidneys. 

Key words: acute poisoning, carbon tetrachloride, 

mushroom (Amanita phalloides), ethylene glycol, hepatic 

and renal damage, extracorporeal elimination therapy 

Introduction 

Simultaneous damage of liver and kidneys in acute poisoning 

occurs very often in present times (Table 1). This fact is very 

important from the diagnostic, differential diagnostic and 

therapeutic point of view (1-6). Besides that a damage of 

other organs after acute poisoning can cause multiorgan 

failure in patients previously healthy (2,5). This finding is 

very important in patients with preceding illness (2,7,8). 

The aim of the study was the retrospective analysis of 60 

patients after carbon tetrachloride poisoning, of 81 patients 

after mushroom poisoning (Amanita phalloides) and of 20 

patients after ethylene glycol poisoning with the liver and 

kidney damage from the diagnostic, differential diagnostic, 

conservative, intra- and extracorporeal elimination treatment 

Table 1. Simultaneous damage of liver and kidneys in acute 

poisonings 

Acute 

Damage of 

poisoning Liver Kidneys 

1. Carbon tetrachloride + + 

2. Dichlorethane + + 

3. Ethylene glycol + + 

4. Paraquat, Diquat + + 

5. Mushroom + + 

(Amanita phalloides) 

6. Arsenic hydride + + 

7. Mercury chloride - + 

8. Trichloroethylene + -+ 

9. Paracetamol + -+ 

10. Carbamazepine -+ - 

11. Isoniazid + - 

12. Other -+ -+ 

Patients and Methods 

Sixty men, mean age was 41±9 years, after carbon 

tetrachloride poisoning were investigated. Among them 52 

patients were poisoned by inhalation way and 8 patients 

drunk carbon tetrachloride in mistake of other drinks. At the 

beginning of acute poisoning dyspeptic syndrome was 

dominated, especially after oral poisoning. All patients used 

ethylalcohol for „the treatment of acute dyspepsia“. Use of 

ethylalcohol potentiated carbon tetrachloride poisoning 

(2,7,8). Clinical and laboratory signs after acute carbon 

tetrachloride poisoning from liver damage were present 

earlier ( several hours or days) in comparison to the signs of 

kidney damage. From that reason one third of poisoned 

patients was admitted to the Clinic for Infection Diseases in 

Faculty Hospital, Košice or to the other Departments of 

Infection Diseases in Eastern Slovakian region. That 

diagnostic mistake was caused by insufficient anamnesis, 

minimization of the contact with carbon tetrachloride by 

patients or deliberate concealment of the contact with that 

poison. Carbon tetrachloride is a very good lipid solvent and 

in the past it was used in home setting and in many 

workplaces (1,7,8). Except this carbon tetrachloride was 

used as a filling in fire-extinguishers (2). 

Eighty-one patients were investigated after mushroom 

poisoning (Amanita phalloides), mean age was 30.5±9 years. 

Among them were 28 children and 53 adult patients. Clinical 

and laboratory signs after acute poisoning of Amanita 

phalloides were present first of all during the several hours as 

an acute dyspepsia with the rapid increase of serum bilirubin 

and of activity of serum transaminases. Acute renal failure 

developed later or was not present. All acute poisoning 

caused by Amanita phalloides were accidental except one 

point of view. 

______________________ 

Correspondence to: M. Mydlik, Nephrological Clinic, Faculty Hospital of 

L. Pasteur, Rastislavova 43, 041 90 Košice, Slovak Republic; E-mail: k.derzsiova@ fnlp.sk


young lady, who ate Amanita phalloides in attempted suicide 

(3,9,10). 

The third group of patients were 20 patients after ethylene 

glycol poisoning. Mean age was 41± 8 years, among them 

were 2 women and 18 men. Ethylene glycol was the most 

frequent oral poisoning in a form of antifreeze agent which 

was used in the cars. That poison was used frequently in the 

change for ethylalcohol or from „ absence of ethylalcohol 

drinks“ in three soldiers. Ethylene glycol is a very dangerous 

poisoning nowadays and it is relatively frequent in our 

country in present times (3,5,6,11,12). 

In three groups of patients with those poisonings all available 

diagnostic and therapeutic methods were used including 

various forms of intra-and extracorporeal elimination 

methods (1-8,10-16). In the treatment of carbon tetrachloride 

poisoning we did not use a hyperbaric oxygen therapy (17). 

Results 

Clinical signs and some laboratory parameters, conservative 

and intra-and extracorporeal elimination treatment in the 

patients after carbon tetrachloride, mushroom (Amanita 

phalloides) and ethylene glycol poisonings were described in 

Tables 2-4. 

Table 2. Liver damage in acute poisoning 

Poisoning Number of Hepatomegaly Bi Serum AST Coma 

patients 

(µmol/L) ALT (µkat/L) 

1. Carbon tetrachloride 60 57 78.4±20.5 16.2±3.3 - 4 

2. Mushroom 

81 81 76.8±14.1 89.5±12.4 88.7±12.8 16 


3. Ethylene glycol 20 16 17.5± 2.0 1.23± 0.45 1.12±0.4 5 

Bi-bilirubin 

Table 3. Kidney damage in acute poisoning 

Poisoning Number of 

patients 

Proteinuria 

(g/24h) 

K 

(mmol/L) 

Serum 

Urea 

Creatinine 

(µmol/L) 

Metabolic 

acidosis (pH) 

1. Carbon tetrachloride 60 57 78.4±20.5 16.2±3.3 - 4 

2. Mushroom 

81 81 76.8±14.1 89.5±12.4 88.7±12.8 16 


3. Ethylene glycol 20 16 17.5± 2.0 1.23± 0.45 1.12±0.4 5 

Table 4. Intra-and extracorporeal elimination therapy in acute poisoning 

Poisoning 

Number of Intra-and extracorporeal elimination therapy 

patients PD HD ET HP PF 

Mortality 

1. Carbon tetrachloride 60 8 81 6 - 4 2 (3.3%) 

2. Mushroom 

81 - 20 2 128 23 24 (29.6%) 


3. Ethylene glycol 20 - 28Ac+147Bic - 5 - 3 (15%) 

PD-peritoneal dialysis, HD-haemodialysis, Ac-acetate, Bic-bicarbonate, ET-exchange transfuzion, 

HP-haemoperfusion, PF-plasmapheresis 

Discussion 

In differential diagnosis of the patients with simultaneous 

damage of the liver and kidneys, a direct anamnesis of 

patients is very important, but also an indirect anamnesis 

from family members or from co-workers. It is necessary to 

distinguish acute viral hepatitis from acute carbon 

tetrachloride poisoning, from poisoning by dichloroethane, 

mushroom poisoning (Amanita phalloides), arsenic hydride, 

ethylene glycol and from acute leptospirosis (1-3, 5,6,8- 

10,12,16,18). After acute paraquat poisoning and 

simultaneously with a damage of the liver and kidneys, 

the damage of lung and heart is developed. The patient who 

is insufficiently treated after paraquat poisoning is dying 

during the following 10-14 days. Cause of death after that 

poisoning is alveolocapillary block in the lung (19,20). Acute 

arsenic hydride poisoning in our department was not present 

during the last 35 years (5,21-23). Acute mercury chloride 

poisoning leads to acute renal failure without liver damage 

(5). Acute trichloroethylene poisoning is manifested by 

cerebral and liver damage. Damage of the kidneys is very 

rare but it was described (24). Acute paracetamol poisoning 

leads to liver damage, but kidney damage in some patients 

was also described (25-27). After oral carbamazepine 

poisoning important liver damage was observed (28). Liver 

damage after isoniazid poisoning was also observed. High 

intravenous dose of pyridoxine (5g/24 h) as an antidote in 

that poisoning was recommended (5). We successfully used 

this therapy in one our patient after severe isoniazid 

poisoning. 

Acute toxic hepatitis dominated in the first group of patients 

who were poisoned by carbon tetrachloride, in four of them 

developed liver coma. Two patients suffering from preceding 

chronic hepatitis died in liver coma despite of the use of 

repeated haemodialyses and exchange transfusions. 

Anamnestic serum titres against Leptospira grippotyphosa 

were found in one patient suffering from acute carbon 

tetrachloride poisoning and in another patient with the same 

poisoning acute leptospirosis developed caused by Leptospira 

sejr. Dynamics of titres testified to acute illness (2). 

Determination of serum titres against leptospirosis was very 

important especially in the patients from agricultural region. 

Uremic syndrome as a sign of acute renal failure in acute 

carbon tetrachloride poisoning developed relatively slowly in 

comparison to other acute poisonings (2). Duration of acute 

carbon tetrachloride poisoning until to restore to normal of 

hepatic tests and of renal functions was in average 39±9 days. 

The treatment of carbon tetrachloride poisoning included: use


of hepatotropic drugs, forced diuresis, repeated 

haemodialyses, peritoneal dialysis and in patients in liver 

coma we used exchange transfusions and later 

plasmaphereses (2,8). Occurrence of acute tetrachloride 

poisoning is very rare in the last years, because it was due to 

prohibition to use of carbon tetrachloride in fireextinguishers, 

in industry and in various chemical 

laboratories. 

In the second group of 81 patients, who were poisoned by 

mushroom (Amanita phalloides), acute toxic hepatitis 

developed rapidly until to liver insufficiency and coma. From 

this group of patients 24 patients (29,6%) died in liver coma. 

Damage of the kidneys after Amanita phalloides poisoning 

was less frequent, despite that in 16 patients acute tubular 

necrosis with kidney failure developed. No one patient died 

caused by acute renal failure. That dissociation of liver and 

kidney damage is characteristic for hepatotoxic type of acute 

mushroom poisoning. Conservative treatment including of 

hepatic drugs and of forced diuresis and especially of 

extracorporeal elimination therapy we used relatively late for 

the late admission of the patients to our renal unit (in average 

53 hours after poisoning). 

During many years we tried to use early haemodialysis, 

charcoal haemoperfusion, Amberlite XAD-2 and XAD-4 

haemoperfusions and in the last years plasmapheresis which 

is the bridge to liver transplantation. Haemodialysis was used 

on the influence of uremic syndrome (3,5,9,10,13,14). 

In the third group of patients with acute ethylene glycol 

poisoning dominated severe metabolic acidosis, leucocytosis, 

oxaluria and various neurological symptoms. Leucocytosis 

was present from the onset of acute poisoning caused by 

ethylene glycol irritation of the bone marrow and duration of 

that laboratory sign was in average 7 days. Damage of the 

kidneys with relatively slow development of acute renal 

failure showed for the important disturbances of renal 

functions. Damage of the liver with hepatomegaly and 

increase of serum activity of alanine aminotransferase were 

less important and occurred in 16 patients (11,12). 

Dissociation of the liver and kidney damage was present after 

ethylene glycol poisoning in disadvantage for the kidney 

damage. In the medical treatment of ethylene glycol 

poisoning were applicated hepatic drugs including of the 

group of vitamin B. The therapeutic method of choice in 

ethylene glycol poisoning was in the last years bicarbonate 

haemodialysis using 100 mg% ethylalcohol in dialysis 

solution. Ethylalcohol was used as a blocker of 

alcoholdehydrogenase in metabolic transformation from 

ethylene glycol to glycol aldehyde. Two patients died after 

ethylene glycol poisoning before the possibility to use 

bicarbonate haemodialysis and one patient died for the late 

admission to our dialysis unit (11,12). 

At the end of this retrospective study it is neccessary to say, 

that the review of simultaneous damage of the liver and 

kidneys after carbon tetrachloride poisoning, mushroom 

poisoning (Amanita phalloides) and ethylene glycol 

poisoning contributed to diagnosis, differential diagnosis and 

also to the use of various forms of extracorporeal elimination 

therapy. In the near future several new forms of 

extracorporeal elimination therapy will be used in the 

treatment of acute poisoning i.e. albumin dialysis and the use 

of new sorbents for toxins removal mainly for toxins with 

high albumin and other proteins binding in plasma (29-31). 

References 

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200 p. 

2. Mydlik M, Takac M, Melnicak P et al. Diagnosis and therapy 

of carbon tetrachloride poisonong. Vnitr Lek 25, 1979; 7: 672-9 

3. Mydlik M, Derzsiova K. Use of hemoperfusion in acute 

poisoning: Clinical analysis of 229 patients. In: D. 

Falkenhagen, H. Klinkmann, E. Piskin, K. Opatrny, Jr. (eds): 

Blood-Material Interaction. A basic guide from polymer 

science to clinical application. S 149- S 153. INFA 1998, 

Glasgow, Krems, 156 p. 

4. Mydlik M, Derzsiova K, Mizla P. Use of plasmapheresis in 

dialysis centre - - analysis of 105 patients. Akt Nefrol 8, 2002; 

1: 9-13 

5. Seyfart G. Poison Index. The treatment of acute intoxication. 

1st ed. Pubst Science Publisher, Lengerich, Berlin, Düsseldorf 

et al. 1997; 664 p. 

6. Sevcik M. Practical toxicology. IIIrd ed. Státní zdravotnické 

nakladatelství, Prague 1968; 350 p. 

7. Mydlik M, Halko N. Use of exchange transfusion in acute 

poisonings. Prakt Lék 59, 1979; 22: 846-9 

8. Mydlik M, Derzsiova K, Mizla P. Diagnosis and treatment of 

carbon tetrachloride poisoning – analysis of 60 patients. 

Abstract of the XIIth International Congress of Nephrology, p. 

286. Jerusalem, Israel, 1993; 13-18 

9. Mydlik M, Derzsiova K, Mizla P et al. Use of haemoperfusion 

in acute mushroom poisoning. Clinical analysis of 58 patients. 

Cas Lek Ces 132, 1993; 15: 464-7 

10. Mydlik M, Derzsiova K, Klan J et al. Haemoperfusion with 

alpha-amanitin – an in vitro study. Int J Artif Organs 20, 1997; 

2: 105-7 

11. Mydlik M, Melnicak P, Derzsiova K et al. Dialysis therapy of 

acute renal failure. Analysis of 230 patients. Cas Lek Ces 118, 

1979; 18: 559-63 

12. Mydlík M, Derzsiová K, Mizla P et al. Diagnosis and therapy 

of acute ethylene glycol poisoning – analysis of 20 patients. 

Vnit Lék 48, 2002; 11: 1054-9 

13. Enjabert F, Rapior S, Nouguier-Soule J et al. Treatment of 

amatoxin poisoning: 20–-year retrospective analysis. J Toxicol 

Clin Toxicol 40, 2002; 6: 715-57 

14. Galmarini D, Tarenzi L, Cantaluppi P et al. Acute hepatic 

failure and renal damage. Experimental and clinical studies. 

Minerva Med 30, 1978; 69: 1417-26 

15. Markiewicz K, Trznadel K, Szeszko A. Acute carbon 

tetrachloride poisoning. Med Pr 28, 1977; 1: 15-20 

16. Svendsen B S, Gjellestrand A, Eivindson G et al. Serious 

mushroom poisoning by Cortinarius and Amanita virosa. 

Tidsskr Nor Laegeforen 122, 2002; 8: 777-80 

17. Burkhart K K, Hall A H, Gerace R et al. Hyperbaric oxygen 

treatment for carbon tetrachloride poisoning. Drug Saf 6, 1991; 

5: 332-8 

18. Nehoda H, Wieser C Koller J et al. Recurrent liver failure with 

severe rhabdomyolysis after liver transplantation for carbon 

tetrachloride intoxication. Hepatogastroenterology 45, 1998; 

19: 191-5 

19. Fernandez P, Bermejo A M, Lopez-Rivandulla M et al. A fatal 

case of parenteral paraquat poisoning. Forensic Sci Int 49, 

1991; 2:215-24 

20. Melnicak P, Mydlik M. Poisoning by Gramoxon (paraquat). 

Proceedings of the „Industrial toxicology 78“, Bratislava 21.- 

24. VI. 1978, p. 147-57, Zilina, House of Technique SVTS 

Žilina – 55/78, 308 p. 

21. Labadie H, Stoessel P, Callard P et al. Hepatic venoocclusive 

disease and perisinusoidal fibrosis secondary to arsenic 

poisoning. Gastroenterology 99, 1990; 4: 1140-43 

22. Lin J M, Jiang CQ. Clinical manifestation and ultrasonic 

characteristic of five patients with acute arsenic poisoning. 

Zhonguhua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 21, 2003; 

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23. Mathieu D, Mathieu-Nolf M, Germain-Alonso M et al. 

Massive-arsenic poisoning--effect of hemodialysis and 

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1: 47-50 

24. David NJ, Wolman R, Milne FJ et al. Acute renal failure due to 

trichlorethylene poisoning. Brit J Ind Med 46, 1989; 5:347-9 

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paracetamol – an in vitro study. Akt Nefrol 8, 2002; 4: 129 –32 

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592-5 

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Clin Pharm Ther 18, 1993; 1: 5-8 

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hepatitis. Report of two cases. Acta Med Scand 210, 

1981; 4: 333-5 

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critically ill patients with liver failure. Int J Artif Organs 27, 

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Vascular calcifications in patients on hemodialysis 

T. Damjanovic¹, N. Markovic², S. Djorovic³, T. Djordjevic¹ and N. Dimkovic¹ 

¹Center for Renal Diseases, ²Cardiology Department, ³Radiology Department, 

Zvezdara University Hospital, Belgrade, Serbia and Montenegro 

Abstract 

Vascular calcifications (VC) in ESRD patients are strongly 

associated with cardiovascular morbidity and mortality. 

There are two different types of VC: arterial media 

calcifications (AMC), a non occlusive condition, related to a 

mineral metabolism disturbances, and atherosclerosis with 

intima calcifications (AIC) of atherosclerotic plaques. The 

aim of this study was to evaluate arterial and intima 

calcifications by plain radiography and B-mode 

ultrasonography in HD patients, and to analyze potential risk 

factors on their appearance. Study included 73 HD patients 

(33 males), mean (±SD) age 54,30±8,52 years, and HD 

duration of 115,56±60,32 months. AMC were detected by 

plain radiography of pelvis, hands and region of vascular 

access. AIC were detected by B-mode ultrasonography of 

common carotid arteries. The influence of potential risk 

factors (age, gender, HD duration, diabetes, Ca, P product, 

plasma phosphorous, total cholesterol and albumen) on the 

appearance of overall AMC, AMC in different regions and 

AIC was also analyzed. 

AMC were detected in 41(56,2%) patients: in pelvis 

26(35,3%), hands 7(9,6%), and in region of vascular access 

28(38,4%). In group of patients with overall VC detected by 

plain radiography, there was a strong association with male 

gender (p


Results 

The frequency of owerall arterial media calcifications 

evaluated by plain radiography was in 56% of HD patients 

(Figure 1). 

Fig 1. Frequency of arterial media calcifications evaluated by plain 

radiography 

w/o AMC 

44% 

AMC 

56% 

access were significantly younger (51,68±8,71 vs. 

55,82±8,07; p


Is there a seasonal variation in mortality in hemodialysis patients? 

J. Popovic, N. Dimkovic, Z. Djuric, G. Popovic and N. Lazic 

Centre for Renal Diseases, Zvezdara University Hospital, Belgrade, Serbia and Montenegro 

Abstract 

Mortality rates among hemodialysis (HD) patients differ 

considerably between and within countries. 

The aim of this study was to analyze overall and specific 

mortality in patients on regular hemodialysis during the last 8 

years (1997-2004) and to investigate possible monthly and 

seasonal fluctuations in deaths. 

Baseline demographic characteristics together with primary 

renal disease, previous treatment with peritoneal dialysis, 

cause as well as month and season of death were observed. 

Seasons were divided into four periods: December-February 

(winter), March-May (spring), June-August (summer) and 

September-November (autumn). 

Total of 254 HD patients (59 % males) who died from 1997 

to 2004 were included; overall mean age was 62.1 ± 11.2 

years (range 27-88), with 47.6% aged over 65 (60% in 2004); 

age significantly increased from 59.4 ± 13.1 years in 1997 to 

66.0 ± 7.5 years in 2004 (p< 0.05). Even so, mortality rate 

decreased from 17.2% in 1997 to 9.2% in 2004. The mean 

HD duration was 72.6 ± 68.9 months; 18.9% of patients were 

previously on peritoneal dialysis (highest distribution in 2004 

i.e. 25%). The difference in seasonal mortality throughout the 

whole observation period was statistically insignificant. Still, 

higher frequency was observed in summer and winter periods 

(28.3% and 27.2% respectively) with mortality peaks in June 

and December (11% each). The leading cause of death in 

both months was cardiac disease (50% and 46.4%). 

Throughout the whole period cardiac diseases were the main 

cause of death (46.1%, X²=230.08; p


Table 1. Baseline demographic characteristics by the year 

PATIENTS 1997 

N=39 

1998 

N=30 

1999 

N=33 

2000 

N=32 

2001 

N=37 

2002 

N=26 

2003 

N=37 

2004 

N=20 

TOTAL 

N=254 

Age (years ± SD) 59.4 ± 

13.1 

62.9 ± 

11.7 

62.7 ± 

11.6 

62.5 ± 

12.8 

60.5 ± 

12.2 

63.0 ± 

7.7 

62.5 ± 

9.8 

66.0 ± 

7.5* 

62.1 ± 

11.2 

Age (range) 32- 88 43– 87 44– 79 27– 81 29– 79 48– 81 36– 78 49– 77 27 – 88 

% 65 + 38.5 50 51.5 50 46 38.5 51.4 60 47.6 

Sex (M/F) 18/21 19/11 20/13 15/17 26/11 16/10 22/15 14/6 150/104 

Months on HD ± 

SD 

56.4 ± 

61.6 

64.7 ± 

73.6 

66.8 ± 

69.1 

98.9 ± 

78.5 

85.0 ± 

86.1 

65.3 ± 

39.0 

84.6 ± 

65.3 

47.8 ± 

45.3 

72.6 ± 

68.9 

Range 3-267 4-281 4-250 4-344 4-328 5-146 6-258 4-165 3-344 

% prior on PD 17.9 13.3 24.2 15.6 21.6 11.5 21.6 25 18.9 

* t = 2.049 ; p< 0.05 

Results 

Demographic characteristics 

Baseline demographic characteristics of 254 deceased HD 

patients observed in the period from 1997 to 2004 are listed 

in Table 1. 

HD patients that died in 1997 were younger than in following 

years (significantly so in comparison to 2004, p< 0.05). 

Despite that mortality rate decreased from 17.2% in 1997 to 

9.2% in 2004. Highest percentage of patients aged over 65 

was also observed in 2004 (60%; overall for the whole period 

47.6%). Patients deceased in 2004 were also older at the 

initiation of HD. The frequency of patients previously treated 

with peritoneal dialysis (PD) varied from 13.3% to 25% 

(overall 18.9%). Higher proportion of males for the whole 

period was observed (59%). 

Fig 1. Percentage of deceased by seasons 

Number of deaths 

% 

30 

25 

20 

15 

10 

5 

Seasonal mortality 

p=ns 

Seasonal analysis of deaths 

The seasonal peaks of deaths for the whole 8-year period 

were found in summer and winter months (28.3% and 27.2% 

respectively). The difference between seasons was 

statistically insignificant (X²= 4.075; p=ns). 

The frequency of deaths by seasons is shown on Figure 1. 

Analysis of causes of death for the whole observed period 

revealed cardiac causes as the main cause of death in HD 

population (46.1% of deaths), followed by cerebrovascular 

(11%) and infection-sepsis accounting for 9.8% of deaths. 

The difference is highly significant (X²=230.08; p


April (lowest, i.e. 5.1%), followed by the increase to the 

highest percentage in June (11%) with the drop in summer 

and autumn months characterized by narrow variations, and a 

peak again in December (11% -equal to the one in June). 

(Figure 3). 

Fig 3. Monthly deaths in HD population 

Percentage of death in HD patients 

per month 

12 

10 

% 

8 

6 

4 

2 

0 

1 2 3 4 5 6 7 8 9 10 11 12 

Month 

Leading cause of death in both peak months was cardiac 

(50% in June and 46.4% in December, slightly above the 

overall percentage for this cause). The occurrence of 

cerebrovascular deaths was more prominent in June (17.9%), 

much more than overall, and especially so than in December 

(3.6%). Infection was equally distributed in both peak months 

(10.7%), i.e. slightly above the overall frequency. 

The analysis of sex difference in seasonal mortality revealed 

higher proportion of males in summer months (65.3%) in 

comparison to overall Figure, almost equal gender 

distribution in spring (52% male, 48% female) and similar 

frequency in gender distribution as in the whole observed 

period for winter and autumn months (55.1% and 61.9% 

respectively). Statistically, difference in gender mortality by 

seasons was insignificant (X² = 2.994; p > 0.05). 

Deceased in winter and autumn months (64.0 and 63.1) were 

older than in spring and summer (both 60.6), but without 

significance (F=1.46; p > 0.05). 

Patients who died in autumn had been receiving hemodialysis 

longer (82.5 months) than the ones deceased in winter (66.6), 

spring (75.0) and summer (68.1).The difference was not 

significant (H=1.990; p > 0.05). 

Deceased patients with hypertensive nephrosclerosis as the 

primary renal disease represented the highest proportion for 

the whole observed period (38.6%), followed by diabetes 

(18.9%), pyelonephritis (14.2%), glomerulonephritis (10.2%) 

and polycystic kidney disease (7.5%). The difference is 

highly significant (X² = 216.029 ; p< 0.01). Analysis of renal 

diagnoses in relation to seasonal variations of deaths did not 

show significant difference for none of the primary etiologies 

of ESRD. 

Discussion 

Mortality rate among our HD population in the first year 

analyzed (1997) was somewhat higher than reported by 

European DOPPS and ERA-EDTA for the similar period 

(1,2) and lower than the rate in Yugoslavia (5). Although 

mortality rate varies across countries this speaks more in 

favor of the variations across facilities, even more so for the 

following years. 

Mortality rate in our HD population dropped to 9.2% in 2004 

despite significant increase in age from 1997 to 2004. Also, 

60% of patients died in 2004 were aged over 65 years. 

Numerous studies showed the association between older age 

and increased risk of mortality (1,3,6,7), with the risk of 

death increasing by 3% to 4% per year (1). USRDS (12) 

reported expected remaining life span for dialysis patients 

from 7 to 10 years in aged 40 to 44, and 4 to 5 years for those 

60 to 64 years of age. As for the analysis by the seasons, 

patients that died in autumn and winter were older, but 

insignificantly. 

Our results did not show significant difference in mortality 

between males and females. In DOPPS (1) gender also did 

not predict survival, contrary to the findings of Marcelli et al 

(7) where women had better survival rate. 

We observed significant difference in mortality when 

analysing primary etiologies of ESRD, leading diagnosis 

being hypertensive nephrosclerosis (38.6%). This is almost 

identical to the prevalence of hypertensive nephrosclerosis in 

our HD population (constantly between 30% and 38.8%) (8). 

Diabetes as the cause of renal failure represented the second 

largest proportion of deceased (18.9%) which is in contrast to 

their prevalence in our Centre in observed period (despite its 

steadily increase from 7.9% to 11.7%) (9). DOPPS (1) 

identified diabetes as one of co-morbid factors significantly 

associated with mortality. Five-year survival in patients with 

diabetic nephropathy as an underlying renal disease is only 

20% (13,14). Ten-year survival is less likely for diabetics in 

comparison to nondiabetics (4% vs. 11-14%) (12). Lower 

percentage in deaths than its prevalence in our HD population 

was observed for following diagnoses: glomerulonephritis 

(10.2% vs. 16.8%) and polycystic kidney disease (7.5% vs. 

10.7%). Studies reported the best five-year survival in 

dialysis patients with respect to underlying renal disease in 

glomerular diseases and polycystic kidney disease, 

intermediate with hypertension-induced renal disease, and 

worst with diabetic nephropathy (12,13). There was no 

seasonal variation in deaths with respect to primary etiologies 

of ESRD in our patient group. 

Association between longer dialysis duration and enhanced 

risk of death is well established. Each year on dialysis 

increases the risk of death by approximately 6% (19). Our 

data showed insignificant difference between seasons with 

respect to length of HD. 

Higher percentage of deaths occurred in winter and summer 

but seasonal variations in deaths were insignificant. The 

analysis of monthly deaths revealed two peaks - June and 

December. Similar results were reported for HD patients by 

UK survey with the peak only in December (11). It was 

concluded that the earlier peak in deaths on dialysis 

compared to the general population was probably due to a 

cardiac peak as the main cause of death in the dialysis 

population. Studies (12,15-18) have identified major causes 

of death in dialysis population as: cardiovascular disease, 

infection, and withdrawal from dialysis. Cardiac cause was 

also the leading cause of death in our HD population 

(46.1%), somewhat more so than overall in both peak 

months. USRDS (12) reported that cardiovascular disease 

accounts for approximately 50% of deaths, without the 

tendency to decline. Cerebrovascular death, as the second 

overall single cause in our population, occurred more 

frequently in June. Infection, mostly related to vascular 

access, is responsible for 9.8% of deaths in our group, less 

common than 15-20% reported elsewhere (15,17). It was 

equally represented in both peak months. Withdrawal from 

dialysis is by no means feature in our HD population.


Analysis of seasonal fluctuations of deaths by cause did not 

show significant difference. 

Conclusions 

Mortality rate in our Centre decreased considerably in 

observed period despite the increase in patients` age. 

Insignificant seasonal variations were observed for sex, 

primary renal disease, age and HD duration. 

This mortality analysis in our HD center revealed different 

percentage of deaths between the seasons with higher 

occurrence in summer and winter. Two months were 

especially pinpointed- June and December. The reasons for 

this have yet to be elicited. 

It is advisable to keep elderly patients with cardiovascular 

diseases in focus during autumn and winter, at least in our 

region. 

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5. Djukanovic LJ, Hadzi-Djokic J, Djordjevic V, Radovic M. 

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1997. Belgrade 1998. 

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57: 1176


Propyl gallate-induced platelet aggregation in patients with end stage renal 

disease – The influence of the hemodialysis procedure 

T. Eleftheriadis 1 , G. Antoniadi 1 , V. Liakopoulos 2 , A. Tsiandoulas 1 , K. Barboutis 1 and I. Stefanidis 2 

1 Department of Nephrology, General Hospital of Serres, Serres; 2 Department of Nephrology, University of Thessaly, 

Larissa, Greece 

Abstract 

Platelet dysfunction is a well-established disturbance in 

hemodialysis (HD) patients. Propyl gallate is a synthetic 

platelet activator with the property to stimulate platelet 

aggregation. The aim of this study was to evaluate the 

influence of a single hemodialysis session on propyl gallateinduced 

platelet aggregation. 

Thirty-nine HD patients were enrolled in the study and 20 

healthy volunteers were used as controls. Cellulose diacetate 

(CD) dialysers were used in 20 patients and polysulfone (PS) 

dialysers in 19. HD was performed via an A-V fistula in 27 

patients and via an intravenous catheter in 12. Erythropoietin 

was administered in 37 patients (epoietin-alpha in 24 and 

darbepoietin in 13). Thirty-four were receiving the low 

molecular weight heparin tinzaparin. Propyl gallate slide 

aggregometry was used for evaluating platelet aggregation. 

In HD patients, platelet aggregation was impaired before as 

well as after the HD session. No effect of the HD procedure, 

type of vascular access, adequacy of HD or type of 

erythropoietin on the propyl gallate-induced platelet 

aggregation was detected. Platelet aggregation was higher 

when CD dialyser was used. A negative correlation between 

the time needed for platelet aggregation to occur and 

tinzaparin dose was found. 

Propyl-gallate induced platelet aggregation in HD patients is 

impaired. Platelet aggregation was higher in patients dialyzed 

with CD membrane than in those dialyzed with PS 

membrane. The higher was the dose of tinzaparin, the higher 

the platelet aggregation. The clinical significance of the 

above results needs further evaluation. 

Keywords: hemodialysis, platelet aggregation, propylgallate, 

erythropoietin, low molecular weight heparin 

Introduction 

Chronic hemodialysis (HD) treatment improves uremia 

induced bleeding diathesis but only partially (1,2). The 

bleeding diathesis observed in HD patients is of 

multifactorial origin but platelet dysfunction induced by 

uremia seems to play a central role. Uremic 

thrombocytopathy is attributed to acquired defects in specific 

receptors that impair platelet binding to fibrinogen and von 

Willebrand factor (3,4), lower than normal content of ADP 

and serotonin in platelet -granules (5) and impaired 

arachidonate metabolism (6). Guanidinosuccinic acid, a low 

molecular weight uremic toxin related with an increase in NO 

production by uremic vessels, is implicated in platelet 

dysfunction (7). 

Recombinant human erythropoietin (rHuEPO) administration 

in HD patients improves bleeding diathesis because it 

increases red cells blood count and ameliorates blood 

rheology abnormalities (8,9). On the other hand, there is 

controversy regarding the effect of rHuEPO administration 

on platelet aggregation (9-16). 

Heparin, the commonly used anticoagulant in HD procedure, 

activates platelets and increases platelet aggregation (18,19). 

A few studies have evaluated the effect of the more recently 

used low molecular weight heparins (LMWH) on platelet 

aggregation in HD patients (20,21). In non-uremic subjects 

LMWH seems to increase platelet aggregation, although to a 

lesser extend than unfractionated heparin (22-25). 

Some authors have found out that HD procedure per se 

enhances (3,26) platelet activation and aggregation, while 

others reached different conclusions (27,28). Finally, the type 

of the dialysis membrane used seems to play an important 

role in platelet activation or aggregation during the dialysis 

session. However, the results of different studies are 

controversial (27, 29-31). 

The aim of our study was to evaluate the impact of HD 

procedure and its different aspects on platelet aggregation. 

The impact of the medications commonly used in HD 

patients, i.e. rHuEPO and LMWH, on platelet aggregation, 

was also assessed. For this purpose the simple and 

inexpensive propyl gallate (PG) slide aggregometry test was 

used. This test has been shown to be more sensitive in 

detecting most of the abnormal platelet aggregation 

conditions than the commonly used platelet aggregating 

reagents (32). In addition, this procedure shows strong 

correlation with the bleeding time test (33). To our 

knowledge this is the first time that PG was used as platelet 

aggregation agonist in order to evaluate platelet aggregation 

in HD patients. Impairment in arachidonate metabolism, 

adenosine phosphate receptors or ADP and glycoprotein 

IIb/IIIa (GpIIb/IIIa) receptor availability can contribute to 

platelet dysfunction in HD patients (3-6). Propyl gallate can 

concurrently detect disorders in all these factors (34). This 

fact makes it probably the most suitable reagent for an overall 

evaluation of platelet aggregation in HD patients, in contrast 

with other commonly used platelet aggregation agonists like 

arachidonate, ADP, epinephrine, fibrinogen and others, 

which evaluate only specific platelet aggregation pathways 

(35). 

______________________ 

Correspodence to: T. Eleftheriadis, Department of Nephrology, General Hospital of Serres, Serres, Greece



Patients 

Thirty-nine patients (19 males, 20 females, mean age 62±11 

years) with end stage renal disease undergoing chronic HD 

(mean HD duration 63±61 months) were studied. The cause 

of end-stage renal disease was primary glomerulonephritis in 

13 patients, diabetes mellitus in 5 patients, hypertension in 4 

patients, polycystic kidney disease in 3 patients, Alport’s 

syndrome in 2 patients, analgesic nephropathy in 1 patient, 

primary amyloidosis in 1 patient, and unknown in 10 

patients. Patients underwent regular HD with a bicarbonate 

buffer for 4 hours each day, 3 days a week. Vascular access 

was A-V fistula in 27 patients and a permanent double central 

venous catheter in 12 patients. Thirty-seven patients were 

receiving i.v. erythropoietin (rHuEPO), 24 epoietin-alpha 

(Eprex ® ; Johnson & Johnson, Manati, PR) and 13 

darbepoietin-alpha (Aranesp ® ; Amgen, Thousand Oaks, CA). 

During treatment the LMWH tinzaparin sodium (Innohep ® ; 

Leo Pharmaceuticals, Ballerup, Denmark) was used for 

anticoagulation in 34 patients, unfractionated heparin 

(Heparin ® , Leo Pharmaceuticals, Ballerup, Denmark) in 2 

patients, while 3 patients did not receive anticoagulation. 

Cellulose diacetate (CD) dialysers (FB-T series ® ; Nipro 

Corporation, Osaka, Japan) were used in 20 patients and 

polysulfone (PS) dialysers (F low-flux series ® ; Fresenius 

Medical Care, Bad Homburg, Germany) in the other 19 

patients. 

Twenty healthy volunteers (9 males, 11 females, mean age 

54±16 years) were studied as a control group. Except heparin 

during HD none of the patients or the healthy volunteers was 

receiving coagulation related medication. In particular none 

received antiplatelet agents or had been taking non-steroidal 

anti-inflammatory drugs for at least 20 days prior to the 

study. An informed consent was obtained from each 

individual enrolled into the study and the hospital ethics 

committee gave its approval to the study protocol. 

Methods 

Blood samples for the assessment of platelet aggregation 

were drawn from the dialysis access site immediately before 

and shortly after the 2 nd HD session of the week. Blood was 

then collected with citrate (1 part sodium citrate 3.8 %, 9 

parts blood) in a polypropylene tube and centrifuged for 5 

minutes at 500RCF in order to obtain platelet rich plasma 

(cPRP). The supernatant was transferred with a plastic pipette 

in a plastic test tube. 

Platelet aggregation was evaluated by means of a slide 

platelet aggregation test kit (SPAT ® ; Analytical Control 

Systems, Fishers, IN, USA), which includes PG as platelet 

aggregation agonist. On a plastic microscope slide 100l 

cPRP were mixed with 100 l SPAT reagent (Analytical 

Control Systems, Fishers, IN, USA). A stopwatch was started 

and the slide was rocked continuously until aggregation, in 

the form of many visible white clumps of platelets, occurred. 

The time needed for platelet aggregation to occur (platelet 

aggregation time) is as measure of platelet aggregability. All 

tests were done in duplicate at room temperature, and the 

mean value was obtained. One of the authors, blinded 

regarding the subjects’ identity, performed all the tests in the 

same laboratory environment. 

Concurrently, platelet count, hemoglobin (Hb), international 

normalized ratio (INR) and activated partial thromboplastin 

time (aPTT) were examined in blood samples obtained before 

the start of HD. Blood urea was assessed before the start and 

after the end of HD in order to estimate KT/V according to 

the second generation natural logarithmic formula, developed 

by Daugirdas based on the single pool urea kinetic model 

(36). The weekly dose of rHuEPO (units per Kg of patient’s 

dry body weight) was registered for epoietin-alpha (Epoa/bw) 

and darbepoietin-alpha (Dar-a/bw) and for both 

together (Epo/bw). Multiplying the dose of darbepoietinalpha 

in micrograms by a factor of 200 gave its equivalent in 

weekly epoietin-alpha dose. Finally, the dose of tinzaparin 

per Kg of patient’s dry body weight given in the examined 

hemodialysis session (LMWH/bw) was registered (Table 1). 

Table 1. The values of factors that are evaluated for effect on 

platelet aggregation, expressed as mean±SD 

platelet count (platelets/l) 249871±66976 

Hb (g/dl) 10.9±1.15 

INR 0.91±0.09 

aPTT (patient/control sec) 1.22±0.75 

KT/V 1.18±0.44 

Epo/bw (units/Kg) 189.64±148.43 

Epo-a/bw (units/Kg) 208.61±139.88 

Dar-a/bw (g/Kg) 0.948±0.802 

LMWH/bw (units/Kg) 40.48±19.47 

Statistical analysis 

Results are expressed as a mean value and standard deviation 

(SD). Normality of the evaluated variables was assessed with 

the single sample Kolmogorov-Smirnov test. Unpaired t-test, 

paired t-test and Pearson correlation test were used where 

appropriate. P-values < 0.05 were considered as statistically 

significant. 

Results 

Propyl gallate-induced platelet aggregation time, as it is 

assesses by the time needed for aggregation to occur, before 

the start of the HD session (PLA1) was significantly impaired 

in comparison with the control group (PLAc). PLA1 was 

62.74±15.80sec while PLAc was 43.66±11.25sec (p


Platelet aggregation, as it is assesses by the time needed for 

aggregation to occur, before the start of the HD session 

(PLA1) was significantly impaired in comparison with the 

control group (PLAc). PLA1 was 62.74±15.80sec while 

PLAc was 43.66±11.25sec (p


arachidonate metabolism is incriminated for defective platelet 

aggregation in HD patients. Recently, Xiao et al 

demonstrated that PG-induced platelet aggregation and 

tyrosine phosphorylation of multiple proteins were 

substantially abolished by aspirin, apyrase, and abciximab, 

suggesting that PG is associated with activation of platelet 

cyclooxygenase 1, adenosine phosphate receptors, and 

GpIIb/IIIa, respectively (34). Abnormal function of all these 

factors has been implicated in platelet dysfunction in HD 

patients (3-6). Propyl-gallate is possibly a suitable reagent for 

a global evaluation of platelet aggregation in HD patients, in 

contrast with other commonly used aggregation reagents like 

arachidonate, ADP, epinephrine, fibrinogen and others, 

which are proper for specific, but only partial evaluation of 

platelet aggregation (35). 

As expected, impaired platelet aggregation was detected in 

HD patients. Interestingly, platelet aggregation impairment 

did not show any improvement after the HD procedure. This 

is in contrast with other studies that detected enhancement of 

platelet aggregation after the HD procedure (3,26). On the 

other hand, there are studies, which indicated that HD 

procedure reduces platelet aggregation (27,28). The 

difference among these studies could be attributed to the 

different HD conditions (i.e. HD membrane composition or 

type of anticoagulant) or to the different platelet aggregation 

agonists used. In our study PG was used for the first time in 

order to evaluate platelet aggregation in HD patients. As 

already noted, PG is a platelet agonist that can detect 

impairments in many pathways leading to platelet 

aggregation. Consequently, our study assessed platelet 

aggregation more globally than others, which used more 

specific platelet aggregation agonists that are capable for 

evaluating only one pathway of platelet aggregation each 

(35). 

Regarding the HD procedure per se, we failed to detect any 

difference. Type of dialysis access (A-V fistula or central 

intravenous catheter) did not affect platelet aggregation. Two 

kinds of dialysis membranes were used in our study, CD and 

PS. There are many studies that evaluate the effect of 

membrane composition on platelet activation or aggregation. 

Most of them evaluated the fibrinogen binding to GpIIb/IIIa 

receptor but the results are contradicting. In one study the 

level of bound GPIIb/IIIa fibrinogen receptor was increased 

when PS membrane was used but there was no significant 

change with cellulose triacetate membrane (29). In contrast, 

another study indicated that the degree of GPIIb/IIIa 

activation was greater during hemodialysis with regenerated 

cellulose than PS membrane (30). In a paper comparing the 

influence of cuprophan, hemophan, and PS on fibrinogen 

binding on platelets, Gawaz et al concluded that it is 

increased only when cuprophan membrane was used (31). 

Finally, another study comparing polymethylmethacrylate, 

cuprophan, and PS membranes did not demonstrate any effect 

of the type of dialysis membrane on platelet aggregation (27). 

In our study, we detected that PG induced platelet 

aggregation time before HD session was higher when PS 

membrane was used in comparison with CD membrane. That 

was a long-term effect since there was not difference in the 

change in platelet aggregation time before and after HD if PS 

or CD dialysers were used. The fact that PG induced platelet 

aggregation is totally abolished by the GpIIb/IIIa inhibitor 

abciximab in relatively low concentrations (34), in 

combination with the results of the above studies, raises the 

possibility that PS membrane induces less GpIIb/IIIa 

activation than CD membrane. 

Dialysis adequacy (as expressed by KT/V) did not have any 

effect on platelet aggregation either. This means that the 

clearance of the low molecular weight toxins by HD does not 

influence platelet aggregation. On the other hand, the role of 

some low molecular weight toxins, especially of 

guanidinosuccinic acid, in uremic platelet dysfunction is well 

established (7). It is possible that the difference in urea 

clearances, among our patients was not big enough in order to 

induce difference in platelet aggregation. Standard HD, 3 

times a week, 4 hours per session, seems adequate in 

removing enough guanidinosuccinic acid or other low 

molecular weight uremic toxins, responsible for platelet 

dysfunction. 

Nowadays rHuEPO is administered in most HD patients. The 

beneficial effect of rHuEPO on hemostasis is well 

established. One certain mechanism is improvement in blood 

rheology due to correction of anemia (9). Many studies, using 

various platelet agonists, have evaluated the effects of 

rHuEPO on platelet aggregation in HD patients. Again the 

results were conflicted, as some authors detected an increase 

in platelet aggregation after rHuEPO administration (10-14), 

while others failed to do so (9,15-17). In our study we did not 

detect any correlation between platelet aggregation time and 

the weekly dose of rHuEPO per Kg of dry body weight 

(Epo/bw). There was no statistically significant difference in 

platelet aggregation if epoietin-alpha or darbepoietin-alpha 

was administered. It is notable that in another study platelet 

aggregation responses to thrombin did not differ between the 

two treatments (39). 

In most HD patients, extracorporeal blood circulation is 

maintained with the administration of unfractionated heparin, 

or more recently with the administration of LMWH. Studies 

in non-uremic subjects indicated that unfractionated heparin 

activates platelets and increases platelet aggregation. In nonuremic 

subjects LMWH also seems to increase platelet 

aggregation, although to a lesser extend than unfractionated 

heparin (22-25). There are some studies in HD patients that 

confirm the platelet aggregation enhancement by 

unfractionated heparin (18,19). On the other hand, there are 

quite a few studies that evaluate the effect of the more 

recently used LMWH on platelet aggregation in HD patients. 

In one study LMWH did not alter ADP or collagen induced 

platelet aggregation during dialysis (20), and in another one 

no differences between unfractionated heparin and LMWH 

were observed regarding the platelet aggregation in HD 

patients (21). In our unit the major anticoagulant used was the 

LMWH tinzaparin sodium. Platelet aggregation before HD 

was positively, although weakly, correlated with the dose of 

tinzaparin sodium (units/kg of dry body weight). This 

positive correlation became much stronger after HD, i.e. near 

tinzaparin administration, indicating a short-term effect of 

that LMWH on platelet aggregation. The positive impact of 

LMWH on platelet aggregation could be the result of the less 

microthrombus formation and consequently of the less 

fibrinolytic mechanism activation and fibrinogen fragments 

production during HD procedure. Accoring to a study by 

Sreedhara et al, platelet dysfunction in chronic HD patients 

results from decreased GpIIb/IIIa availability due to receptor 

occupancy by fibrinogen fragments (40). Additionally, Sobel 

et al have detected that unfractionated heparin directly binds


to GpIIb/IIIa modulating its function (41). This aspect has not 

been evaluated yet for LMWH. 

Finally, no correlation was detected in our study among 

platelet aggregation and Hb, INR or aPTT, indicating, as 

expected, that PG induced platelet aggregation is independent 

of other factors that are correlated with blood rheology or 

coagulation. In contrast, also as expected, PG induced platelet 

aggregation was positively correlated with the whole blood 

platelet count. 

Conclusions 

In conclusion, our study demonstrates that PG induced 

platelet aggregation in chronic HD patients is decreased in 

comparison with non-uremic subjects. The HD procedure 

does not influence platelet aggregation neither does the 

dialysis access type (A-V fistula or central intravenous 

catheter) or the low molecular weight uremic toxins 

clearance. Different types of dialysis membranes have 

different effects on platelet aggregation. Polysulfone dialyser 

prolongs the PG-induced platelet aggregation time in 

comparison to CD dialyser. Finally, from the medications 

usually administered in HD patients, only LMWH had a 

positive effect on PG-induced platelet aggregation. The 

clinical significance of the above results, in relation with the 

bleeding or thrombotic episodes in HD patients, needs further 

evaluation. 

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decreased shear-induced platelet aggregation mediated by 

decreased availability of glycoprotein IIb-IIIa receptors. Am J 

Kidney Dis 1996; 27(3): 355-364 

41. Sobel M, Fish WR, Toma N, Luo S, Bird K, Mori K, 

Kusumoto S, Blystone SD, Suda Y. Heparin modulates integrin 

function in human platelets. J Vasc Surg 2001; 33(3): 587-594


Serum cystatin C as an endogenous marker of kidney function in elderly 

with chronic kidney failure 

H. Resic 1 and A. Mataradzija 2 

1 Center for Haemodialysis, 2 Nephrology Institute, Clinical Center, Sarajevo, Bosnia and Herzegovina 

Abstract 

Concentration of serum cystatine C primarly dependes on the 

glomerular filtration rate (GFR), and cystatin C concentration 

in serum can be used as an endogenous marker of kidney 

function. Use of cystatin C in the assesment of GFR in 

elderely, childern and pre - dialysis patients could be usful. 

The aim of the study was to evaluate the use of cystatin C as 

a renal marker of the glomerular filtration rate (GFR) in 

patients with various degrees of kidney failure. 

The study icluded the total of 104 patients (various etiology 

of kidney disease) with different degrees of kidney failure. 

All of them were on conservative tretmant and 10 healtly 

patients will comprised the control group. Mean values of 

cystatin C and creatinine in serum hes been measured and 

compared to endogeneus cretainine clearense. 

There were 104 patients testid in total with various etiology 

of kidney disease. Mean age of patients receiving 

conservative treatment was 68, 4 ± 5, 06 years and controls 

69 ± 3, 66 years. Significant correlation have established 

between creatinine clearence and creatinine r = 0, 663 p < 0, 

001, and between creatinine clearence and cystatin C r = 0, 

765, p < 0, 001 in patients will different degrees of chronic 

kidney failure (CKF). Correlation between creatinine 

clearence and cystatin C was significantly better than 

between serum creatinine p < 0, 05. 

According to results from our study the level of cystatin C in 

serum is better marker of kidney function than the level of 

creatinine in serum. 

Hering in mind that this is faster and cheaper method it could 

find wider application in every day clinical practtise, 

especially in elderly where is often impossible to accurately 

collect 24 - hour - urine (inconontinence) 

Key words: serum cystatin C, chronic kidney failure, 

clearence creatinin 

Introduction 

In clinical practice the glomerular filtration rate (GFR) is 

assessed with various methods. The most accurate method is 

the radioisotopic measurement of inulin clearance (1). 

However, in everyday clinical practice radioisotopic methods 

are expensive, time-consuming and they expose patients to 

radiation, the reason of their substitution with cheaper and 

quicker methods such as the measurement of creatinine and 

creatinine clearance levels in serum (2). As of recent many 

studies have evaluated the use of cystatin C in the assessment 

of GFR in elderly and children, especially in group of predialysis 

patients because the cystatin C value does not depend 

on age, gender of muscle weight. 

Cystatin C is a cystatin protease inhibitor and is a part of 

intracellular protein catabolism. It is non-glycolysed, lowmolecular 

protein (133kD) which comprises 120 amino acids. 

Protein is created in each nucleus, freely passes glomerular 

membrane and almost completely is reabsorbed and 

catabolised in proximal tubular cells (3). Due to these facts 

concentration of serum cystatine C primarily depends on the 

glomerular filtration rate (GFR), and cystatin C concentration 

in serum can be used as an endogenous marker of kidney 

function (4). Many authors, (Schuck O, Teplan V et al., 

2003) agree that cystatin C, as a new endogenous marker, can 

have advantages in early recognition of damaged kidney 

function (5-7). 

The aim of this study was to compare both serum markers: 

creatinine and cystatin C with creatinine clearance in elderly, 

and to evaluate the use of cystain C as a renal marker of the 

glomerular filtration rate (GFS) in patients with various 

degrees of kidney failure. 


The study included the total of 104 patients (various etiology 

of kidney disease), with different degrees of kidney failure. 

All of them were on conservative treatment (I degree of 

creatinine clearance – mean value 72.48 ± 17.62 ml/min – 42 

patients, II degree – creatinine clearance 34.53 ± 9.92 ml/min 

– 44 patients, III degree 11.63 ± 3.71ml/min – 18 patients) 

and 10 healthy patients which comprised the control group. 

Mean values of cystatin C and creatinine in serum had been 

measured and compared to endogenous creatinine clearance. 

Dade Behring nephelometer has been used for cystatin C 

measurement by immunonephelometric method, urea with 

uresis method, creatinine with kinetic method on the same 

machine, and creatinine clearance had been calculated using 

formula: urine creatinine x diuresis / serum creatinine x f (f = 

84.400). 

Patients were divided according to the values of creatinine 

clearance in three groups. During the study they were under 

the standard treatment of a kidney disease: antihypertensives, 

drugs lowering fat, vitamins, and Fe preparations. 

Manuel statistical data processing has been used: 

percentages, arithmetic mean, standard deviation, Chi-square 

test, t-test, differentiation and correlation coefficients. 

Results 

There were 104 patients tested in total with various etiology 

of kidney disease: 46 patients – Chronic Pyelonephritis (CP); 

______________________ 

Correspodence to: H. Resic, Center for Haemodialysis, Clinical Center, Sarajevo, Bosnia and Herzegovina


25 patients – Obstructive nephropathy (ON); 27 patients – 

Polycystic kidneys (PK); 6 patients – Other etiology: 

Nephrectomy, unknown etiology (Other). 

Discussion 

Accurate measurement of kidney function on daily basis in 

clinical practice is essential in order to be able to continually 

follow up differential treatments aimed to maintain or 

improve kidney function, and in time set to begin with kidney 

function replacement therapy in pre-dialysis patients (1). 

Accurate methods such as inulin clearance, radioisotopic 

contrast techniques are often expensive, time-consuming and 

inaccessible, therefore in routine clinical practice more 

suitable are measurement of creatinine concentration in 

serum and creatinine clearance (2). These methods have their 

disadvantages in pre-dialysis patients and in elderly because 

they are not absolute indicators of the GFR (4,5). The 

creatinine level is actually lower because a part of creatinine 

is eliminated by intestinal degradation, then in uremia with 

lower ingestion of proteins occur muscle degradation, which 

in turn lowers the production of creatinine and its level in 

serum. Many substances such as keto acids, glucose, and 

bilirubin can interfere with measurement of creatinine in 

serum (6). Thus the serum level of creatinine is lower than it 

is expected according to the decrease of the GFR. On the 

other hand, the creatinine clearance measurement depends on 

muscle weight of a patient, on the feasibility of adequate 24- 

hour urine collection and tubular secretion which makes the 

method deficient (7). 

As of recent cystatin C has proven to be not only better, but 

ideal, endogenous marker of kidney function since it is a 

constant value that does not depend on gender, patient’s 

muscle weight, accompanying infections or malignancies (3- 

6). It is produced in every nucleus as a constant value, freely 

passes glomerular membrane and it is reabsorbed and 

completely metabolized in proximal tubules. Its plasma level 

linearly correlates with the GFR, i.e. its plasma level 

increases proportionally to the decrease of the GFR which 

makes it more sensitive marker than creatinine (6-10). 

Besides, it does not take long time to prepare 24-hour urine 

for the cystatin C measurement. It is believed to show the 

kidney function damage much earlier than creatinine. 

Serum cystatin C has been measured with immunoassay 

technique which is fast, accurate and suitable for use in 

clinical practice (3,10-12). There are not any drugs yet known 

to affect the serum level of cystatin C in clinical settings, as 

the available literature suggests (11-14). 

In our study we have established significant correlation 

between creatinine clearance and creatinine r = - 0,663, 

p


Fig 2a Control group – distribution according to gender 

CONTROL GROUP – DISTRIBUTION 

ACCORDING TO GENDER 

Females 

60% 

Males 

40% 

Fig 2b Conservative treatment according to gender 

Females 

50% 

Conservative treatment 

according to gender 

Males 

50% 

Table 3. Mean cystatin C values (mg/l) 

Number of 

patients (n) 

CONSERVATIVE TREATMENT 

Arithmetic 

mean x 

Standard 

deviation SD 

I DEGREE 42 1,015 0,279 

II DEGREE 44 1,898 0,677 

III DEGREE 18 3,931 0,897 

TOTAL 104 1,893 1,181 

CONTROL GROUP 10 0,648 0,131 

Test results of significant differencies in mean creatinine values, 

according to the type of treatment compared to control group 

I DEGREE II DEGREE III DEGREE 

CONTROL GROUP 5,495 11,351 15,239 

SIGNIFICANCE p


Table 5. Coefficients of linear correlation between creatinine and 

creatinine clearance 

COEFIFCENT OF LINEAR 

CORREALATION (r) 

SIGNIFICAN 

CE 

CONSERVATIVE TREATMENT 

I DEGREE r= -0,302 p


Thyroid disfunction and ultrasonographic abnormalities in uremic patients 

undergoing conservative management and haemodialysis 

A. Mataradzija 1 , H. Resic 2 and E. Kucukalic-Selimovic 3 

1 Nephrology Department, 2 Center for Haemodialysis, 3 Nuclear Medicine Department, Clinical Center, University of Sarajevo 

Abstract 

Morphologic abnormalities of thyroid gland are ordinary in a 

form of thyroid nodules, goiter or thyroid carcinoma in 

patients with chronic renal failure (CRF). The aim of our 

study was to examine the prevalence of morphologic and 

functional abnormalities of patients with CRF at conservative 

management and haemodialysis. 

The study was conducted in 81 patients (40 with CRF, 31 

haemodialaysis patients and 10 healthy controls. All patients 

were evaluated by ultrasonography of the thyroid gland and 

blood sample was drawn for a various biochemical 

parameters of thyroid function. The duration of the study was 

12 months. 

The study cohort with mean age 56,20±11,80 years, was 

predominated by female patients (61.7%), on a conservative 

treatment (72.5%). Disease duration at conservative 

management was significantly longer in comparison with 

patients on haemodialysis (11.3±6.46 vs. 8.45±5.61 years). 

Ultrasound revealed an irregular assessment of thyroid gland 

in 52.1% of the patients. Significantly lower mean values of 

thyroid hormones was obtained in the evaluated groups as 

compared with the controls. 

A substantial number of our patients have been presented 

with changes of one or both lobules of thyroid gland. The 

mean values of serum thyroid hormones were significantly 

lower in patients with CRF at conservative management and 

haemodialysis treatment in compare with patients of control 

group. 

Key words: thyroid disfunction, dialysis, ultrasonography 

Results 

All results are presented within the following tables and 

figures. 

Introduction 

Many authors have agreed that morphologic abnormalities of 

thyroid gland are ordinary in a form of thyroid nodules, goiter 

or thyroid carcinoma in patients with chronic renal failure 

than in the general population (1-5). In the same time, we 

found out lower serum thyroid hormone values, and these 

patients were clinically euthyroid. Modern literature 

describes these phenomena as “Sick euthyroid syndrome” 

(12,13). 

The aim of the study was to examine the prevalence of 

morphologic and functional abnormalities of patients with 

chronic renal failure (CRF) at conservative management and 

haemodialysis. 


Patients 

This study totally involves 81 patients. 40 patients with CRF 

at conservative management, (with clearence creatinine in a 

range of 80-10 ml/min), 31 patients at haemodialaysis 

treatment (clearence creatinine underlying 10 ml/min), and 

10 healthy patients with regular kidney and thyroid function 

used as control group. 

Methods 

All patients were evaluated by ultrasonography of the thyroid 

gland (Siemens ultrasonograph with linear sonde of 70 

MHz). Free and total serum thyroid hormone values have 

been calculated using Radioimmunoassay methods, and FT3, 

FT4 by Chemiluminescent technique. Duration of study has 

been 12 months, using standard statistical methods for 

manually elaboration of information, with no use of 

computer. 

Table 1. Gender distribution 

Number of 

Therapy type 

males 

Number of 

females 

Males in 

percentages 

Females in 

percentages 

Total 

number 

p-values 

Conservative management 

I degree 1 8 11,10% 88,90% 9 n.s. 

II degree 2 5 28,60% 71,40% 7 n.s. 

III degree 8 16 33,30% 66,70% 24 n.s. 

Total 11 29 27,50% 72,50% 40 n.s. 

Haemodialysis 15 16 48,40% 51,60% 31 n.s. 

Control group 5 5 50% 50% 10 n.s. 

Total 31 50 38,30% 61,70% 81 p< 0,001 

Demographic data (61,7% patients were females and 38,3% were males out of total 81 patients) 

______________________ 

Correspodence to: A. Mataradzija, Nephrology Department, Clinical Center, University of Sarajevo, 

Bosnia and Hercegovina


Fig 1a In a group of patients at conservative management, female population was predominant 


27,50 % 

72,50 % 

Males 

Females 

1 2 

Fig 1b. The same pattern of female predominance was observed also in haemodialysis treatment 

Haemodilaysis 

51,60% 

Female 

48,40% 

Males 

1 2 

Table 2. Average age 

Number of 

Therapy type 

patients 


Mean age in years 

Standard 

deviation 

Standard 

omission 

p-values 

I degree 9 62,44 9,96 3,32 n.s. 

II degree 7 57,57 12,37 4,68 n.s. 

III degree 24 58,71 9,00 1,84 n.s. 

Total 40 59,35 10,03 1,59 n.s. 

Haemodialysis 31 51,55 11,78 2,12 p


Table 4. Ultrasonic assessment of thyroid gland 

Regular 

assessment of % 

thyroid gland 

Conservative 

management 

Irregular 

assessment of 

thyroid gland 

% 

Total number 

of patients 

I degree 2 22,2 7 78,8 9 100 

II degree 3 42,9 4 57,1 7 100 

III degree 10 29,4 24 70,6 34 100 

Total 15 37,5 25 62,5 40 100 

Haemodialysis 19 61,3 12 38,7 31 100 

Total 34 47,9 37 52,1 71 100 

% 

Ultrasonic assessment of thyroid 

gland (conservative management) 

Ultrasonic assessment of thyroid 

gland (haemodialysis treatment) 

Regular 

assessment 

38% 

Irregular 

assessmnet 

Irregular 

assessment 

39% 

Regular 

assessment 

62% 

61% 

Fig 2a 

Fig 2b 

Ultrasonic assessment was irregular in 62% of patients at conservative management (Figure 2a), and in 39% patients 

at haemodialysis treatment (Figure 2b) 

Table 5. Average values of thyroid hormone in different groups of patients 

TT4 nmol/l TT3 nmol/l FT4 pmol/l FT3 pmol/l TSH mlU/l TBG mg/l 

Total 

proteins 

I degree 128,00±38,59 2,18±0,82 15,43±3,49 4,43±1,02 2,48±1,32 26,87±2,3 70,22±3,91 

II degree 123,40±57,23 2,33±0,93 15,40±2,65 4,62±1,43 1,96±0,90 23,51±3,5 72,14±5,59 

III degree 99,42±28,62 2,09±0,47 13,43±2,36 4,23±0,84 3,03±1,67 20,58±7,8 73,71±4,48 

Total 110,10±26,86 2,15±0,67 14,25±2,76 4,28±1,01 2,72±1,54 22,51±6,8 72,65±4,79 

Hemodialysis 93,13±23,94 2,11±0,76 11,86±2,29 4,35±1,02 2,13±1,45 22,55±7,6 72,32±4,95 

Control group 123,09±3,51 2,94±0,14 17,72±1,59 5,66±0,21 2,54±0,87 23,22±3,4 75,70±4,24 

p values P


Average values of TT4 

in groups of patients 

Conservative 

ti 

Haemodialysis 

Control 

1 2 3 

110,10 93,13 123,09 

Fig 3. Significantly lower average values of hormones TT4 in evaluated groups of patients at conservative 

management and haemodialysis in comparison with the control group 

Average values of TT3 

in groups of patients 

2,94 

3,00 

2,15 2,11 

2,00 

1,00 

0,00 

Total conservative 

Haemodialysis 

Control group 

Fig 4. Significantly lower average values of TT3 in evaluated groups of patients in comparison with the control group 

FT4 average values in groups of patients 

20,00 

10,00 

0,00 

14,25 

11,86 

17,72 


Haemodialysis 

Control group 

Fig 5. Significantly lower value of serum hormone FT4 in evaluated groups in comparison with the control group


Average values of FT3 in groups of patients 

6 4,28 

5 

4 

3 

2 

1 

0 

Total conservative management 

Haemodialysis 

5,66 

4,35 

Control group 

Fig 6. Significantly lower values of serum hormone FT3 in patients at conservative management 

and haemodialyis treatment in comparison with the control group 

Average values of TSH in groups of patients 

3,00 

2,50 

2,00 

1,50 

1,00 

0,50 

0,00 

2,72 2,13 2,54 

Total conservative management Haemodialysis Control group 

Fig 7. TSH value was not significantly lower in evaluated groups in comparison with the controls 

Average values of TBG in groups of patients 

22,51 22,55 

23,22 

1 2 3 

Fig 8. There are no significant differences between TBG values in evaluated groups of patients in comparison with controls 

Discussion 

Higher rates of goiter, hypothyroidism, thyroid nodules and 

thyroid carcinoma could be more typical at CRF patients than 

at general population (1-5). In our study 62,5% patients of 

conservative management and 38,7% of haemodialysis 

treatment had thyroid nodules in one or both thyroid lobes. 

All patients of evaluated groups had lower serum values of 

total proteins in compare with control group (6-11), 

confirming theory of malnutrition (6,7). Patients were 

clinically euthyroid, which correspons to the ”Sick euthyroid 

syndrome”, according to available literature (12,13). Average 

ages of patients in evaluated groups correspond to world 

information and its amount is 59 yrs.


Conclusions 

Ultrasonic nodules changes one or both lobules of thyroid 

gland were recorded at 52,1% of examined patients. Average 

values of serum concentrations TT4, TT3, FT4, and FT3 

were significantly lower in patients with CRF at conservative 

management and haemodialysis treatment in compare with 

patients of control group. Duration of haemodialysis 

treatment is inversely proportionally serum value TT3 in 

evaluated patients. Serum concentrations TSH and TBG were 

same in patients of evaluated groups in compare with control 

group. Serum value of total proteins was significantly lower 

of tested patients in compare with control group. 3% out of 

total number of 71 patients with CRF had clinically 

hypotiroidsm, and required substitutive therapy. 97% were 

clinically euthyroid (“Sick Euthyroid syndrome”). 

References 

1. Miki H, Oshimo K, Inque H, Kawano M, Tanaka K, Komaki 

K, Uyama T, Kawauchi M, Minakuchi J, Kawashima S, 

Monden Y. Thyroid nodules in female uremic patients on 

maintenance hemodialysis. J Surg Oncol 1993; 54: 216-218 

2. Prinz RA, Barbato AL, Lawrance AM, Paloyan E. Simultaneus 

primary hyperparathyroidism and nodular thyroid disease. 

Surgery 1982; 92: 454-458 

3. Kantor AF, Hoover RN, Kinlen LJ, McMullan R, Fraumeni JF. 

Cancer in patients receiving long-term dialysis treatment. Am J 

Epidemiol 1987; 126: 370-376 

4. Penn I. The effect of immunosuppresion on pre-exiting cancers. 

Transplantation 1993; 55: 742-747 

5. Brunner FP, Landais P, Selwood NH. Malignancies after renal 

transplantation: the EDTA-ERA registry experience. Nephrol 

Dial Transpl 1995; 10(suppl.): 74-80 

6. Owens WF, Lew NL, Liu Y, Lowrie EG, Lazarus JM.The urea 

reduction ratio and serum albumin concentration as predictors 

of mortality in patients undergoing hemodialysis. N Engl J Med 

1993; 329: 1001-1006 

7. Lim VS, Fang VS, Katz Al, Refetoff S. Thyroid disfunction in 

chronic renal failure: A study of the pituitary-thyroid axis and 

peripheral turnover kinetics of thyroxine and triiodthyronine. J 

Clin Invest 1977; 60: 522-534 

8. Oppenheimer JH, Schwartz HL, Mariash CN, and Kaiser FE. 

Evidence for a factor in the sera of patients with non-thyroidal 

disease which inhibitsiodothyronine binding by solid matrices, 

serum proteins, and rat hepatocites. J Clin Endocrinol Metab. 

1982; 54: 757-766 

9. Chopra IJ, Van Herle AJ, Chua Teco GN, Nguyen AH. Serum 

free thyroxine in thyroidal and nonhyroidal illnesses: A 

comparison of measurement by radioimmunoassay, equilibrium 

dialysis, and freethyroxine index. J Clin Endocrinol Metab. 

1980; 51: 135-143 

10. Nelson JC, Wilcox RB, Pandian MR. Dependence of free 

thyroxine estimates obtained with equilibrium tracer dialysis on 

the concentration of thyroxine- binding globulin. Clin Chem 

1992; 38: 1294-1300 

11. Robey C, Shreedhar K, Batuman V. Effect of chronic 

peritoneal dialysis on thyroid function tests. Am J Kidney Dis 

1989; 13: 99-103 

12. Lim VS, Henriwuez C, Seo H, Refetof S, Martino E. Thyroid 

function in a uremic rat model: Evidence suggesting tissue 

hypothyroidism. J Clin Invest 1980; 66: 946-954 

13. Lim VS, Flanigan MJ, Zavala DC, Freeman RM. Protective 

adaptation of low serum triiodothyronine in patients with 

chronic renal failure. Kidney Int 1985; 28: 541-549 

14. Owen WF Jr, Lew NL, Liu Y, Lowrie EG, Lazarus JM. The 

urea reduction ratio and serum albumin concentration as 

predictors of mortality in patients undergoing hemodialysis. N 

Engl J Med 1993; 329: 1001-1006


Prednisone/Cyclophosphamide treatment in adult-onset autosomal 

dominant familial focal segmental glomerulosclerosis (FSGS 1) 

L. Grcevska 1 , S. Dzikova 1 , V. Ristovska 1 , M. Milovanceva Popovska 1 , G. Petrusevska 2 

and M. Polenakovic 1 

1 Department of Nephrology, Clinical Center Skopje, 2 Institute of Pathology, Faculty of Medicine Skopje, Republic of Macedonia 

Abstract 

Familial FSGS is a heterogeneous disease characterized by 

proteinuria and an unremitting deterioration of the excretory 

function. FSGS 1 is a less severe form of familial FSGS with 

autosomal-dominant inheritance, adult onset and slow 

progression to end-stage renal failure. Previous studies 

showed steroid unresponsiveness or variable response to 

steroids, cyclophosphamide and ACE inhibitors, and low rate 

of recurrence after transplantation. 

We treated with steroids/cyclophosphamide 8 patients (3 

families) with FSGS1 (aged 24±3,11) and compared the 

results to 20 patients with idiopathic FSGS (aged 35±2), and 

6 untreated patients with familial form. The treatment 

included prednisone 0,5mg/kg/daily for 4 months with slow 

tapping over to 20mg daily for further 6 months and 

cyclophosphamide 50mg daily 6 months, alternate use after. 

The follow-up period was two years. 

None of the patients with idiopathic FSGS experienced endstage 

renal failure during follow-up (serum creatinine 

115±10, to 183,4±65 mol/l) and proteinuria decreased 

(5,42±0,84 to 4,68±1,2g/d). One FSGS 1 patient died because 

of intracranial hemorrhage, 2/8 started dialysis within two 

years, but 5/8 presented similar results as control group: slow 

increase of creatinine form 130,62±15,91 to 215,78±63,62 

mol/l and slight decrease of proteinuria from 4,69±0,81 to 

3,28±0,24g/d. All untreated patients with FSGS1 experienced 

end-stage renal failure within two years. 

Both forms of adult-onset FSGS, idiopathic and familial 

presented slow deterioration of the renal function under 

immunosuppression, but we can not say that they are good 

responders: complete or partial remission was not noted. The 

number of treated patients is small to make exact 

conclusions. 

Key words: familial focal segmental glomerulosclerosis, 

prednisone, cyclophosphamide 

Introduction 

The discovery of molecular effects of podocyte components 

causing familial forms of nephrotic syndrome suggested the 

role of podocytes as the site of permselectivity in the kidney 

(1). Since then, knockout models of podocyte components 

and further molecular genetic screenings definitely 

consolidated this concept (1,2). Nephrin (NPHS1) was the 

first podocyte protein to be found mutated in association with 

a rare form of congenital nephrotic syndrome with autosomal 

recessive inheritance. Podocin (NPHS2) was the second 

recognized protein to cause proteinuria in familial cases with 

recessive inheritance and in sporadic patients (1,2,3). The 

clinical picture of nephrotic syndrome caused by podocin 

mutations ranges from an early onset, resembling congenital 

nephrotic syndrome to a late onset in the second decade of 

life, resembling idiopathic focal segmental 

glomerulosclerosis (1,5). Finally, -actinin 4 was the most 

recognized structural component of the podocyte causing 

proteinuria in rare cases of dominant nephrotic syndrome in 

adults. So, the familial forms of FSGS are caused by 

mutations in genes at (1,6): 

- 1q25-31 for NPHS2, autosomal recessive form, and 

- 11q21-22, and 19q13 for -actinin 4, autosomal 

dominant form of FSGS (FSGS1) 

Patients with autosomal recessive form do not respond to 

standard steroid treatment of nephrotic syndrome and exhibit 

a decreased risk of FSGS recurrence after kidney 

transplantation (7-9). 

The aim of our study was to analyze the responsiveness to 

prednisone/cyclophosphamide treatment in two groups of 

patients: (I) patients with adult-onset autosomal dominant 

familial FSGS (1) and (II) patients with idiopathic FSGS. 


Selection of the patients 

3 groups of patients were included in the study. 

1. 8 patients (3 families) with FSGS1 aged 24±3,11, treated. 

2. 20 treated patients with idiopathic FSGS aged 35±2 

3. 6 untreated patients with FSGS 1 (treated only with 

supportive treatment), aged 26±4,2. 

Treatment and follow-up 

Prednisone 0,5mg/kg/daily was performed for 4 months with 

slow tapping over to 20mg daily for the further 6 months, 

then continuing with this dosage to the end of the study. The 

daily dosage of cyclophosphamide for the first 6 months was 

50mg, alternate day use was performed later. The whole 

follow-up period was two years. Laboratory investigations 

were performed monthly. 

______________________ 

Correspodence to: L. Grcevska, Department of Nephrology, Clinical Center Skopje Vodnjanska 17, Skopje, 

Republic of Macedonia; Phone: +389 23 123 334; E-mail: maknefpo@mt.net.mk


Results 

Idiopathic FSGS 

None of the patients experienced end-stage renal failure 

during follow-up. Serum creatinine presented slight, non 

significant increase, from 115±10, to 183,4±65 mol/l, 

p>0,05. Proteinuria decreased, but also non-significantly, 

from 5,42±0,84 to 4,68±1,2g/daily, p>0,05. So we did not 

noted partial or complete remission of the nephrotic 

syndrome. 

FSGS1 patients – treated 

One of the patients, 20-year old, female, died because of 

intracranial hemorrhage due to severe form of hypertension. 

2/8 patients started dialysis within two years, one after 18 

months, the other after 6 months. The second patient (male, 

18 years) was transplanted after two months dialysis 

treatment without evidence of recurrence during the followup. 

5/8 patients presented similar results as idiopathic form. 

Slight, but significant increase of serum creatinine was noted 

in this group of patients, from 130,62±15,91 to 215,78±63,62 

mol/l, p0,05), from 4,69±0,81 to 3,28±0,24g/daily, so it was still 

in nephrotic ranges. 

FSGS1 patients – untreated 

The results at the end of the study were catastrophic. At start, 

their mean value of serum creatinine was not significantly 

different comparing to previous two groups: 167,92±45,21 

mol/l. All six patients experienced end-stage renal failure 

within two years. 

Discussion 

There are many studies on genetic disorders in familial forms 

of FSGS (1-3,10-13), but the experience with the treatment of 

these cases is poor, so it is very difficult for us to compare 

our results. It was accepted that nephrotic syndrome in 

familial forms of FSGS is steroid resistant (2,7,11,13). 

Patients with homozygous or compound heterozygous 

mutations in NPHS2 exhibit primary steroid resistance and a 

decreased risk of FSGS recurrence after kidney 

transplantation (7). There was published a study in 2004 

which tested this hypothesis comparing the results between 

two groups of patients: 1)190 patients with familial FSGS 

and 2) 124 patients with idiopathic FSGS (7). Recurrence of 

the disease was noted in only 8% of familial form, but in 

35% of idiopathic form. Because of steroid unresponsiveness 

of familial form, the patients in this study were treated with 

cyclosporine A and cyclophosphamide with partial response. 

Besides small number of patients we noted some encouraging 

results: 5/8 treated patients did not develop end stage renal 

failure after two years and all untreated ones did. 

Conclusions 

The small number of patients did not allow us to perform 

standard statistic methods in order to make exact conclusions. 

It is also unclear what can be the mechanism of the drug 

acting in the patients who responded and did not worsen their 

renal function. 

References 

1. Caridi G et al. Broadening the spectrum of diseases related to 

podocin mutations. J am Soc Nephrol 2003; 14: 1278-1286 

2. Pollak MR. The genetic basis of FSGS and steroid-resistant 

nephrosis. Semin Nephrol 2003; 23: 141-146 

3. Winn MP. Approach to the evaluation of heritable diseases and 

update of familial focal segmental glomerulosclerosis. Nephrol 

Dial Transplant 2003; 6: vi14-20 

4. D’agati V. Pathologic classification of focal segmental 

glomerulosclerosis. Semin Nephrol 2003; 23: 117-134 

5. Winn MP et al. Clinical and genetic heterogeneity in familial 

focal segmental glomerulosclerosis. International collaborative 

group for therapy of familial focal segmental 

glomerulosclerosis. Kidney Int 1999; 55: 1241-1246 

6. Rana K et al. Clinical, histopathologic, and genetic studies in 

nine families with focal segmental glomerulosclerosis. Am J 

Kidney Dis 2003; 41: 1170-1178 

7. Ruf RG et al. Patients with mutations in NPHS2 (podocin) do 

not respond to standard steroid treatment of nephrotic 

syndrome. J Am Soc Nephrol 2004; 15: 722-732 

8. Weber S et al. NPHS2 mutation analysis shows genetic 

heterogeneity of steroid resistant nephrotic syndrome and low 

post-transplant recurrence. Kidney Int 2004; 66: 571-579 

9. Yorgin PD, Belson A, Higgins J, Alexander SR. Pulse 

methylprednisolone, cyclosporine, and ace inhibitor therapy 

decreases proteinuria in two siblings with familial focal 

segmental glomerulosclerosis. Am J Kidney Dis 2001; 37: E44 

10. Conlon PJ et al. Spectrum of disease in familial focal and 

segmental glomerulosclerosis. Kidney Int 1999; 56: 1863-1871 

11. Ekim M et al. Three siblings with steroid-resistant nephritic 

syndrome: new NPHS2 mutations in a Turkish family. Am J 

Kidney Dis 2004; 44: 10-12 

12. Koziell A et al. Genotype/phenotype correlations of NPHS1 

and NPHS2 mutations in nephrotic syndrome advocate a 

functional inter-relationship in glomerular filtration. Human 

Molecular genetics 2002; 11: 379-388 

13. Karle SM et al. Novel mutations in NPHS2 detected in both 

familial and sporadic steroid-resistant nephritic syndrome. J Am 

Soc Nephrol 2002; 13: 388-393


Microalbuminuria – The new marker for Balkan Endemic Nephropathy? 

G. Imamovic 1 , S. Trnacevic 1 , M. Tabakovic 1 , E. Mesic 1 , M. Uzeirbegovic 2 , M. Malohodzic 3 , 

A. Halilbasic 1 , V. Habul 1 , D. Tulumovic 1 , E. Hodzic 1 , M. Atic 1 , S. Lekic 1 M. Dugonjic 1 , 

A. Becirovic 1 and E. Hasanovic 

1 Clinic for nephrology, urology, dialysis and renal transplantation, 2 Central laboratory, 3 Nuclear medicine department, University 

medical center Tuzla, Bosnia and Herzegovina 

Abstract 

Several criteria are necessary to meet in order to have the 

diagnosis of Balkan endemic nephropathy established. One of 

them is tubular proteinuria type that may be found, but not in 

the early stage of the disease. Beta 2 microglobulin may be 

found in the early stage, but its determination is rather 

cumbersome and not suitable for a daily routine. Therefore, 

urinary albumin/creatinine ratio was determined in 8 patients 

(all females, aged 58,37±4,37 years) from the town of Šamac 

region (Bosnia and Herzegovina) as a measure of albumin 

excretion in order to establish useful marker in the early stage 

of the disease. Increased urinary albumin/creatinine ratio, was 

found in 50% of BEN patients. According to these 

preliminary results, microalbuminuria could be used as the 

reliable marker for the early detection of BEN. 

Key words: Balkan endemic nephropathy, microalbuminuria, 

urinary albumin/creatinine ratio 

Introduction 

Balkan endemic nephropathy (BEN) is an unknown disease 

that ultimately leads to terminal uremia. It affects rural 

population of Croatia, Bosnia and Herzegovina, Serbia, 

Romania and Bulgaria. It is characterized primarily with 

tubulo-interstitial changes (1) and predominant tubular 

proteinuria type has been reported by now (2). Beta 2 

microglobulin (B2M) has been used as a marker for early 

detection of the disease, but its use is rather complicated and 

not applicable for a daily routine. Quantitative measurement 

of urinary albumin as a marker of an early tubular changes 

has not been done by now. Therefore, we tested nonproteinuric 

urine samples of patients suffering from early 

stages of BEN for microalbuminuria in order to suggest 

possible new reliable BEN marker. 


Urinary albumin to creatinine ratio was determined in 

random urine samples in eight patients suffering from the 

early stage of BEN (all females, aged 58,37±4,37 years, 

range 51-63) from the town of Šamac region, Bosnia and 

Herzegovina 

Patients were selected on the basis of established criteria. 

Half of them had biopsy-proven lesions compatible with 

BEN in a study done 17 years ago (1). Hypertension, diabetes 

mellitus, heart failure and urinary tract infection have been 

ruled out because those conditions give rise to 

microalbuminuria themselves. 

Random urine samples were first tested on proteinuria with 

20% sulfosalycilic acid (SSA) test. The SSA test is 

performed by mixing one part urine supernatant (e.g., 2.5 

mL) with three parts 3 percent sulfosalicylic acid, and 

grading the resultant turbidity according to the following 

schema (the numbers in parentheses represent the 

approximate protein concentration): 0 = no turbidity (0 

mg/dL), trace = slight turbidity (1 to 10 mg/dL), 1+ = 

turbidity through which print can be read (15 to 30 mg/dL), 

2+ = white cloud without precipitate through which heavy 

black lines on a white background can be seen (40 to 100 

mg/dL), 3+ = white cloud with fine precipitate through 

which heavy black lines cannot be seen (150 to 350 mg/dL), 

4+ = flocculent precipitate (>500 mg/dL). Only those who 

proved non-proteinuric were tested on microalbuminuria 

using urinary albumin/creatinine ratio. 

Albumin was determined by immunonephelometric method 

using BN System (Dade Behring) on the basis of scattered 

light that has passed through immune complexes made up of 

albumin and specific antibodies. Intensity of scattered light 

was proportional to the quantity of the albumin in a sample. 

Results were evaluated by comparing them with the standard 

of known concentration. 

Creatinine in serum and urine was determined using autoanalyzer 

Dimension RxK (Dade Behring) with kinetic 

method by Jaffe, modified by Larsen (3). 

Albumin to creatinine ratio was then calculated. The cutpoint 

for females is > or = 2,7 mg/mmol (25 mcg/mg) (4) 

Diethylen Threeamino Penta Acetate (DTPA) clearance rate 

was determined by computerized method after the patients 

had been overloaded with fluid (1 L two hours prior to the 

test) and after they had voided. Tc99m DTPA was then 

applied in a dose of 185 MBq. Radioactivity was measured, 

memorized in a computer and clearance rate calculated. 

The statistical analysis was done using chi square test. 

P


microalbuminuric range in US (5,6) and between 5% and 7% 

in Europe (7,8). 

Results 

Urinary albumin/creatinine ratios in individual samples are 

shown in Table 1. Increased urinary albumin/creatinine ratio 

was found in 50% of BEN patients, what was statistically 

significant result (p


Lymphatic neoangiogenesis in human neoplasia and transplantation as 

experiments of nature 

D. Kerjaschki 

Department of Pathology, Medical University of Vienna, Austria 

Introduction 

A plexus of lymphatic vessels guides interstitial fluid, 

passenger leukocytes, and tumor cells towards regional 

lymph nodes. Microvascular endothelial cells of lymph 

channels (LECs) are difficult to distinguish from those of 

blood vessels (BECs) because both express a similar set of 

markers, such as CD31, CD34, podocalyxin, von Willebrand 

factor (vWF), etc. Analysis of the specific properties of LECs 

was hampered so far by lack of tools to isolate LECs. 

Recently, the 38 kD mucoprotein podoplanin was found to be 

expressed by microvascular LECs but not BECs in vivo. Here 

we isolated for the first time podoplanin + LECs and 

podoplanin - BECs from dermal cell suspensions by 

multicolor flow cytometry. Both EC types were propagated 

and stably expressed VE-cadherin, CD31, and vWF. 

Molecules selectively displayed by LECs in vivo, i.e., 

podoplanin, the hyaluronate receptor LYVE-1, and the 

vascular endothelial cell growth factor (VEGF)-C receptor, 

Flt-4/VEGFR-3, were strongly expressed by expanded LECs, 

but not BECs. Conversely, BECs but not LECs expressed 

VEGF-C. LECs as well as BECs formed junctional contacts 

with similar molecular composition and ultrastructural 

features. Nevertheless, the two EC types assembled in 

vascular tubes in a strictly homotypic fashion. This EC 

specialization extends to the secretion of biologically relevant 

chemotactic factors: LECs, but not BECs, constitutively 

secrete the CCR7 ligand SLC/CCL21 at their basal side, 

while both subsets, upon activation, release MIP-3a/CCL20 

apically. These results demonstrate that LECs and BECs 

constitute stable and specialized EC lineages equipped with 

the potential to navigate leukocytes and, perhaps also, tumor 

cells into and out of the tissues. 

Lymphatic Neoangiogenesis in Human Cancer. 

Formation of lymphatic metastasis is the initial step of 

generalized spreading of tumor cells, and predicts poor 

clinical prognosis. Lymphatic vessels generally arise within 

the peritumoral stroma, although the lymphangiopoetic 

vascular endothelial growth factors (VEGF)–C and -D are 

secreted in apparently small amounts by tumor cells. In a 

carefully selected collective of human cervical cancers (stage 

pT1b1) we demonstrate by quantitative 

immunohistochemistry and in situ hybridization that density 

of lymphatic microvessels is significantly increased in 

peritumoral stroma, and that a subset of stromal cells express 

large amounts of VEGF-C and -D. The density of cells 

producing these vascular growth factors correlates with 

peritumoral inflammatory stroma reaction, lymphatic 

microvessel density, peritumoral carcinomatous 

lymphangiosis, and with the frequency of lymph node 

metastasis. The VEGF-C and -D producing stroma cells were 

identified in situ as a subset of activated tumor associated 

macrophages (TAMs) by expression of a panel of 

macrophage specific markers, including CD68, CD23, CD14 

and others, in double-immunofluorescence confocal 

microscopy. These group of TAMs also expressed the 

VEGF-C and -D specific tyrosine kinase receptor flt-4. As 

TAMs are derived from monocytes in the circulation, a 

search in peripheral blood for candidate precursors of flt-4 

expressing TAMs revealed a subfraction of flt-4 expressing, 

CD14-positive monocytes, that, however, failed to express 

VEGF-C and -D. Only after in vitro incubation with TNFα, 

LPS or VEGF-D these naive monocytes started to synthesise 

VEGF-C de novo. In conclusion VEGF-C expressing TAMs 

play a novel role in peritumoral lymphangiogenesis, and 

subsequent dissemination in human cancer. 

Lymphatic Neoangiogenesis in Human Renal Transplants. 

Renal transplant rejection is caused by a lymphocyte rich 

inflammatory infiltrate that attacks cortical tubules and 

endothelial cells. Immunosupressive therapy reduces the 

number of infiltrating cells, however, their exit routes are not 

known. Here we demonstrate by immunohistochemistry on 

human renal transplant biopsies, a > 100 fold increase of 

lymphatic vessel density over normal kidneys in grafts with 

nodular mononuclear infiltrates, using antibodies to the 

lymphatic endothelial marker protein podoplanin. Nodular 

infiltrates are constantly associated with newly formed, Ki-67 

expressing lymphatic vessels and contain the entire repertoire 

of T- and B-lymphocytes to provide specific cellular and 

humoral alloantigenic immune responses, including Ki-67 + 

CD4 + and CD8 + T-lymphocytes, S100 + dendritic cells, as 

well as Ki-67 + CD20 + B-lymphocytes and l- and k-chainexpressing 

plasmacytoid cells. Numerous chemokine receptor 

CCR7 + cells within the nodular infiltrates are apparently 

attracted by secondary lymphatic chemokine (SLC/CCL21) 

that is produced and released by lymphatic endothelial cells 

in a complex with podoplanin. Thus, lymphatic 

neoangiogenesis contributes not only to the export of the 

rejection infiltrate, but is also involved in the maintainance of 

a potentially detrimental alloreactive immune response in 

renal transplants, and provides a novel therapeutic target. 

______________________ 

Correspodence to: D. Kerjaschki, Department of Pathology, Medical University of Vienna, Austria


A Novel Role for Podoplanin in Lymphangiogenesis. 

In order to analyze podoplanin function in the development 

of the lymphatic vasculature, we generated podoplanin -/- mice 

in 129S/v : Swiss background. 

The lack of podoplanin resulted in loss of about 30 % of the 

embryos. Within the first week, approximately 55% of born 

podoplanin -/- mice died, but 20% of born mice survived till 

adulthood. With respect to the lymphatic vasculature, young 

podoplanin -/- mice showed enlarged and tortuous lymphatic 

vessels, while the lymphatic network was impaired. 

Additionally, throughout the small intestine a network of 

dilated, tortuous and leaky lymphatic vessels resulting in the 

chylous ascitis and hyperemic Peyer´s patches were found. 

Lymphatic fluid transport, as revealed by intradermal 

injection of Chicago sky blue dye, was impaired in the 

knockouts. These data indicate that formation of the 

lymphatic vessel system is erroneous in lymphoid and nonlymphoid 

tissues in podoplanin -/- mice. The most impressive 

alkteration of the lymphatic vessels in Podoplanin -/- mice, 

however, was their lack of separtion from the blood 

vasculature during their entire life span. We have 

demonstrated that this separation defect is due to the platelet 

aggregating effect of podoplanin on LEC surfaces. Thus, 

platelets that come in contact with LECs in the embryonic 

lymphatic saccules aggregate and degranulate, and induce 

constriction of the blood vascular-lymphatic anastomosis 

(10). 

References 

1. Kerjaschki D, Regele HM, Moosberger I, Nagy-Bojarski K, 

Watschinger B, Soleiman A, Birner P, Krieger S, Hovorka A, 

Silberhumer G, Laakkonen P, Petrova, T, Langer B, Raab I. 

Lymphatic neoangiogenesis in human kidney transplants is 

associated with immunologically active lymphocytic infiltrates. 

J Am Soc Nephrol 2004; 15: 603-12 

2. Schoppmann SF, Birner P, Stockl J, Kalt R, Ullrich R, Caucig 

C, Kriehuber E, Nagy K, Alitalo K, Kerjaschki D. Tumorassociated 

macrophages express lymphatic endothelial growth 

factors and are related to peritumoral lymphangiogenesis. Am J 

Pathol 2002; 161: 947-56 

3. Petrova TV, Makinen T, Makela TP, Saarela J, Virtanen I, 

Ferrell RE, Finegold DN, Kerjaschki D, Yla-Herttuala S, 

Alitalo K. Lymphatic endothelial reprogramming of vascular 

endothelial cells by the Prox-1 homeobox transcription factor. 

EMBO J 2002; 21: 4593-9 

4. Saaristo A, Veikkola T, Enholm B, Hytonen M, Arola J, 

Pajusola K, Turunen P, Jeltsch M, Karkkainen MJ, Kerjaschki 

D, Bueler H, Yla-Herttuala S, Alitalo K. Adenoviral VEGF-C 

overexpression induces blood vessel enlargement, tortuosity, 

and leakiness but no sprouting angiogenesis in the skin or 

mucous membranes. FASEB J 2002; 16: 1041-9 

5. Kang DH, Kanellis J, Hugo C, Truong L, Anderson S, 

Kerjaschki D, Schreiner GF, Johnson RJ. Role of the 

microvascular endothelium in progressive renal disease. J Am 

Soc Nephrol 2002; 13: 806-16 

6. Kriehuber E, Breiteneder-Geleff S, Groeger M, Soleiman A, 

Schoppmann SF, Stingl G, Kerjaschki D, Maurer D. Isolation 

and characterization of dermal lymphatic and blood endothelial 

cells reveal stable and functionally specialized cell lineages. J 

Exp Med 2001; 194: 797-808 

7. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R, 

Amann G, Kriehuber E, Diem K, Weninger W, Tschachler E, 

Alitalo K, Kerjaschki D. Angiosarcomas express mixed 

endothelial phenotypes of blood and lymphatic capillaries: 

podoplanin as a specific marker for lymphatic endothelium. Am 

J Pathol 1999; 154: 385-94 

8. Matsui K, Breiteneder-Geleff S, Kerjaschki D. Epitope-specific 

antibodies to the 43-kD glomerular membrane protein 

podoplanin cause proteinuria and rapid flattening of podocytes. 

J Am Soc Nephrol 1998; 9: 2013-26 

9. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P, 

Poczewski H, Kalt R, Schaffner G, Kerjaschki D. Podoplanin, a 

novel 43-kd membrane protein of glomerular epithelial cells, is 

down-regulated in puromycin nephrosis. Am J Pathol 1997; 

151: 1141-52 

10. Uhrin P, Zaujec J, Bauer M, Hilpert M, Kowalski H, Furtbauer 

E, Hannes Stockinger H, Kerjaschki D, Binder B R. Podoplanin 

induced platelet aggregation mediates separation of blood and 

lymphtic vessels. "submitted"


Cytokines and acute renal failure 

K. Cakalaroski 

University Clinic for Nephrology, Clinical Centre, Skopje, Republic of Macedonia 

Introduction 

The crucial question in the settings of acute renal failure 

(ARF) is to elucidate the role of cytokines in the acute and 

chronic issue of the disease: death or survival with normal 

kidney function or inevitable evolution to chronic kidney 

disease (CKD) and terminal renal insufficiency. 

Topics for Discussion – Field of controversy 

Many years ago the relation between ischemic (mainly 

septic) ARF in the clinical settings and serum level of 

pro/anti inflammatory cytokines (IFNγ, TNFα, IL-1β, IL- 

6,IL-8,IL-10,ICAM –1,VCAM), is the field of controversy. 

Intrinsic ARF, as a special form of acute renal insufficiency 

is also associated with the cytokines production (their blood 

levels, kidney tissue and urine concentrations). The evolution 

of obstructive type of ARF may be related with the serum and 

tissue presence of anti and pro-inflammatory cytokines. ARF 

after kidney grafting is a differential diagnostic problem 

between s.c. initial tubulopathy and toxic effects of drugs 

used in the first few days after kidney transplantation. 

Plasma levels of pro and anti-inflammatory cytokines and 

adequate conservative measures on the one hand, and the 

mortality ratio in ARF on the other, may be strongly related. 

Elimination of cytokines from the blood and tissues 

compartments via extracorporeal procedures may have a 

beneficial effect on clinical evolution of ARF. 

Chronically elevated levels of pro-inflammatory cytokines 

once the ARF is over, are the signs of inflammation and 

progression to CKD. The limitations of the cytokines plasma 

dosing may compromise theirs clinical benefit. 

Sepsis related ischemic ARF 

The endotoxins (bacterial corporal lipopolysacharids, LPS s ) 

via CD-14, activate and deliver cytokines (TNFα, IL-1β,IL- 

6) from polymorphonuclear cells (PMNC s ) in ARF patients, 

via the process named “cytokines deliberation” with 

consecutive higher plasma concentration of all measured 

cytokines when compared to CKD patients (1). The impaired 

monocyte’s cytokines production in critically ill patients with 

ARF is presented with a dip fall of the blood levels of TNFα 

and IL-6 (84% vs 45% respectively) (2). The elevated serum 

concentrations of sTNFα R I, II are predictive for septic 

shock induced ARF (3). The IL-6 enhances the renal injury, 

organs dysfunction and inflammation (producing T-cells 

activation and elevation of CRP plasma levels) caused by the 

whole body ischemia-reperfusion in patients with ARF due to 

______________________ 

Correspodence to: K. Cakalaroski, University Clinic for Nephrology, Clinical Centre, 

Skopje, Republic of Macedonia 

systemic shock or cardiac arrest. The lesions correlate with 

the plasma level of IL-6: for each 100 pg/ml increase for IL- 

6, patients are 2.8 times likely to develop organ dysfunction 

(4). Endotoxemic ARF is caused by direct action of TNFα on 

TNFαR-1 in the kidney (proximal tubular epithelial cells, 

PTEC s ). In this case the urinary elimination of TNFα is 

increased (5). The Fas, FaSL and caspase mediated 

apoptopsis of PTEC s by inflammatory cytokines or LPS s , can 

be possible mechanism of endotoxic renal dysfunction (6). 

The cytokines stimulate PMNC s attraction, aggregations, 

adhaezion (via ICAM-1 and VCAM), activation and 

degranulation with lysosims liberation (7). The endothelial 

lesions, disseminated intravascular coagulation (DIC) via 

sepsis induced TF, fibrinolysis and prolonged 

vasoconstriction (because ET-1 production) – lead to tissue 

hypoxia, inflammation, Tc-mediated injury and ARF (8). 

Intrinsic ARF 

Many factors are included in the genesis of intrinsic ARF 

beginning with the obstetric shock and finishing with the 

acute tubulointerstitial nephritis. The obstetric shock is 

related to PAI-1 activation, renal DIC and a possible 

successive partial or total cortical necrosis of the kidney (9). 

Necrotic vasculitis mainly presented by a glomerulitis or 

pneumonitis is due to the activation of Th 1 and mediators of 

tissue injury like leucotriens, TxA 2 , PAF, reactive oxygen 

and nitrogenous metabolites (ROM, RNO), complement, 

intravascular activation of coagulation and secondary 

fibrinolysis. Interleukins (especially IL-2,IL-6,IL-8 and 

IFNγ), via nature killers cells (NK) and macrophages (M 

induce vascular (mainly capillary) leak syndrome because 

elevated plasma concentration of nitrogen oxide (NO). The 

consequences of capillary wall lesion are extension of 

interstitial volume, depletion of effective arterial blood 

volume, arterial hypotension and DIC with effective 

thrombocytopenia (under 60 000/cmm) and ARF (namely, 

acute tubular necrosis, ATN) (10). The acute tubulointerstitial 

nephritis is mediated by elevated plasma levels of 

interleukins (from IL-1 to IL-7), IFN s (α,β,γ), platelet derived 

growth factor –A, B, epithelial growth factor, fibrocyte 

growth facror-2, TNFα,β and TGFα,β generated by 

infiltrating and somatic renal cells (11,12). 

Tubular obstruction and ARF 

The endotoxins and the same cited pro-inflammatory 

cytokines provoke direct vasoconstrictive effects in the 

kidney, enhancement of tubular damage and delay of


recovery phase. This type of ARF in the clinical practice is 

known as “nephrohydrosis” (13). 

Acute renal failure after kidney grafting 

The persistent urinary excretion of IL-6 and IL-8 means 

organic sustained ARF. Urinary TNFα and LDH are reduced 

in severe and increased in mild ARF (14). Severe ARF 

correlates with increased plasma concentration of TNFα,s 

TNFαR s 55,75. If the plasma levels of s TNFα R s diminish, 

the renal function improves explicating the better urinary 

excretion of TNFα R s (7). Uromodulin decreases in urine in 

the case of ARF and augments if acute rejection is no longer 

questionable (15). 

Plasma levels of pro and anti-inflammatory cytokines and 

mortality in ARF 

Many plasma cytokines (IFNγ, TNFα, IL-1β, IL-6,IL-8,IL- 

10) may predict mortality and correlate with the lethality rate 

in patients with ARF (2). Proinflammatopry cytokines 

(especially IFNγ, TNFα, IL-1β, IL-6, IL-8) lead to systemic 

inflammatory response syndrome (SIRS) and contrary antiinflammatory 

response syndrome (CARS) precipitating the 

lethal issue of ARF (following the results of PICARD study 

group). The plasma levels of IL-6 (not-survivors: survivors = 

235:114, pg/ml), IL-8 (not-survivors : survivors = 36:21, 

pg/ml) and IL-10 (not-survivors : survivors = 3.1 : 2.4, p/ml) 

are more increased in patients with ARF not-survivors when 

compared to survivors (16). 

Chronically elevated levels of pro-inflammatory cytokines 

as a sign of inflammation and progression to CKD 

The perpetual production of cytokines (IL-1, IL-6, IL-7, 

TNFα) is a central event in the patophysiology of cell 

mediated damage by resident mesangial, interstitial or 

infiltrating cells (17). The vascular endothelial dysfunction 

and accelerated atherosclerosis are related to acute phase 

proteins (CRP, SAP) and plasma cytokines demonstrating a 

chronic inflammatory state after ARF (18). Proteinuria 

transcripts the genes of pro-inflammatory cytokines (mainly 

TNFα gene) (19). The plasma levels of IL-1, TNFα and IFNγ 

directly correlate with ICAM-1 and VCAM-1 expression 

(20). The IFNγ / IL-10 ratio is greater in patients with severe 

nephrosclerosis due to lupus nephritis (21). The Th 1 /Th 2 renal 

parenchymal predominance support cell-mediated immune 

response and produce pro-inflammatory cytokines (IFNγ, 

TNFα, IL-12) as a mediators responsible for progression of 

renal lesions (22). 

Conservative therapeutic anti-cytokines options 

Many conservative therapeutic anti-cytokines procedures are 

trailed in the ARF patients with variable, mainly nonsatisfactory 

results. In the following text will be presented the 

most acceptable anti-cytokines conservative treatment. 

The inhibition of LPS activity is a theoretically very 

attractive therapeutic modality of septic induced ARF. The 

use of anti LPS monoclonal (E-5 murine, human anti lipid A 

region and HA-1A) and polyclonal antibodies (J5) in 

combination with the anti CD 14 Ra on the Mis effective 

in the experimental setting, but without efficacy in clinical 

medicine (23). 

The inhibition of IL-1 with r hu IL1Ra blocks the basic signal 

transduction and reduces interstitial cells infiltration (24). 

The inhibition of TNFα with murine anti TNFα monoclonal 

antibodies (afelimomab) in i.v continuous infusion, prevents 

endothelial lesions and reduce mortality in experimental and 

clinical cases (25). 

The inhibition of IL-6 with IL-6 monoclonal antibodies 

reduces the renal injury, kidney dysfunction and 

inflammation caused by ischemia-reperfusion via reduction 

of immuno-histochemical markers (ICAM-1, P-selectin, 

nitrothyrosin, ROM and RNM) (4). LPS may induce renal IL- 

6 m RNA synthesis that may be inhibited by a single dose of 

ACE inhibitors (like enalaprilate) and in this way, to prevent 

cytokines-induced renal damage during endotoxemia (31). 

There are other therapeutic conservative modalities used 

more or less successfully in patients with ARF like thyroid 

hormones (26), growth factors (EGF, HGF, IGF-1) (27), 

soluble P-selectin GP ligand –1 (28) and R hu PAF Ra (29). 

The blockade of sepsis induced tissue factor (TF) with site 

inactivated F VIIa (s.c.TF-F VIIa-complex) is a mode to 

attenuate kidney injury in septic shock (32). With Zn-bis DL- 

OH aspartat may be stimulated the production of heat shock 

proteins (mainly HSP-70) and the regeneration of necrotic 

tubular cells (33). The BMP-7 reduces the harmful effects of 

pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNFα, MCP- 

1) on kidney proximal tubular cells after inflammation related 

to ischemic injury and stimulates a tubular cells proliferation 

(42). The monomethyl arginin (L-NMMA) as a NO synthase 

(NOS) – blocker (34), vasodilative atrial natriuretic peptides 

(ANP s ) (35), anti-endothelin – 1 monoclonal antibodies and 

receptor antagonists (Anti ET-1 Mo abs; ET-1 Ras) (36,37), 

anti ICAM-1 monoclonal antibodies (38) and Arg-Gly-Asp 

(RGD) peptides (39) - are also trailed in clinical studies. 

Active therapeutic anti-cytokines options 

The aim of active therapeutic procedures is to eliminate or 

diminish the production of pro-inflammatory cytokines 

related to different haemodialysis membranes or to promote 

the elimination of active reactants from the blood, via extracorporeal 

devices (membranes, adsorbens). 

Today it is largely accepted that the cuprophan membrane 

enhances activation of various pathways of tubular cells 

injury during the haemodialysis procedure like ROM, RNM, 

cytokines, eicosanoides, complement production and PMNC s 

activation, as compared to synthetic membranes (40). 

The hydrophobic biocompatible high flux dialyzers 

membranes (PAN, PS, PA, PMMA, PAES) do not activate 

the M PMNC s and do not deliberate cytokines and other 

active chemical reactants (41). 

Elimination of active reactants via extracorporeal devices 

(membranes, adsorbens) today is accepted as an attractive 

therapeutic modality. Classical haemodialysis (HD) not 

influence the plasma levels of many cytokines (IL-1β, IL-2R, 

IL-6, TNFα), but reduces the blood levels of hemokine IL-8 

for 50% (43). CVVH ± HD improve hemodinamics in septic 

shock related ARF with or without modifying plasma 

concentration of cytokines (IL1β, TNFα) except IL-6, 

although the clearances for two basic pro-inflammatory 

cytokines is about the same (Clearance for TNFα = 21 

ml/min; clearance for IL-6 = 25 ml/min) (44).


High permeability hemofiltration (HPHF) using polyflux 

hemofiltres with nominal cut-off point of 60 kD and 

intermittent hemoperfusion – hemofiltration (HP-HF), 

eliminate TNFα (Clearance = 15-28 ml/min), IL-6 (Clearance 

= 23-42 ml/min) and IL-1 Ra (Clearance = 25-54 ml/min) in 

septic patients with ARF (45). Plasmaferesis eliminates 

endotoxins (LPS s ), cytokines (IL-1β, IL-6, TNFα) soluble 

cytokines-binding proteins, C 3 and Ca ++ (30). 

The molecular adsorbens recycling system (MARS) 

eliminates albumin-binding toxins.water soluble toxins, NO, 

IFNγ, TNFα, IL-6 and IL-8 in sever liver failure and Multi 

Organs Dysfunction Syndrome (MODS) with marked 

decrease of sequential organ failure assessment (Sequential 

Organ Failure Assessment, SOFA score reduction from 9.7 to 

7.0). MARS may increase the serum levels of IL-6 by 

unknown mechanism (47). 

Limitations of cytokines plasma dosage 

The soluble receptors have almost all pro- inflammatory 

cytokines (IL-1, IL-2, IL-6, TNFα, IFNγ); that’s the active 

interleukins fraction is difficult to know. The cytokines 

retention may be due to reduced renal clearance a blood 

accumulation. The tissue (namely effective) cytokine 

concentration differs from the plasma levels for the same 

cytokine and the conclusion about his efficiency following 

the blood cytokines presence is inconclusive. Finally, the 

cytokines network is interrelated and may compensate for 

each other. 

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6. Jo SK, Cha DR, Cho WY, Kim HK, Chang KH, Yun SY, Won 

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7. Hoffman JN, Hartl WH, Deppisch R, Faist E, Jochum M, 

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729-737


Amyloidosis 

M. Tunca 

Dokuz Eylül University School of Medicine, Department of Internal Medicine, Izmir, Turkey 

Abstract 

Amyloidosis is a group of diseases due to pathologic 

deposition of various insoluble fibrilllar proteins having an 

identical secondary structure (ß-pleated sheet) in the 

extracellular compartments of the body. When diabetes 

mellitus type II and Alzheimer’s disease are taken into 

account, the prevalence of amyloidosis is obviously quite 

high. There are four types of amyloidosis: AL (light chain) 

amyloidosis, AA amyloidosis (secondary to chronic 

inflammation), Aß 2 M amyloidosis (secondary to long-term 

hemodialysis) and heredofamilial (ATTR and other) 

amyloidoses. The process may be focal, local or generalized 

whose clinical consequences are reflections of the involved 

organ(s). Some clinical features such as macroglossia (AL), 

nephrotic syndrome (AA), arthropathy (Aß 2 M ) or familial 

peripheral neuropathy (ATTR) may imply the type of deposit 

to a certain extend. A definitive diagnosis requires not only 

tissue confirmation (Congo Red staining with green 

birefringence under polarized light) but also genomic DNA 

and protein analyses and immunohistochemistry. Suitable 

tissue samples can be obtained from subcutaneous fat pad 

aspiration, rectal mucosa or renal biopsy. Neural or 

myocardial biopsies are rarely necessary. Serum amyloid P 

component (SAP) scintigraphy can visualise and demonstrate 

the amyolid burden of the patient; this procedure may also be 

useful to measure the response to treatment. 

Amyloidosis is not regarded as an irreversible pathology 

anymore. Detection of the precursor protein such as SAA or 

mutated transthyretin is an important step in therapeutic 

decision making since further progression of the deposits can 

thus be halted. There are several treatment options available 

to the clinicians, while the efficacy of some of these 

modalities has not yet been definitely established. 

Keywords: amyloidosis, pathogenesis, treatment, familial 

mediterranean fever 

Introduction 

Amyloidosis is a heterogeneous group of diseases. 

Misfolding and deposition of certain soluble proteins as 

insoluble fibrils in the extracellular compartments of the body 

cause variety of organ dysfunctions. They have an identical 

secondary structure (ß-pleated sheet) but there are 21 

different proteins causing amyloidosis. The majority of 

amyloid cases are due to acquired pathologic processes but a 

substantial minority is hereditary. 

Historically, the term “amyloid” (starch-like) was first used 

by Schleiden, a German botanist, in 1838. Virchow 

introduced it to medical jargon in 1858 for the autopsy 

findings which were later demonstrated that they had nothing 

to do with starch but were proteinaceous in nature. Congo 

Red staining was first used by Divry in 1927 and the 

ultrastructure of amyloid fibrils was identified by Cohen and 

Calkins in 1959 (1). 

Types of amyloidosis and their prevalence 

Amyloidosis may be localized or systemic. There are four 

types of systemic amyloidosis: 

1. AL amyloidosis is due to plasma cell dyscrasia 

related to multiple myeloma where the precursor 

protein is either or immunoglobulin. 

2. AA amyloidosis is secondary to chronic 

inflammation and the precursor protein is serum 

amyloid A protein. 

3. Aß 2 M amyloidosis is secondary to long-term 

hemodialysis and the precursor protein is ß 2 

microglobulin. 

4. ATTR and other heredofamilial amyloidoses where 

the precursor protein is usually abnormal 

transthyretin (previously called prealbumin). The 

precursor of other rarer forms may be apolipoprotein 

A-I, lysozyme, or fibrinogen A -chain. 

It is estimated that amyloidosis is the cause of death of about 

6,6/10,000 people in the United Kingdom annually (2) and 

age-adjusted incidence of AL amyloidosis is around 8 per 

million person-years in USA where the ratio of ATTR 

amyloidosis rate is 10-20 percent of AL cases (3). AA type is 

relatively more prevalent in under-developed countries since 

untreated chronic infections such as tuberculosis are still not 

exceptional; other chronic inflammatory diseases 

(rheumatoid arthritis etc) are outstanding sources of new 

cases universally. Familial Mediterranean fever (FMF) needs 

mentioning since it is quite prevalent in Turkey (0,1 percent) 

and in Greece and may be encountered in other Balkan 

countries to a certain extend. Migration of populations from 

the Middle East in the twentieth century has transformed 

FMF into a universal disease. It was recently observed that 

about 13 percent of nearly 3000 FMF patients had AA 

amyloidosis in Turkey and it is the most frequent cause of 

amyloidosis in that country (4, 5). Aß 2 M amyloidosis is 

potentially important source because there are approximately 

1 million people on dialysis programs worldwide and the 

development of amyloidosis approaches to 100 percent after 

20 years (6). 

______________________ 

Correspodence to: M. Tunca, Dokuz Eylül University School of Medicine, Department of Internal Medicine, 

Balcova, 35350 Izmir Turkey; E-mail: mehmet.tunca@deu.edu.tr


When diabetes mellitus type II and Alzheimer’s disease are 

taken into account, the prevalence of amyloidosis is 

obviously quite high. Amyloidosis is not systemic but 

localized to pancreatic islet cells and cerebrum in these 

diseases and their pathogenetic implications are not well 

established. 

Pathogenesis 

The newly synthesized polypeptides enter a funnel-like 

pathway of conformational modification in the cytoplasm 

under physiological conditions. Some polypeptides may 

however misfold and aggregate as insoluble fibrils, which is a 

pathologic process leading to amyloidosis. The first step in 

amyloidogenesis is the change in the precursor protein that 

starts the process of fibrillar autoaggregation. Production of 

the precursor protein is quantitatively and/or qualitatively 

pathologic. There are specific amino acid substitutions in the 

precursor light chain in AL amyloidosis or transthyretin in 

ATTR and it is thought that such changes destabilize the 

molecule and facilitate fibrillogenesis. A third mechanism is 

proteolytic remodelling of the precursor protein. 

Since not every person with the same mutation develops 

amyloidosis and there is a very wide time range in AA 

amyloidosis, other genetic and macro and 

microenvironmental factors may be operational. Certain 

genotypes such as being M694V homozygote and SAA1 

alpha/alpha and a positive family history for amyloidosis 

have significantly higher risk of amyloidosis in FMF (7). The 

concentration of urea in the inner renal medulla or the pH 

level of the tissues may enhance fibrillogenesis. It is also well 

recognized that once amyloid deposition starts, progression is 

easier (“amyloid begets amyloid”). This may explain the 

concentration of amyloid in certain organs but the tissue 

specificity of each protein (joints, peripheral nerves etc.) is 

not well understood (1, 3, 8). 

Diagnosis 

Every patient must be evaluated for extend of organ 

involvement and family history and the type of precursor 

protein must be determined. A definitive diagnosis requires 

not only tissue confirmation (Congo Red staining with green 

birefringence under polarized light) but also genomic DNA 

and protein analyses and immunohistochemistry, otherwise 

some hereditary forms may be misdiagnosed as AL type (2, 

9). Suitable tissue samples can be obtained from 

subcutaneous fat pad aspiration, rectal mucosa or renal 

biopsy. Neural biopsy is not very reliable because of the focal 

distribution of amyloid. Myocardial biopsy is rarely 

necessary. Serum amyloid P component (SAP) scintigraphy 

can visualise and demonstrate the amyolid burden of the 

patient; this procedure may also be useful to diagnose subclinical 

disease in progress or measure the response to 

treatment (1, 10). 

Clinical aspects 

Some clinical features such as macroglossia (AL), nephrotic 

syndrome (AA), arthropathy (Aß 2 M) or familial peripheral 

neuropathy (ATTR) may imply the type of deposit to a 

certain extend but the clinical picture is rarely conclusive. 

AL type is the most diverse in clinical picture but the central 

nervous system is spared. Kidney and heart are frequently 

involved and the absence of hypertension despite severe renal 

failure should alert the physician. Heart failure is particularly 

an ominous sign. Easy bruising and spontaneous periorbital 

purpura (“racoon eyes”), hard hepatomegaly with elevated 

alkaline phosphatase and macroglossia (about 20 percent of 

the patients) are prominent features (3). 

AA amyloidosis almost always presents with proteinuria 

which may be intermittent initially. Other organ 

involvements such as hepatomegaly, splenomegaly, 

malabsorption, thyroid or heart failure are exceptional 

features of the average patient. 

Aß 2 M amyloidosis is always secondary to prolonged dialysis 

and the commonest complaint is severe joint pain in the 

absence of radiological changes of arthritis, the shoulders 

usually being the most affected. Carpal tunnel syndrome 

(usually bilateral) is also frequently encountered (5). 

Heredeofamilial amyloidosis is probably more frequent than 

perceived (2). Peripheral and autonomous nervous system 

involvements are outstanding features while renal failure is 

rare and macroglossia has not been reported. The most 

frequent mutation of ATTR causes cardiac conduction 

disturbances while other mutations may cause cardiac failure 

indistinguishable from AL type. Generally heart failure is 

less common and more benign in ATTR type (3). 

Treatment 

Amyloidosis is not regarded as an irreversible pathology 

anymore. Detection of the precursor protein such as SAA or 

mutated transthyretin is an important step in therapeutic 

decision making since further progression of the deposits can 

thus be halted. Another main objective is to support the 

failing organ while transplantation may be the definitive 

therapy (11, 12). 

AL amyloidosis may respond to oral melphalan and 

prednisone but high dose IV melphalan with autologous stem 

cell transplantation is more effective. The trials with IDOX 

(4’-iodo-4’-deoxydoxorubicin) have been disappointing. 

AA amyloidosis can respond favourably to specific treatment 

of the primary disease. SAA determinations with aim of 

values lower than 10 mg/L would be appropriate. It has been 

shown that colchicine can slow the progression of 

amyloidosis and regress the proteinuria in FMF (13, 14), 

where drug dosage must be escalated to tolerated maximum 

which is usually 2-2.5 mg/day. Patients with amyloidosis 

secondary to inflammatory arthritides may respond to anti- 

TNF treatment (infliximab or etanercept) particularly when 

combined with methotrexate (15). CPHPC is a promising 

drug which removes SAP from the circulation and amyloid 

deposits (9, 11). Sodium-I,3-propane-disulfonate (Fibrillex) 

is an investigational drug whose phase II/III results will soon 

be available. 

Liver transplantation is the definitive therapy of ATTR 

amyloidosis. If the liver of the affected patient is transplanted 

to another patient (so-called “domino liver transplantation”) 

systemic ATTR amyloidosis may sometimes develop in the 

recipient. Renal transplantation is the only effective treatment 

of Aß 2 M amyloidosis.


References 

1. Hawkins PN. The diagnosis, natural history and treatment of 

amyloidosis. The Goulstonian Lecture 1995. J R Coll 

Physicians Lond 1997; 31: 552-560 

2. Lachmann HJ, Booth DR, Booth SE, Bybee A, Gilbertson JA, 

Gillmore JD, Pepys MB, Hawkins PN. Misdiagnosis of 

hereditary amyloidosis as AL (primary) amyloidosis. N Engl J 

Med 2002; 346: 1786-1791 

3. Falk RH, Comenzo RL, Skinner M. Medical Progress: The 

Systemic Amyloidoses. N Engl J Med 1997; 337: 898-909 

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(FMF) in Turkey: results of a nationwide multicenter study. 

Medicine (Baltimore) 2005; 84: 1-11 

5. Tuglular S, Yalcinkaya F, Paydas S, et al. A retrospective 

analysis for aetiology and clinical findings of 287 secondary 

amyloidosis cases in Turkey. Nephrol Dial Transplant 2002; 

17: 2003-2005 

6. Kurer MH, Baillod RA, Madgwick JC. Musculoskeletal 

manifestations of amyloidosis. A review of 83 patients on 

haemodialysis for at least 10 years. J Bone Joint Surg Br 1991; 

73: 271-276 

7. Delibas A, Oner A, Balci B, et al. Genetic risk factors of 

amyloidogenesis in familial Mediterranean fever. Am J Nephrol 

2005; 25: 434-440 

8. Merlini G, Bellotti V. Mechanisms of disease: Molecular 

mechanisms of amyloidosis. N Engl J Med 2003; 349: 583-596 

9. Hazenberg BP, van Gameren II, Bijzet J, Jager PL, van 

Rijswijk MH. Diagnostic and therapeutic approach of systemic 

amyloidosis. Neth J Med 2004; 62: 121-128 

10. Hawkins PN, Aprile C, Capri G, Vigano L, Munzone E, Gianni 

L, Pepys MB, Merlini G. Scintigraphic imaging and turnover 

studies with iodine-131 labelled serum amyloid P component in 

systemic amyloidosis. Eur J Nucl Med 1998; 25: 701-708 

11. Hirschfield GM, Hawkins PN. Amyloidosis: new strategies for 

treatment. Int J Biochem Cell Biol 2003; 35: 1608-1613 

12. Grateau G, Jeru I, Rouaghe S, et al. Amyloidosis and autoinflammatory 

syndromes. Curr Drug Targets Inflamm Allergy 

2005; 4: 57-65 

13. Oner A, Erdogan O, Demircin G, Bulbul M, Memis L. Efficacy 

of colchicine therapy in amyloid nephropathy of familial 

Mediterranean fever. Pediatr Nephrol 2003; 18: 521-526 

14. Livneh A, Zemer D, Langevitz P, Laor A, Sohar E, Pras M. 

Colchicine treatment of AA amyloidosis of familial 

Mediterranean fever. An analysis of factors affecting outcome. 

Arthritis Rheum 1994; 37: 1804-1811 

15. Gottenberg JE, Merle-Vincent F, Bentaberry F, et al. Antitumor 

necrosis factor alpha therapy in fifteen patients with AA 

amyloidosis secondary to inflammatory arthritides: a followup 

report of tolerability and efficacy. Arthritis Rheum 2003; 48: 

2019-2024


Oxidative stress evaluation in uraemic patients undergoing continuous 

ambulatory peritoneal dialysis 

G. Mircescu 1 , C. Capusa 1 , I. Stoian 2 , D. Lixandru 2 , E. Rus 3 , L. Coltan 2 and L. Gaman 2 

1 “Dr Carol Davila” University Hospital of Nephrology, Bucharest, Romania 

2 Biochemistry Dept. “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 

3 NephroCare “Dr Carol Davila” Dialysis Centre, Bucharest, Romania 

Abstract 

Since the great bulk of evidences regarding oxidative stress 

associated with chronic renal failure (CRF) came from 

haemodialysis patients and only scant data is available 

concerning peritoneal dialysis, we investigated the oxidative 

status in CRF patients on continuous ambulatory peritoneal 

dialysis (CAPD). 

Plasma lipid peroxidation assayed as thiobarbituric acid 

reactive substances (TBARS; nmol/g protein), plasma total 

free thiols (Pt-SH; mcmol/g protein) as marker of 

extracellular antioxidant status and parameters of 

enzymatic/non-enzymatic intracellular antioxidant defence 

(erythrocyte superoxide dismutase (SOD; U/g haemoglobin), 

glutathione peroxidase (EGPx; U/g haemoglobin) and nonprotein 

thiols (E-SH; mcmol/g haemoglobin)] were measured 

by spectrophotometry in 18 CAPD patients, 16 non-dialysed 

CRF patients and 12 healthy subjects. 

Increased TBARS were found only in CAPD patients versus 

Controls (58.3±19.8 vs. 42.7±12.4, p= 0.01). Also, they had 

lower activity of erythrocyte SOD (164.2±47.5 vs. 

463.2±77.5 in CRF group, p


median volume of ultrafiltration per day was 1000mL 

(between 300-1800mL). 

Sixteen age- and gender-matched non-dialysed CRF patients 

(mean serum creatinine 4.5±2.7mg/dL) were randomly 

selected from those attending the Nephrology Clinic for 

routine periodical examination over a period of six months 

and composed the control patients group. They were not 

considered candidates for RRT in the near future. 

The CRF groups had a similar distribution according to 

primary renal disease, with primary chronic 

glomerulonephritis being the main cause of CRF (Table I). 

There were no patients with diabetic nephropathy. 

Exclusion criteria for the patients were inflammatory and 

infectious disorders (including exit or tunnel infections and/or 

peritonitis two months before enrolment), malignancies, 

diabetes mellitus, active liver diseases, severe anaemia, and 

medications with antioxidative potentialities (vitamin C or E, 

folic acid, allopurinolum). None of the patients required 

blood transfusions in the preceding two months. 

Normal control group consisted of twelve matched healthy 

volunteers with normal routine biochemistry and urine 

analyses. They did not take any medication or nutritive 

supplements. 

An informed consent was obtained from all participants. 

Methods 

Blood samples were drawn after overnight fasting into 

standard vacuum tubes with lithium heparin, were transported 

on ice, in the dark, and were processed within 30 minutes of 

collection by centrifugation (3000 g for 10 minutes at room 

temperature). 

The plasma aliquots were stored at -70°C until analysed for 

oxidative stress determinations or used immediately for 

routine laboratory parameters. Erythrocytes were washed 

three times with isotonic saline solution and lysed in cold 

ultrapure water. The red cell stroma was removed by 

centrifugation (10000 g for 15 minutes at 4°C) and the clear 

supernatant was stored at -70°C until erythrocytes antioxidant 

parameters were assayed. Reagents and ultra pure water were 

treated with Chelex 100 in order to bind transitional metals. 

Usual biochemistry parameters, C-reactive protein included, 

were determined by means of routine automated procedures 

on multiparameter analyser and haemoglobin concentrations 

were measured by automated spectrophotometric method. 

All the oxidative stress parameters were determined by 

spectrophotometry and the measurements were carried out on 

triplicate samples. 

Plasma thiobarbituric acid reactive substances (TBARS), 

measured according to the method described by Esterbauer 

(4), were used to assess plasma lipid peroxidation. Plasma 

aliquots were treated with butylated hydroxytoluene in order 

to prevent artifactual oxidation during the assay procedure 

and mixed with 0.67% thiobarbituric acid (TBA) and 20% 

trichloroacetic acid. Blank samples from distilled water and 

the same reagents were prepared. The reaction mixture was 

heated at 100°C for one hour and then cooled with tap water. 

The pink TBA-reacting substances adduct was extracted in n- 

butanol and the absorbance of the organic layer was read at 

532nm, after centrifugation. The concentration of lipid 

peroxidation products was calculated as malondialdehyde 

(MDA) equivalents using the extinction coefficient for the 

MDA-TBA complex of 153000 M -1 cm -1 and expressed as 

nmol/g protein. 

Plasma total free thiols (Pt-SH) assayed by the Ellman’s 

method (5), served as marker of extracellular antioxidant 

status. Briefly, aliquots of plasma were mixed with 5mM 

phosphate buffer (pH=8) and 10% sodium dodecil sulphate. 

Then Ellman’s reagent was added and samples were 

incubated at 37˚C for one hour. The absorbance was read at 

412nm against a reagent blank. Results were calculated using 

a calibration curve with glutathione as standard, and 

expressed as mcmol/g protein. 

Erythrocyte superoxide dismutase activity (SOD) was 

assessed by analysing the rate of inhibition of pyrogallol 

auto-oxidation, according to Marklund and Marklund (6). 

The erythrocyte lysate was treated with a mixture of 

ethanol/chloroform and was centrifuged for 20 minutes at 

6000 g. The supernatant fluid was mixed with 0.2mM 

pyrogallol and TRIS buffer (pH = 8.2), in the presence of 

oxygen. The rate of change in the absorbance at 420nm was 

monitored for 3 minutes against the blank sample. SOD 

activity was expressed as U/g haemoglobin. 

Erythrocyte glutathione peroxidase activity (EGPx) was 

determined indirectly by measuring the rate of consumption 

of NADPH during the regeneration of reduced glutathione 

from the oxidised glutathione produced by EGPx in the 

presence of oxidising substrate (7). The erythrocyte lysate 

was mixed with TRIS buffer and the enzymatic reaction was 

initiated by adding 1mM tert-butyl hydroperoxide. The 

conversion of NADPH to NADPH + was followed by 

recording the decrease in the absorbance at 340nm for 3 

minutes. The enzyme activity was calculated using the molar 

extinction coefficient for NADPH of 6220 M -1 cm -1 and was 

expressed as U/g haemoglobin. 

Erythrocyte non-protein thiols (E-SH), which consist mainly 

of reduced glutathione (GSH), were measured according to 

the method of Beutler et al (8). The erythrocyte lysate was 

mixed with a protein precipitating solution (1.67 g 

metaphosphoric acid, 0.2 g disodium ethylenediaminetetraacetic 

acid and 30 g sodium chloride in 100 ml distilled 

water). After 5 minutes, the mixture was filtered and 0.3M 

sodium phosphate and Ellman’s reagent were added. The 

absorbance was read at 412nm against a reagent blank. The 

results were calculated using the molar extinction coefficient 

for GSH of 13600 M -1 cm -1 and were expressed as mcmol/g 

haemoglobin. 


Results were expressed as mean ± standard deviation and 

were compared using Student’s t test or ANOVA (for 

normally distributed variables). Median and interquartile 

range wase used to express the non-parametric variables. 

Standard regression analysis and Pearson r correlation 

coefficient were used to evaluate the relationship between 

pairs of variables. 

A p value < 0.05 was considered significant.


Results 

Table 1. Main characteristics of the subjects 

Parameter CAPD Group CRF non-D group Controls 

Number 18 16 12 

Main causes of CRF: 

primary chronic 

glomerulonephritis (%) 

44 38 - 

tubulo-interstitial nephritis (%) 28 25 - 

vascular nephropathies (%) 22 25 - 

polycystic kidney disease (%) 0 12 - 

other (%) 6 0 - 

Mean age (years) 55.6±16.9 55±15.7 45.5±12.1 

Gender ratio (M/F) 8/10 10/6 5/7 

Smokers in (%)] 3 (16.7) 3 (18.8) 1 (8.3) 

Body mass index (kg/m 2 ) 

‡ 26.2±3.7 

† 22.8±3.1 25.6±3.0 

Serum iron (mcg/dL) 58.6±28.4 54.8±24.7 73.1±24.7 

Serum cholesterol (mg/dL) 225.8±57.0 191.9±55.8 229.5±48.1 

Serum albumin (g/dL) 

† 3.7±0.4 

† 3.9±0.6 4.6±0.3 

Haemoglobin (g/dL) 

† 9.7±2.3 

† 9.6±2.3 14.1±1.3 

Serum creatinine (mg/dL) 

†‡ 9.4±2.9 

† 4.5±2.7 0.9±0.1 

Serum uric acid (mg/dL) 

† 6.1±0.8 

† 6.8±1.5 4.7±1.5 

p


renal replacement therapy, but more probably it is related to 

the low sensibility and specificity of the used assay. 

Alternatively, the serum levels of TBARS might be kept in 

the normal range by some protective mechanisms like hepatic 

microsomes that are capable of both TBARS generation and 

degradation, as supported by the study of Gotoh et al (12). 

Previously, Malgorzewicz et al also discovered higher plasma 

content of MDA in peritoneal dialysis compared to healthy 

group, especially in severely malnourished patients and 

supposed that the increase in oxidative stress may be related 

to a degree of malnutrition (13). Our findings support the 

presence of oxidative stress in uraemic patients on CAPD 

with no apparent deficiency of the nutritional status, at least 

as reflected by the BMI and serum cholesterol (that did not 

differ from Controls), or serum albumin (that was similar to 

CRF group). Furthermore, the CRF group who had lower 

BMI and serum albumin than normal controls showed no 

differences in TBARS levels compared to healthy subjects. 

However, the present study does not provide enough data to 

sustain or deny the role of malnutrition in augmenting 

oxidative stress because the evaluation of nutritional status 

was not a primary objective and its casual assessment relied 

on few, unspecific parameters. 

Indirect evidences of oxidative stress can also be obtained 

from the investigation of antioxidant parameters. The 

decrease of plasma total free thiols levels observed in all 

studied CRF patients compared to healthy individuals, which 

reflects a deficiency of the extracellular antioxidant defence, 

could be speculated in favour of an increased consumption 

for counteracting the potential oxidative burden associated 

with chronic renal failure. The presumable contribution of 

hypoalbuminaemia to the lower plasma thiols appears 

implausible in our patients since no significant correlation 

was found between serum albumin and Pt-SH in either CRF 

groups (r = 0.27, confidence interval 95% = -0.3 ÷ +0.7; p = 

0.4 in CRF non-dialysed group, and r = -0.13, confidence 

interval 95% = -0.47 ÷ +0.32; p = 0.5 in CAPD group, 

respectively). Therefore, we interpreted the reduced plasma 

thiols as an indirect proof of augmented oxidative stress in 

CRF patients, dialysed or not. Similar results have been 

reported by Tarng et al, who demonstrated the decrease of the 

whole-blood reduced glutathione as well as of the plasma 

ascorbate and alfa-tocopherol in chronic peritoneal dialysis 

patients and in non-dialysed patients with advanced renal 

failure (mean creatinine clearance 11.8±4.6mL/min) (3). 

The deficiency of antioxidant systems in peritoneal dialysis 

patients is further sustained in our results by the marked 

decline of erythrocyte SOD activity. Therefore, one can 

assume that both extracellular and enzymatic intracellular 

defence against oxidative stress are defective in uraemic 

patients undergoing CAPD. These findings are in agreement 

with some other studies that reported reduced enzymatic 

erythrocyte defence (superoxide dismutase and catalase) in 

CAPD group (14,15), although discrepant results were also 

obtained. For example, Bonnefont-Rousselot et al did not 

detect any abnormalities in erythrocyte SOD activity in 

CAPD-treated patients in comparison with age-matched 

control group (16). However, they included older subjects 

than ours and this might allow to speculate the lack of 

difference on the account of advanced age, which impairs the 

antioxidant systems by itself and, possible, uraemia or 

dialysis procedure were not able to add further deterioration 

in this distinctive age group. 

Concerning the other two studied erythrocyte antioxidant 

parameters, contrary to previous published data that 

described unchanged EGPx and reduced erythrocyte 

glutathione (11), significantly higher values were noticed in 

both CRF groups. We hypothesised that an enzymatic 

induction occurs in response to oxidative stress and accounts 

for the increased EGPx activity and the regeneration of the 

intracellular pool of reduced glutathione (GSH). Indeed, the 

ability of glutathione peroxidase to adapt its activity 

according to oxidative stress was demonstrated in chronic 

haemodialysis children (17) and was supposed by Canestrari 

et al in order to explain the increased EGPx found in CAPD 

subjects (cited by (16)). Also, it was stated that the oxidantsinduced 

up-regulation of glucose-6-phosphate dehydrogenase 

(G6PD) might play a role in the increase of intracellular 

GSH. In experimental setting, on culture of human cells, the 

authors showed that G6PD expression is rapidly enhanced in 

response to oxidative stress and it controls intracellular 

reduced glutathione without interfering with the activity of 

the other enzymes involved in the peroxide/hydroperoxidedetoxifying 

pathway (18). As a result, cells overexpressing 

G6PD had an overall reduced state, although the precise 

biochemical mechanism underlying the enzyme’s antioxidant 

properties is still debated. Until now, two alternative 

explanations were proposed: either G6PD influences the 

output of GSH and thus contribute to the maintaining of an 

intracellular redox potential, or it is implicated in a 

mechanism that promotes glutathione storage (18). Anyway, 

these experimental inferences support our interpretation of 

the increased EGPx and E-SH in uraemic patients as an 

adaptive reaction to enhanced oxidative stress. 

Conclusions 

We concluded that peritoneal dialysis patients are exposed to 

oxidative stress, evidenced by both an increase in plasma 

lipid peroxidation (TBARS) and a suppressed activity of 

antioxidants (erythrocyte SOD). Also, the impairment of 

extracellular antioxidant system expressed by the lower levels 

of plasma free thiols, probably accounted for by the enhanced 

consumption in order to offset the oxidative burden, plead for 

an imbalance between reactive species production and 

antioxidant defence mechanisms in CAPD patients. The 

enhancement of erythrocyte glutathione-dependent 

antioxidants might reflect rather an adaptive response to 

oxidative processes and, therefore, indirectly argues in favour 

of oxidative stress occurring before initiation of dialysis and 

augmented thereafter. 

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damage to peripheral blood leukocyte DNA in chronic 

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274(5): 2750-2757


Hyperhomocysteinemia in ESRD 

A. F. Perna 1 , C. Lombardi 1 , R. Capasso 1 , F. Acanfora 1 , E. Satta 1 , D. Ingrosso 2 , M. G. Luciano 1 

and N. G. De Santo 1 

1 First Division of Nephrology/Department of Pediatrics, 2 Department of Biochemistry and Biophysics “F. Cedrangolo”, 

Cardiovascular Research Center, School of Medicine, Second University of Naples, Naples, Italy 

Abstract 

In chronic renal failure and in uremia, the role of uremic 

toxins and in particular of protein-bound molecules, such as 

AGEs, phenols, and homocysteine, is attracting much 

attention from scientists interested in understanding the 

mechanisms leading to the dramatic increase in 

cardiovascular risk and mortality typical of the condition. 

Homocysteine is a cardiovascular risk factor in the general 

population and in uremia. It displays several effects at the 

genomic level and on protein function, and it represents an 

interesting candidate in the study of the pathogenesis of the 

cardiovascular disease specific of uremia. Several aspects of 

hyperhomocysteinemia, such as clinical epidemiology, 

therapeutic aspects, and malnutrition, will be discussed. 

Key words: homocysteine, folate receptors, uremia, chronic 

renal failure, hypomethylation, uremic toxins 

Homocysteine metabolism 

Homocysteine is a sulfur amino acid whose metabolism is 

related to methionine, an essential amino acid, contained 

either in the normal diet or originating from protein 

breakdown. Methionine, when it is not employed in protein 

biosynthesis, is condensed with ATP to form S- 

adenosylmethionine (AdoMet), a sulfonium compound. 

AdoMet in turn donates, after decarboxylation, the propyl 

amino moiety in polyamine synthesis, while its methyl group 

is utilized in the transmethylation pathway to methylate 

various methyl acceptors (proteins, DNA, and small 

molecules, such as guanidino acetate, in creatine 

biosynthesis). AdoMet demethylated product is S- 

adenosylhomocysteine (AdoHcy). AdoHcy is hydrolyzed to 

adenosine and homocysteine in a reversible reaction, which is 

inhibited by AdoHcy itself (in a competitive product type of 

inhibition). Homocysteine is then metabolized to 

cystathionine in the transsulfuration pathway, where CBS is 

the rate-limiting enzyme. The remethylation pathway leads to 

methionine formation from homocysteine, which receives a 

methyl group from methyltetrahydrofolate (MTHF), in a 

reaction catalyzed by methionine synthase. MTHF reductase 

is the enzyme which catalyzes the reduction of 

methylenetetrahydrofolate to MTHF, thereby irreversibly 

committing one carbon units to MTHF. This represents a 

“folate trap”, because MTHF can be used in this and only this 

remethylation reaction, while folates in less reduced forms 

can be used in other reactions and in particular by thymidilate 

synthase in the synthesis of this DNA precursor. 

Hyperhomocysteinemia and the cardiovascular disease in 

uremia 

An increase in plasma homocysteine levels is associated with 

an elevation of cardiovascular risk (1). The inherited 

enzymatic defect of CBS represents the most common form 

of homocystinuria, in which affected patients, who display 

high homocysteine levels in blood, used to die of premature 

cardiovascular disease (2, 3). In the general population, even 

a mild or moderate increase in blood homocysteine levels is 

associated with an increase in cardiovascular risk (4-7). In 

uremic patients, both those under conservative treatment and 

undergoing dialysis, there is an increase in cardiovascular 

risk, and actually uremia represents a model of generalized 

atherosclerosis. In addition, traditional risk factors or those 

typical of uremia can hardly explain this increase. 

Hyperhomocysteinemia is highly prevalent in this population. 

Recent studies show that hyperhomocysteinemia predicts 

mortality and cardiovascular events in uremic patients on 

hemodialysis (8-12). However, it has been recently proposed 

that low levels of homocysteine are linked to an increase in 

cardiovascular risk, so called reverse epidemiology (13-14). 

A recent study by Ducloux et al (15) clarifies once and for all 

this controversy. In this work, 459 patients, stratified in 

relation to the presence or absence of a chronic malnutritioninflammation 

state (CISM), were followed prospectically. 

Homocysteine levels > 30 micromolar were associated with a 

significantly higher risk of global mortality (Hazard ratio 

1.55, CI 1.12-4.72) in patients without CISM, and not in 

patients with CISM. Also taking into exam cardiovascular 

mortality, homocysteine levels > 30 micromolar were kinked 

to an increase in risk (Hazard ratio 1.91, CI 1.23-3.23) in 

patients without CISM, and not in those with this 

confounding factor. The conclusion is that 

hyperhomocysteinemia is a strong risk factor in patients 

without CISM, while in those with CISM, the association is 

masked by the combination of malnutrition-inflammation. In 

this case, in fact, homocysteine levels are lower, an 

expression of a reduced amino acid pool, and of 

hypoalbuminemia. Reverse epidemiology is so explained in 

patients with CISM, while in patients without CISM, the 

direct relation between homocysteine and mortality is 

confirmed. 

______________________ 

Correspodence to: A. F. Perna, MD, PhD, Division of Nephrology/Department of Pediatrics, Second University 

of Naples, Via Pansini 5, Ed. 17, Naples, Italy 80131; Phone: +39-081-5666651; 

Fax +39-081-5666655; E-mail: alessandra.perna@unina2.it


Causes of hyperhomocysteinemia 

As for the cause, this could be theoretically linked to 

increased production, reduced removal, or reduced excretion. 

The latter can be ruled out because urinary homocysteine 

excretion is negligible (16). Considering the possibility of an 

increase in homocysteine production: homocysteine comes 

from the hydrolysis of AdoHcy, the demethylated product of 

AdoMet, the methyl donor in transmethylations. However, 

the accumulation of homocysteine leads to an increase of its 

precursor AdoHcy, due to the slowing down of the hydrolysis 

reaction, and this in turn leads to transmethylation inhibition, 

and in particular of certain methyltransferases. We have 

shown, and findings were then confirmed by Loeher et al. 

and van Guldener et al. (17-19), that this is the case in CRF 

and uremia. At the high concentrations of AdoHcy present in 

uremia, and considering the Km and Ki of the various 

methyltransferases, several methyltransferases are affected, 

such as DNA methyltransferase, or the protein L-isoaspartate 

methyltransferase, a repair enzyme of damaged L-isoaspartyl 

residues, while others are not, such as the quantitatively most 

important one, guanidinoacetate methyltransferase. This 

methyltransferase leads to the formation of creatine, and then 

creatinine forms spontaneously from creatine. 

In 1980, Mitch and coworkers administered radiolabeled 

creatinine intravenously to subjects with severe CRF, and 

were able to show that the average rate of creatinine 

production was 150 micromoles/kg/day, similar to the 

production present in normal subjects (20). Guttormsen et al 

(21) showed that the average net influx of homocysteine into 

plasma was the same in controls and renal failure patients. 

Stam and coworkers (22) using stable isotope techniques 

have shown that transmethylations are reduced, as well as 

homocysteine clearance, in the whole body of CRF patients. 

All in all, it can be said that in uremia some 

methyltranferases are inhibited by AdoHcy, while some are 

relatively affected, and continue to produce AdoHcy and 

homocysteine, which will lead to AdoHcy accumulation, thus 

producing a vicious cycle. So, at present current evidence is 

in favor of the idea that no increased production of 

homocysteine is present in uremia. 

The most likely possibility, at this point of studies, is: 

decreased metabolic removal, either from the kidney, or from 

an extrarenal source. Studies from independent groups have 

shown that there is no difference in homocysteine 

concentration between the renal artery and the renal vein in 

humans (23, 24). However, this does not rule out the 

possibility that the kidney metabolizes homocysteine, 

because limitations of the measurement, expressing 

themselves in a relatively high coefficient of variation, could 

be an issue, that is small differences could still be important, 

and also we don’t know what happens in the fed state. 

Glomerular filtration of homocysteine is restricted because of 

protein binding, however, homocysteine could still be 

removed through peritubular uptake. Peritubular uptake refers 

to the transport of aminoacids, peptides and proteins which 

are taken up from the arterial capillaries coming from the 

efferent arteriole, surrounding the proximal tubules, and go 

into tubular cells, where they are either secreted into the 

tubular lumen, or remain into cells and are metabolized. This 

concept is supported by findings by Garibotto et al (25). In 

this paper, they show that the fractional extraction of 

homocysteine is positively linked to renal plasma flow, with 

a net uptake occurring when renal plasma flow is above 500 

ml/min. In addition, they show that homocysteine renal 

clearance is linked to renal plasma flow, that is, in these 

subjects, homocysteine clearance goes down to 0 when renal 

plasma flow is reduced. So at this point, we know that a 

reduction in homocysteine removal is found in CRF, but little 

more than that, and the matter is still controversial. 

Metabolic consequences of hyperhomocysteinemia 

Recently, some unforeseen consequences of 

hyperhomocysteinemia, coming from other laboratories, and 

ours, have been explored, which can ultimately affect 

mortality. We have proposed and explored the “unbalanced 

methylation” hypothesis in uremia. The accumulation of the 

homocysteine precursor AdoHcy, occurring when 

homocysteine levels are high, leads to an inhibition of those 

methyltransferases which are more sensitive to the inhibitor 

AdoHcy (high-sensitive methyltransferases, HS-Mtases). The 

low-sensitive methyltransferases (LS-Mtases) will continue 

to consume AdoMet and produce AdoHcy to an almost 

normal extent, thus further maintaining inhibition of the HS- 

MTases. 

For example, we have shown some years ago that 

methylation-dependent membrane protein repair, a process in 

which a methylation reaction is involved, is inhibited in 

erythrocytes of uremic patients (17). 

In addition, we have shown that total DNA methylation is 

reduced in dialysis patients and levels of decrease correlate 

significantly with plasma homocysteine levels (26). 

DNA methylation is viewed as a mechanism for gene 

silencing and regulation, as, for example, in the case of 

“imprinted genes”. Considering the way through which genes 

are passed from one generation to another, the allele coming 

from one of the parents is generally shut off through 

methylation. Under normal conditions, gene expression is 

therefore termed monoallelic for these genes (the gene 

coming from either the mother or the father is expressed, the 

other is silenced in a non-random manner). SYBL1 (a 

pseudoautosomal gene, X or Y inactivated) and H19 (an 

imprinted gene with maternal expression) are regulated in the 

way we just described. The allelic expression of these genes 

was used to test the "functional" outcome of DNA 

hypomethylation in uremic patients. Results show that, for 

SYBL1, gene expression in patients is biallelic, that is both 

alleles are expressed. For H19, only in patients with high 

homocysteine levels (approximately above 60 M) gene 

expression is biallelic. 

After folate therapy, gene expression returns monoallelic and 

total DNA methylation improves in parallel with a decrease 

of homocysteine levels, thus testifying that homocysteine 

modifies DNA methylation in a reversible fashion. So, we 

can state that, in patients with higher homocysteine, there is a 

transcriptional activation of the normally repressed allele, due 

to DNA hypomethylation. Folate treatment is able to revert 

the biallelic expression into monoallelic in the patients who 

had biallelic expression. 

Coming to plasma proteins, plasma proteins in hemodialysis 

patients display a significant increase in the content of L- 

isoaspartyl residues, so they are significantly altered, or 

damaged (27). 

This alteration under normal conditions can be repaired by a 

mechanism depending on a specific methyl transfer reaction.


This particular methyltransferase has been shown to be 

inhibited in uremia and, therefore, this kind of protein 

damage is increased. This inhibition depends partially on 

high homocysteine levels, and therefore methylation 

inhibition, because folate therapy is able to reduce damage 

levels. 

However, the pathogenesis of this alteration, when 

considering the plasma protein compartment, depends mostly 

on uremic toxicity. Several uremic toxins, from different 

chemical groups, can induce protein damage. However, we 

found that guanidine in particular is able to elicit this protein 

damage in a dose-dependent manner. Deamidated albumin, 

that is in vitro damaged albumin, was prepared with a 

standard protocol, and the binding capacity of various 

substances to this damaged albumin was tested. A reduced 

binding of homocysteine to serum albumin was found. We 

can conclude that increased protein damage, due to the 

uremic milieu and hypomethylation, induces protein damage, 

with reduced homocysteine binding to proteins, and possible 

increase in free homocysteine levels. 

Among the possible consequences of hyperhomocysteinemia, 

there is protein homocysteinylation, that is the binding of 

homocysteine to proteins, which occurs basically as a postbiosynthetic 

acylation of free amino groups (protein-Nhomocysteinylation, 

mediated by homocysteine thiolactone). 

This protein modification in in vitro experiments leads to 

functional derangements, such as a loss of enzymatic activity. 

Another type of protein homocysteinylation is through the 

formation of a covalent disulfide bond found primarily with 

cysteine residues (protein-S-homocysteinylation). We have 

been able to demonstrate the presence of a significant 

increase of homocysteinylated proteins in uremia (28). We 

obtained, with a new method combining gel filtration, 

hydrolysis, and HPLC chromatography, data for both protein- 

S-Hcy and protein-N-Hcy, which were significantly increased 

in the plasma of uremic patients on hemodialysis. This type 

of protein alteration occurs in uremia, because of the high 

homocysteine levels present in this condition, thus 

representing another example of a widespread presence of a 

derangement of the peptide link in uremia. Protein 

homocysteinylation could be one of the principal mediators 

of homocysteine toxicity, contributing to determine structural 

and functional alterations at the molecular and cellular level. 

Therefore, it can be stated that in chronic renal failure and 

end stage renal disease, both altered gene expression and the 

alterations in protein structure, dependent on 

hyperhomocysteinemia and acting through an increase of a 

homocysteine-related metabolite, may play a crucial role in 

terms of macromolecule functional derangement. 

Therapy 

In view of the epidemiological data and the high frequency of 

cardiovascular disease in chronic renal failure patients, 

numerous attempts have been made to lower plasma total 

homocysteine concentrations in these patients. In the general 

population, it is possible to reduce homocysteine levels by 

means of dietary intervention or with small folate 

supplementation. In chronic renal failure patients, possible 

tools conducive to a reduction of homocysteine levels are 

folate therapy, therapy with betaine, serine, N-acetylcysteine, 

or B vitamins (vitamin B 6 , B 12 , B 2 ), and improved dialysis. 

Betaine, serine, and B vitamins are either not effective, or can 

add only a modest additive effect. The mainstay of therapy is 

represented by folic acid, or folic acid in its active, 

circulating form, MTHF (29-30 for review). 

Folic acid therapy in chronic renal failure patients have been 

shown to reduce, albeit not to normalize, plasma total 

homocysteine concentrations, particularly in dialysis patients, 

who express therefore a resistance to folates. Folic acid 

supplementation of 1 mg daily, in contrast to what is usually 

observed in the general population, does not have any effect 

on plasma total homocysteine concentration in chronic failure 

failure patients, despite the induction of supernormal plasma 

folate levels. Oral supplementation with high doses of folic 

acid (up to 15 mg daily), which leads to a 20 to 50-fold 

increase of plasma folate concentrations, is only partially 

effective in reducing plasma total homocysteine. This relative 

resistance to folate action does not appear to be caused by 

defects in folate absorption or impairment in folic acid 

conversion in the plasma to the active metabolite MTHF. 

Moreover, active reduced forms of folic acid did not lead to a 

greater decrease in plasma total homocysteine levels than 

those observed with native folic acid supplementation in 

hemodialysis patients. However, MTHF provides a moiety 

which does not need to be further metabolized. This is 

important because a polymorphism, the C677T transition of 

MTHF reductase, is very common in the population (20 % in 

the homozygous, 30-40 % in the heterozygous). Providing 

the active form circumvents the possibility that the specific 

patient genetic pattern could affect folate utilization. 

Other abnormalities in homocysteine metabolism, as for 

instance a relative resistance to vitamin B12 action, have 

been observed in chronic renal failure patients. Theses 

abnormalities may also participate to the genesis of 

hyperhomocysteinemia in these patients. However, as 

mentioned previously, the correction of these abnormalities 

in folate-replete patients has only a partial additional effect 

on fasting total homocysteine in chronic renal failure patients. 

For the sake of completeness, it has to be mentioned that 

more efficient dialysis procedures could allow an improved 

removal of uremic toxins and/or homocysteine. The main 

reason for the genesis of hyperhomocysteinemia, and the 

reduced efficacy of folate therapy in dialysis chronic renal 

failure patients, as mentioned previously, is unclear at 

present. The accumulation of uremic toxins and the decrease 

in homocysteine clearance and metabolism owing to a 

decreased functioning renal mass are the two most probable 

explanations. Standard dialysis procedures using low-flux 

dialysers or high-flux dialysis are unable to remove sufficient 

amounts of homocysteine to maintain total homocysteine 

within the normal range. In contrast, dialysis in super-flux 

mode significantly lowered total homocysteine concentrations, 

possibly due to a greater reduction in uremic toxin concentration. 

This may also be partially due to albumin removal, 

since the major part of circulating homocysteine is protein-bound. 

Recently, it has been also demonstrated that total 

homocysteine levels were significantly lower among patients 

undergoing daily nocturnal HD. 

A displacement of homocysteine from protein-binding sites, 

allowing increased free homocysteine availability for plasma 

clearance by dialysis procedures could be an interesting alternative 

strategy to reduce total homocysteine concentrations. 

It has been reported by Scholze et al (31) that the acute 

intravenous administration of N-acetylcysteine (5 g in 5% 

glucose for 4 hours) during a hemodialysis session, which


presumably can displace homocysteine from protein-binding 

sites, was able to completely normalize total homocysteine 

concentrations at the end of the session, with residual 

efficacy for the next two days. The acute decrease of total 

homocysteine concentrations induced by N-acetylcysteine 

supplementation during the dialysis session has been also 

shown to improve pulse pressure and endothelial function in 

hemodialysis patients. In a previous study, the same group 

has also shown that acetylcysteine reduces cardiovascular 

events, when given 600 mg per os, for two years (32). 

Friedman et al (33) have shown that long-term oral N- 

acetylcysteine administration (1.2 g twice a day) total 

homocysteine levels were reduced by 19 % in hemodialysis 

patients, compared with an 8 % reduction in patients treated 

with placebo (p = 0.07). Patients were vitamin-replete. 

Possibly, this study was underpowered to detect a statistically 

significant difference, and also acetylcysteine was 

administered orally. 

Although these results are promising, the efficacy and safety 

of intravenous administration of N-acetylcysteine needs, 

however, to be evaluated before drawing a definite 

conclusion. In any case, these data suggest that maneuvers 

aimed to displace homocysteine from protein-binding sites 

may represent a valuable strategy to normalize total 

homocysteine in dialysis patients. 

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2003; 41(2): 442-6


C.E.R.A. (Continuous Erythropoietin Receptor Activator): 

A new perspective in anaemia management 

W. Sulowicz 

Jagiellonian University, Krakow, Poland 

Abstract 

Challenges in anaemia management 

Continuous Erythropoietin Receptor Activator (C.E.R.A.), an 

innovative agent with unique receptor activity and a 

prolonged half-life, is currently in development to provide 

correction of anaemia and stable control of haemoglobin (Hb) 

levels at extended administration intervals in patients with 

chronic kidney disease (CKD) on dialysis and not on dialysis. 

Phase II studies suggest that C.E.R.A. can correct anaemia 

and maintain Hb levels when administered intravenously or 

subcutaneously at extended intervals of up to once monthly. 

C.E.R.A. is currently undergoing evaluation for the control of 

anaemia in patients with CKD in a large-scale Phase III 

programme. 

Keywords: anaemia, chronic kidney disease, haemoglobin 

Introduction 

Chronic kidney disease (CKD) is highly prevalent, with 

increasing numbers of patients affected by the disease 

worldwide (1). The progressive nature of CKD and ensuing 

end-stage renal disease is associated with high and increasing 

treatment costs that are creating a significant burden on 

global healthcare resources (2). Forecasts indicate that the 

incidence of CKD is expected to continue rising (3,4), 

reflecting the growing incidence of diabetes and the ageing 

population (1). 

Anaemia is already highly prevalent in the early stages of 

CKD (5–7) and is associated with increased mortality and 

morbidity (8–10). Evidence suggests that patients with CKD 

and anaemia progress more rapidly to dialysis than patients 

without anaemia (11,12). In addition, anaemia negatively 

impacts on cardiovascular disease (13,14), cognitive function 

(15) and quality of life (16,17). 

National and international clinical guidelines exist to 

facilitate the management of anaemia in patients with CKD 

(18–21). A number of studies demonstrate that maintaining 

haemoglobin (Hb) levels within recommended targets is 

associated with positive clinical outcomes (10,22–25). 

Extrapolation of data from the Dialysis Outcomes and 

Practice Patterns Study estimated a potential gain of 23 910 

patient-years in the US haemodialysis population over a 5- 

year period if patients were brought within Hb targets (≥11 

g/dL) (24). 

Despite the proven benefits, management of renal anaemia 

remains suboptimal; anaemia is often under-recognised and 

under-treated (5,26). The use of erythropoiesis-stimulating 

agents (ESAs) before the initiation of dialysis is still 

uncommon in most countries (27,28) with many CKD 

patients only receiving treatment once anaemia is advanced. 

Furthermore, with current treatment, approximately twothirds 

of patients with CKD have Hb levels outside guideline 

targets (29,30). In addition, substantial variability in Hb 

values over short periods of time has been demonstrated in 

many patients with CKD treated with ESAs (29,31–34). This 

variability in ESA response may be due to a range of factors, 

such as dialysis effects, intercurrent illness and iron therapy. 

Effective management of anaemia is already time consuming, 

and the burden on healthcare providers can only increase as 

the prevalence of CKD continues to rise. Currently most 

available ESAs require frequent administration (up to three 

times per week) (19,20). Furthermore, frequent monitoring 

and dose adjustment is required to maintain Hb levels within 

guideline targets with current treatment. New agents that 

offer more effective anaemia management by providing 

stable Hb levels at extended administration intervals may 

reduce the burden on healthcare professionals. 

C.E.R.A. 

Continuous Erythropoietin Receptor Activator (C.E.R.A.), an 

innovative agent with unique receptor activity and a 

prolonged half-life, is currently in development to provide 

correction of anaemia and stable control of Hb levels at 

extended administration intervals in patients with CKD on 

dialysis and not on dialysis. C.E.R.A. is chemically 

synthesised, and differs from erythropoietin through the 

integration of amide bonds between amino groups and 

methoxy polyethylene glycol-succinimidyl butanoic acid 

(35). 

Evidence is accumulating that C.E.R.A. has unique receptor 

properties, acting differently to epoetin at the receptor level 

(36). Data suggest that C.E.R.A. has a much lower affinity 

for the erythropoietin receptor compared with epoetin beta, 

leading to a reduced specific activity in vitro. However, since 

the elimination half-life is so prolonged, C.E.R.A. has 

increased erythropoietic activity in vivo (37). 

Phase I studies in healthy subjects 

Four Phase I studies have been conducted in healthy subjects 

to investigate the pharmacokinetic and pharmacodynamic 

properties of C.E.R.A. In two single ascending dose studies, 

subjects were randomised to receive single intravenous (IV) 

doses of C.E.R.A. (0.4–3.2 µg/kg) or placebo (n=38) or 

______________________ 

Correspodence to: W. Sulowicz, Department of Nephrology, Jagiellonian University, 31-501 Krakow, 

ul. Kopernika 15 c, Poland; Email: wladsul@mp.pl


single subcutaneous (SC) doses of C.E.R.A. (0.1–3.2 µg/kg) 

or placebo (n=70) (38). In two multiple ascending dose 

studies, subjects were randomised to receive three IV doses 

of C.E.R.A. (0.4–3.2 µg/kg) or placebo (n=61) once every 3 

weeks or four SC doses of C.E.R.A. (0.4–3.2 µg/kg) or 

placebo (n=48) once every 2 weeks (38). In the single 

ascending dose studies, a dose-dependent erythropoietic 

response was seen with both IV and SC C.E.R.A. 

administration. C.E.R.A. induced an increase in reticulocyte 

counts that peaked within 10 days and returned to baseline 

after 20 days. Consistent results were obtained in the multiple 

ascending dose studies: a dose-dependent erythropoietic 

response was observed with both routes of administration. 

The half-life for C.E.R.A. was observed to be considerably 

longer than those reported for currently available ESAs 

(Table 1) (39–42). In the multiple ascending dose studies, the 

clearance of both IV and SC C.E.R.A. was low (IV 27.6–44.6 

ml/h; SC 97–347 ml/h) (41). The prolonged half-life and low 

clearance observed with C.E.R.A., together with its unique 

receptor activity, give rise to a different pharmacological 

profile compared with currently available ESAs and suggest 

that extended administration intervals are possible. 

Table 1. Half-lives of C.E.R.A. and currently available 

erythropoietic stimulating agents in healthy volunteers 

(39–42) 

Mean half-life (h) 

IV SC 

C.E.R.A. 133 137 

Epoetin alfa 6.8 19.4 

Epoetin beta 8.8 24.2 

Darbepoetin alfa 25.3 48.8 

IV, intravenous; SC, subcutaneous. 

Phase II studies in patients with CKD and anaemia 

Four Phase II dose-finding studies have investigated the 

feasibility of C.E.R.A. for the correction of anaemia and 

maintenance of Hb levels at extended administration intervals 

in more than 350 patients with CKD. 

Fig 1. Mean (±SEM) haemoglobin (Hb) change from baseline after 

6 weeks’ treatment with C.E.R.A. in A) patients with chronic kidney 

disease receiving dialysis and B) patients with chronic kidney 

disease not receiving dialysis (35). 

A) 

Mean Hb change (g/dL) 

2.0 

1.5 

1.0 

0.5 

0.0 

1x/wk 1x/2wk 1x/3wk 

Administration schedule 

B) 

Mean Hb change (g/dL) 

2.0 

1.5 

1.0 

0.5 

0.0 

1x/wk 1x/2wk 1x/3wk 

Administration schedule 

In two studies, one in CKD patients receiving dialysis (n=61) 

(43) and one in CKD patients not yet receiving dialysis 

(n=65) (44), SC C.E.R.A. was administered for the correction 

of anaemia. All patients were aged 18 years, with Hb 8–11 

g/dL and were ESA-naïve. These studies examined escalating 

doses of C.E.R.A., with once-weekly, once every 2 weeks, 

and once every 3 weeks schedules being assessed in each 

dose group. Mean increases in Hb are shown in Figure 1. 

There was a significant dose response to C.E.R.A. and the Hb 

response was independent of the frequency of administration 

(43,44). These results suggest that C.E.R.A. is capable of 

correcting anaemia to guideline targets within the 

recommended timeframe when administered to ESA-naïve 

CKD patients at extended administration intervals. 

Two Phase II, multicentre, dose-finding studies have been 

conducted to determine the efficacy of C.E.R.A. for the 

maintenance of Hb levels in adult patients with renal anaemia 

(Hb 10–13 g/dL) on dialysis. In one study, 91 haemodialysis 

patients previously maintained on three-times weekly IV 

epoetin alfa were switched to IV C.E.R.A. (45). After a 2- 

week run-in period, patients were randomised to one of three 

C.E.R.A. doses based on their previous epoetin dose and data 

on exposure to C.E.R.A. from healthy subjects; once-weekly 

and once every 2 weeks administration schedules were 

assessed in each dose group for 19 weeks. A significant 

(P


month period (Figure 2); mean Hb levels over time were 

11.51 g/dL (95% confidence interval (CI): 11.31, 11.71), 

11.18 g/dL (95% CI: 10.91, 11.46) and 11.15 g/dL (95% CI: 

10.91, 11.39) for once every 2 weeks, once every 3 weeks 

and once monthly administration schedules, respectively 

(47,48). Data from these long-term extension studies indicate 

that C.E.R.A. can control anaemia when administered at 

extended intervals, maintaining sustained and stable Hb 

levels in dialysis patients. 

A large-scale Phase III programme is underway evaluating 

the efficacy and safety of C.E.R.A. in approximately 2400 

patients with CKD on dialysis and not on dialysis from 29 

countries. 

Fig 2. Mean (±SD) haemoglobin (Hb) over time following once 

monthly subcutaneous administration of C.E.R.A. 

Reproduced with permission from Curr Hematol Rep (35). 

Copyright 2005, Current Science Group 

16 

Hb (mean ± SD, g/dL) 

15 

14 

13 

12 

11 

10 

9 

8 

7 

Tolerability 

1 2 3 4 5 6 7 8 9 10 11 12 

Months of treatment (extension period) 

C.E.R.A. was generally well tolerated in healthy volunteers 

(38,49). Similarly, in patients with CKD, C.E.R.A. was 

generally well tolerated with no unexpected safety concerns. 

Available information indicates that the incidence of adverse 

events (AEs) was in accordance with that expected for this 

study population (50). The most frequent AEs in the two 12- 

month maintenance study extension periods (n=109) were 

hypotension and muscle cramp (8.05% and 4.39% of all AE 

episodes, respectively) (50). The most common serious AEs 

were hypotension (five events), myocardial infarction (five 

events), cellulitis (four events) and pancreatitis (four events). 

In all studies conducted to date (healthy volunteers and 

patients with CKD), there has been no evidence of antibody 

development in any patient treated with C.E.R.A. 

Conclusions 

C.E.R.A.’s pharmacokinetic properties, including a prolonged 

half-life and low clearance, together with its unique receptor 

binding properties, result in a different pharmacological 

profile compared with currently available ESAs and suggest 

that extended administration intervals are feasible. Phase II 

studies in patients with CKD suggest that C.E.R.A. can 

correct anaemia and maintain Hb levels at extended intervals 

of up to once monthly. C.E.R.A. is currently undergoing 

evaluation for the management of anaemia in patients with 

CKD in a large-scale Phase III programme, with preliminary 

results expected soon. The potential for C.E.R.A. to be 

administered at extended administration intervals may 

simplify anaemia management, reducing the burden for 

patients and healthcare professionals. 

Acknowledgements 

Medical writing support was provided by Emma Marshman at 

Prime Medica Ltd during the preparation of this paper, supported 

by F. Hoffmann-La Roche. Responsibility for opinions, conclusions 

and interpretation of data lies with the authors. 

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47. Locatelli F, Villa G, Beyer U, Dougherty FC, on behalf of the 

BA16286 extension study group. Subcutaneous CERA 

(Continuous Erythropoietin Receptor Activator) maintains 

haemoglobin concentrations with dosing intervals up to 4 

weeks in dialysis patients (abstract). Nephrol Dial Transplant 

2005; 20(Suppl 5): v261 

48. Besarab A, Beyer U, Dougherty FC. Long-term intravenous 

CERA (Continuous Erythropoietin Receptor Activator) 

maintains haemoglobin concentrations in hemodialysis patients 

(abstract). Nephrology 2005; 10 (Suppl): A312 

49. Reigner B, Jordan P, Pannier A, Dougherty FC. Phase I studies 

of the new erythropoietic agent, CERA (Continuous 

Erythropoiesis Receptor Activator): demonstration of a dosedependent 

response (abstract). Nephrol Dial Transplant 2003; 

18(Suppl 3): 167 

50. Dougherty FC, Beyer U. Safety and tolerability profile of 

Continuous Erythropoietin Receptor Activator (CERA) with 

extended dosing intervals in patients with chronic kidney: 

disease on dialysis (abstract). Nephrology 2005; 

10(Suppl)A313


Diagnosis and treatment of renal bone disease 

G. B. Spasovski 

Department of Nephrology, University Clinical Center, University of Skopje, Republic of Macedonia 

Abstract 

The abnormalities in bone histology in patients with chronic 

renal failure (CRF), known as renal osteodystrophy (ROD), 

can be observed early in the course of the disease. Patients 

with mild to moderate degrees of CRF rarely experience 

symptoms, although skeletal changes may occur years before 

the symptoms arise. In end stage renal failure (ESRF) when 

patients require chronic maintenance dialysis, nearly all of 

them have abnormal bone histology. Moreover, survival rates 

of patients on dialysis have increased because of therapeutic 

improvement and the resultant increase in duration of dialysis 

has led to a further rise in renal osteodystrophy. Because 

metabolic bone disease can produce fractures, bone pain, and 

deformities late in the course of the disease, diagnosis, 

prevention and early treatment are essential. 

To date, bone biopsy is the most powerful and informative 

diagnostic tool to provide precise information on the type and 

severity of renal osteodystrophy and also useful in research to 

assess the effects of therapies on bone. Alternatives to the 

bone biopsy continue to be searching for, but the noninvasive 

bone markers have not been proven to hold 

sufficient diagnostic performance. Hence, transiliac bone 

biopsy remains the golden standard for the diagnosis of renal 

osteodystrophy. 

The therapy of renal bone disease depends on the particular 

type of ROD. However, some therapies (aluminum hydroxide 

and calcium carbonate) can exacerbate mineral balance and 

lead to the development of low-turnover bone diseases 

(osteomalacia or adynamic bone) and increased levels of 

vascular calcifications. Nowadays, phosphate control has 

been achieved with alternative phosphate binders that are not 

associated with these side effects (Sevelamer hydrochloride - 

RenaGel and Lanthanum carbonat - Fosrenol ). 

Key words: renal failure, renal osteodystrophy, bone biopsy, 

bone markers, calcium carbonate, lanthanum carbonate. 

Bone biopsy 

Double tetracycline labelling is the first prerequisite for an 

informative bone biopsy with an interval of 8 to 12 days (1). 

Phosphate binding treatment is stopped at the time of labeling 

and transiliac bone biopsy is then performed 2 to 6 days after 

the second label, under local anesthesia. 

The possible complications from bone biopsies can include 

pain, haematoma, wound infection, and rarely neuropathy. 

However, studies show that the biopsies of the anterior iliac 

result of procedure (2-4). The operator's experience is 

important in minimizing morbidity and in obtaining an 

adequate specimen. Based on histomorphometric findings, 

two main groups of renal bone disease can be classified: low 

(LTO) and high bone turnover (HTO) (3,5). Adynamic bone 

disease is characterized by a decreased number of osteoblasts 

and osteoclasts with a low bone formation rate (BFR). The 

second type of LTO, osteomalacia (OM), has a superimposed 

mineralization defect producing a great amount of 

unmineralized osteoid. HTO bone disease includes mild and 

severe hyperparathyroid bone disease (HPTH) or osteitis 

fibrosa, characterized by an excessive number of osteoclasts 

and osteoblasts and a high rate of bone formation and 

resorption. Mixed uremic osteodystrophy (Mx) possesses the 

combined features of both HPTH and OM. 

Alternatives to the bone biopsy continue to be searching for, 

but the non-invasive bone markers have not been proven to 

hold sufficient diagnostic performance (6). Hence, transiliac 

bone biopsy remains the golden standard for the diagnosis of 

renal osteodystrophy. 

Clinical application - Because bone biopsies more accurately 

determine the type of renal osteodystrophy and can indicate 

potential aluminum, strontium and lanthanum accumulation 

in dialyzed patients, they also allow tailoring of therapeutic 

measures. Additionally, bone biopsies could be performed at 

an interval of 6-12 months, sufficient for recognition of the 

specific changes of bone remodeling and mineral 

metabolism. 

The biopsy shows the degree of bone turnover helping the 

practitioner to determine the route, aggressiveness, and length 

of phosphate binding and calcitriol therapy. Severe 

hyperparathyroidism with marked bone marrow fibrosis is an 

indication for high doses of calcitriol if the calciumphosphorus 

product can be controlled. In patients with mild 

to moderate increase in bone turnover with or without 

mineralization defect, doses of intravenous or preoperative 

calcitriol and duration of therapy may be adjusted to avoid 

the development of ABD. 

In case of ABD, calcitriol therapy is not desirable because of 

the risk of inducing hypercalcemia, extraosseous 

calcifications and further suppression of parathyroid glands' 

activity. Moreover, the use of a low calcium dialysate is 

recommended, as well as a lesser daily intake of calcium in 

the diet, ommiting the treatment with calcium containing 

phosphate binding agents. 

Clinical experience 

Our bone biopsy study in 84 ESRD patients revealed that 

crest result in very small morbidity and no mortality as a 

______________________ 

Correspodence to: G. B. Spasovski, Department of Nephrology, University Clinical Center, 

Vodnjanska 17, 1000 Skopje, Macedonia; Telephone: + 389 70 268 232; Fax: + 389 2 3231 501; 

E-mail: gspas@sonet.com.mk


62% of a predialysis population had abnormal bone histology 

(3). ABD was found to be the most frequent bone lesion 

observed in 23%, while HPTH (mild form) was diagnosed in 

only 9% of the patients. The majority of patients (38%) in our 

study presented with a normal bone histology. Hence, this 

finding differs considerably from the ROD spectra reported 

previously in non-dialysed renal failure patients where none 

of the patients was reported to have normal bone histology 

(7,8). Furthermore, our study revealed that serum calcium 

levels in 15 out of 19 (79%) patients with ABD appeared to 

be > 2.1 mmol/l; 4 of them were hypercalcemic >2.5 mmol/l 

and 12 (63%) presented with calcium x phosphorus product > 

4.4 mmol/l (55 mg 2 /dl 2 ). These findings were in agreement 

with the afore-mentioned treatment for ABD patients. 

A recent study with lanthanum carbonate (LC) as a new 

phosphate binder, has shown its safety and effectiveness in 

phosphate binding (9). Additionally, we evaluated plasma 

and bone lanthanum levels in 20 incident dialysis patients at 

baseline, after 1 year of LC (n=10) and calcium carbonate 

treatment (CC=10) and after a 2-year of follow-up period 

during which the lanthanum treatment was replaced by CC 

(n=19) (4). 

Baseline plasma lanthanum levels were below 0.03 ng/ml in 

most of the patients of both groups. During lanthanum 

carbonate treatment lanthanum levels increased reaching a 

maximal level of 1.26 ± 1.24 ng/ml at 24 weeks, after which 

they stabilized at a value varying around 0.60 ng/ml. 

Compared to the values noted at cessation of lanthanum 

treatment a significant decline of plasma lanthanum levels 

was noted at 6 weeks of follow-up (0.59 ± 0.52 vs. 0.17 ± 

0.12 ng/ml; P < 0.05). There was no further significant 

decrease in mean plasma lanthanum concentration during the 

further 2-year follow-up (0.09 ± 0.03 ng/ml). 

The mean bone concentration in patients receiving LC 

increased substantially from 0.05±0.03 to 2.3±1.6 mg/g 

(P


Efficiency of mycophenolate-mofetil in resistant nephrotic syndrome – case 

reports 

B. Milic, T. Ilic, I. Mitic, I. Budosan and S. Curic 

Clinic of Nephrology, Institute of Internal Diseases, Clinical Centre, Novi Sad, Serbia and Montenegro 

Abstract 

Patients with nephrotic syndrome (NS) in the primary 

glomerulonephritis are usually treated with standard 

immunosuppressive treatment including steroids and 

cyclophosphamide. This is a case report of two patients 

resistant to standard therapy who showed remarkable 

improvement in their NS when using mycophenolate-mofetil 

(MMF). First patient, (male, 54-year old) was admitted to our 

hospital with a 1-month clinical and laboratory history of NS. 

He was diagnosed with membranoproliferative glomerulonephritis 

after renal biopsy. After three months of standard 

steroid therapy (methylprednisolone 1-2 mg/kg BW) he did 

not achieve remission. Because of that we continued the 

treatment with pulse doses of methylprednisolone, followed 

with cyclophosphamide pulses. During the following two 

years of therapy patient continued to have frequent relapses. 

Proven to be steroid and cyclophosphamide resistant he was 

shifted to MMF therapy (dose of 2g/day). One month after 

starting therapy patient achieved complete remission with no 

side effects. The other patient (male, aged 62) was diagnosed 

with NS (membranous nephropathy) two months before 

admitting in our hospital. Standard therapeutic procedure 

included pulse doses of methylprednisolone during one 

month and then cyclophosphamide per os (2 mg/kg BW) for 

one year but with no respond in decreasing of proteinuria. In 

order to achieve remission, Cyclosporin A (2-5 mg/kg BW) 

was given. Finally, after one month of no response, we 

administered mycophenolate-mofetil when patient promptly 

entered partial remission of nephrotic syndrome. We 

concluded that MMF was found to be effective and beneficial 

drug in cases of resistant primary nephrotic syndrome. 

Key words: resistant nephrotic syndrome, mycophenolatemofetil 

Introduction 

down regulates the expression of cell-surface adhesion 

molecules, has been used successfully for the prevention of 

acute rejection of renal allografts (1,2). Preliminary studies 

suggest that it may also be effective in treatment of the 

resistant NS in the primary glomerulonephritis. 


We report data on two patients with NS who were diagnosed 

with primary glomerulonephritis. First patient, (male, 54- 

year old) was admitted to our hospital with a 1-month clinical 

and laboratory history of NS. He had nephrotic range 

proteinuria (mean proteinuria 8,5 g/24h) and stable serum 

creatinine level and creatinine clearance. Biopsy documented 

membranoproliferative glomerulonephritis by light 

microscopy and immunofluorescence was showed. After 

three months of standard steroid therapy (methylprednisolone 

1-2 mg/kg BW) he did not achieve remission. Because of that 

we continued the treatment with pulse doses of 

methylprednisolone, followed with cyclophosphamide pulses. 

During the following two years of therapy patient continued 

to have frequent relapses (proteinuria range 0,4-15 g/ 24h). 

Proven to be steroid and cyclophosphamide resistant he was 

shifted to MMF therapy (dose of 2g/day). The other patient 

(male, aged 62) was diagnosed with NS (membranous 

nephropathy) two months before admitting in our hospital 

(mean proteinuria of 10 g/24h) with stable serum creatinine 

level and halved creatinine clearance. Standard therapeutic 

procedure included pulse doses of methylprednisolone during 

one month and then cyclophosphamide per os (2 mg/kg BW) 

for one year but with no respond in decreasing of proteinuria. 

In order to achieve remission, Cyclosporin A (2-5 mg/kg 

BW) was given but with no response. Finally MMF was 

administered in standard dose of 2g/day along with 

Cyclosporine A. Both patients received steroids along with 

MMF (median dose was 20 mg/kg/day). Baseline evaluations 

on enrolment consisted of physical examination; serum 

albumin, serum cholesterol, and serum creatinine levels; and 

a 24-hour urine collection for protein and creatinine 

clearance. Patients were followed up for 6 months, and 

evaluated at 1- to 2-month intervals for these parameters. A 

partial remission was defined as at least a 50% reduction of 

proteinuria with a stable serum creatinine level (3). A 

complete remission was defined as proteinuria less than 0,3 

g/24 h with a stable serum creatinine level. Side effects of 

MMF were also observed. 

Patients with nephrotic syndrome (NS) in the primary 

glomerulonephritis are usually treated with standard 

immunosuppressive treatment including steroids and 

cyclophosphamide. However, a small proportion of patients 

run a multiply relapsing course despite treatment. Some go 

into remission with cyclosporine but are dependent on this 

drug, with which prolonged usage can lead to nephrotoxicity. 

Mycophenolate-mofetil (MMF) is a specific inhibitor of 

inosine monophosphate dehydrogenase which is involved in 

de novo purine synthesis. MMF, an immunosuppressive 

agent that preferentially inhibits stimulated lymphocytes and 

______________________ 

Correspodence to: B. Milic, Clinic of Nephrology, Institute of Internal Diseases, Clinical Centre, 



Results 

Both patients experienced remissions following MMF 

therapy. First patient, only one month after starting therapy, 

achieved complete remission (mean proteinuria 0,07 g/24h), 

with stable serum creatinine and increased serum albumin 

level, while the serum cholesterol levels were reduced 

significantly. Other patient promptly entered partial remission 

of NS (mean proteinuria 4 g/24 h), with stable serum 

creatinine and increased serum albumin level while serum 

cholesterol level decreased. There was no improvement in 

creatinine clearance level. In both cases, there was no side 

effect of MMF. 

Conclusions 

MMF is a relatively new immunosuppressive drug now being 

used in the prevention of allograft rejection. Preliminary 

studies have shown that MMF is effective in treatment in 

several types of primary glomerular diseases when 

conventional therapy has failed (4,5). In the majority of 

patients it was well tolerated and achieve the goals of 

improvement of nephrotic syndrome and stabilisation of renal 

function. Although being usually given with variable doses of 

steroid, results demonstrate that MMF has major steroidsparing 

effects and can be even effective as monotherapy. We 

concluded that MMF was found to be effective and beneficial 

drug in cases of resistant primary nephrotic syndrome. 

References 

1. Eugui EM, Mirkovitch A, Allison AC. Lymphocyte-selective 

antiproliferative and immunosuppressive effects of 

mycophenolic acid in mice. Scand J Immunol 1991; 33: 175- 

183 

2. Sintchak M, Fleming MA, Futer O et al. Structure and 

mechanisms of inosine monophosphate dehydrogenase in 

complex with the immunosuppressant mycophenolic acid. Cell 

1996; 85: 921-930 

3. Cattran DC, Greenwood C, Ritchie S, Bernstein K, Churchill 

DN, Clark WF, Morrin PA, Lavoie S. A controlled trial of 

cyclosporine in patients with progressive membranous 

nephropathy. Canadian Glomerulonephritis Study Group. 

Kidney Int 1995; 47:1130-1135 

4. Choi MJ, Eustace JA, Gimenez LF et al. Mycophenolate 

mofetil treatment for primary glomerular diseases. Kidney Int 

2002; 61: 1098-1114 

5. Badid C, Desmouliere A, Laville M. Mycophenolate 

implications for the treatment of glomerular disease. Nephrol 

Dial Transplant 2001; 16: 1752-1756


Bacterial infections associated with double lumen central venous catheters 

M. S. Simin, M. Milosevic, S. Vodopivec, I. Mitic and S. Curic 

Institute for Internal diseases, Clinic for Nephrology and Clinical Immunology, Novi Sad, Serbia and Montenegro 

Abstract 

Central venous catheters were a major advance in end-stage 

renal disease patient care until the burden of catheter related 

complications became obvious. 

A retrospective study on infections complications of the 

double lumen catheterisation of central veins for 

haemodialysis and PD was done in 2004 year. In period from 

January to December 2004, 60 catheters were placed. 55 

patients had double lumen, one for plasmapheresis and 54 for 

haemodialysis. The mean duration of catheters remaining in 

situ was 36.67 days (range 7 - 145 days). There were 7 

double lumen catheters placed with coffs and tunelisation, 

and the other 48 patients had catheters of a short duration. 

Routine smears were taken from the top of catheters after 

extraction, and from the skin surrounding the place of 

catheter insertion. Hemocultures were also taken, and smears 

from periphery (smears from pharynx, nose and 

urinoculture). Reasons for placing catheters were acute renal 

failure in 19.65%, end stage renal disease in 35.71%, and 

complications of permanent vascular access in 44.64%. 

In 2|55 patients, Staphylococcus aureus and Enterococcus 

species were isolated from the top of dialysis catheter, after 

extraction, and without positive blood bacterial cultures. In a 

few patients micro-organisms were isolated from periphery 

on skin changes 2|55, urine 2|55, pharynx 1|55. Prophylaxis 

with antibiotics was administered in 18|55 patients, with 

antibiotics from group of cephalosporin and chinolone over 

next 7 to 14 days. Mortality during the period of observation 

was 7|55 patients. 

The conclusion based on the aforementioned parameters is 

that duration of placed catheters did not correlated with the 

frequency of infections. The respect of the surgical principles 

of asepsis and desinfection has contributed to a decreased 

frequency of catheter infections for dialysis. 

Key words: central venous catheter, bacterial infection, 

hemoculture, urinoculture 

Introduction 

Central venous catheters were a major advance in end-stage 

renal disease (ESRD) patient care until the burden of catheter 

related complications became obvious. Catheter-related 

infection is one of the major causes of morbidity, with 

potential lethal hazards in haemodialysis patients. Today, the 

incidence of bacteraemia ranges from 0, 5 to 13 per 1000 

patient - days with haemodialysis catheters (1). According to 

data from the last register of dialysis treatment and 

transplantation of kidneys in Serbia and Montenegro, an 

increase of hospitalised patients with vascular problems was 

noticed. More precisely, every fifth haemodialysis patient 

was hospitalised due to a vascular problems (A-V fistula 

thrombosis, malfunction, infection or moving from CAPD to 

HD) (2). Haemodialysis catheters are indicated in two types 

of situation: first, short term solution to permit creation, 

maturation or revision of arteriovenous fistula or graft, 

second, long term solution for patients with exhausted 

vascular access sites and with contraindications for 

arteriovenous access due to medical reasons such as heart 

failure, severe limb ischaemia or to provide comfort in 

elderly patients with poor prognosis. Today, an estimatation 

is that 13% of patients with end-stage renal disease are 

regularly treated with permanent or temporary central 

catheters. The catheter insertion site plays a significant role 

in the magnitude of the infectious risk. The femoral route is 

apparently less favourable then subclavian or jugular route, 

(1-3). 


A retrospective study of complications caused by infections 

of central venous catheters for haemodialysis was done in 

University Hospital in Novi Sad, Department of Nephrology 

in 2004. In the period from January to December 2004, 60 

catheters were placed. 55 patients had central venous 

catheter, one of them because of plasmapheresis and 54 

because of haemodialysis. The duration of catheter placement 

averaged 36,67 days (shortest 7 days, longest 145 days). 

There were 7 patients with long term cuffed tunnelled 

catheters and 49 patients had short duration catheters. 

Insertion site of short time catheters was: v. jugularis 28, v. 

subclavia 19, and v. femoralis 2. During the treatment 39 

patients had 1 catheter, in 2 patients 3 catheters were changed 

and in 1 patient 4 catheters were changed. 

Results 

Smears were taken from catheter tips after extraction, and 

from the skin surrounding catheter insertion place. Blood 

cultures and smears were also taken from periphery (smears 

from pharynx, nose and urinoculture). Indications for placing 

catheters were acute renal failure in 19.65%, ESRD in 

35.71%, and complications of permanent vascular access in 

44.64%. In 2/55 patients, Staphylococcus aureus and 

Enterococcus species were isolated, from catheter tips after 

extraction, but without positive blood cultures. In a few 

patients Staphylococcus aureus, Pseudomonas aeruginosa, 

Escherichia coli, and Klebsiella pneumoniae, were isolated 

from periphery (skin changes 2|55, urine 2|55, pharynx 1|55). 

______________________ 


M. S. Simin, Institut for Internal diseases, Clinic for Nephrology and Clinical Immunology, 



Prophylaxis with antibiotics of cephalosporin and chinolone 

groups lasting 7 to 14 days was administered in 18 (32,2%) 

patients. 8/55 patients (14,5%) were treated with antibiotics 

according to antibiogram. 29 patients were left without 

antibiotic therapy. Mortality during this period of 

observation was 7|56 patients. 

Conclusions 

According to presented data, the conclusion is that duration 

of catheterisation period did not correlate with increase of the 

infection frequency. The number of patients dialysed with 

catheters is increased in the last few years, together with 

catheter related complications, especially bacteraemia, that 

became leading cause of morbidity and mortality. Obedience 

of surgical principles of asepsis and desinfection (sterile 

gloves, masks, galoshes, skin preparation for catheter 

insertion, appliance of desinfection means when 

disconnecting and connecting the catheters) evidently 

contribute to reduction of frequency of haemodialysis 

infections. 

References 

1. Bernard C. Haemodialysis catheter-related infection; time for 

action. Nephrol Dial Transplant 1999; 14: 2288-2290 

2. Radovic M, Nesic V. Godisnji izvestaj o lecenju dijalizama i 

transplantacijom bubrega u Srbiji i Crnoj Gori 2002 g. 

3. Hoen B, Paul-Dauphin A, Hestin D, Kessler M. Epibacdial: a 

multicenter prospective study of risk factor for bacteraemia in 

chronic hemodialysis patients. J Am Soc Nephrol 1998; 9(5): 

869-876 

4. Hung KY, Tsai TJ, Yen CJ, Yen TS. Infection associated with 

double-lumen catheterisation for temporary haemodialysis; 

experience of 168 cases. Nephrol Dial Transplant 1995; 10- 

247-251 

5. Tokaras JI, Miller ER, Alter MJ, ArudinoMJ, National 

surveillance of dialysis associated diseases in the UNITED 

states. 1995 ASAIO J 1998; 44(1): 98-107


Early renal protocol biopsies: beneficial effects of treatment of borderline 

changes and subclinical rejections on the histological changes for chronic 

allograft nephropathy 

J. Masin-Spasovska 1 , N. Ivanovski 1 , S. Dzikova 1 , G. Petrusevska 2 , B. Dimova 2 , Lj. Lekovski 3 , 

Z. Popov 3 and G. Spasovski 1 

1 Department of Nephrology, 2 Department of Pathology, 3 Department of Urology, Faculty of Medicine, Skopje, 

Republic of Macedonia 

Abstract 

Protocol biopsies in the early course after renal 

transplantation revealed that chronic allograft nephropathy 

(CAN) starts early. Our study aimed to identify borderline 

changes (BC), subclinical rejections (SR) and histological 

markers of chronic allograft nephropathy (CAN) in protocol 

biopsies at 1 and 6 months after living related kidney 

transplantation, and to determine whether treatment of BC 

and SR at the 1-month postransplant has a beneficial effect 

on graft histology and function at 6 months. 

Forty paired allograft biopsies were evaluated according to 

the Banff scoring schema. 

BC was found in 13/40 (32.5%) and 12/40 (30%), and SR in 

15/40 (37.5%) and 21/40 (52.2%) of patients, on 1 and 6- 

month biopsies, respectively. The mean HI (histological 

index/ sum of scores for acute/chronic changes) and CAN 

score (sum of histological markers for chronicity) increased 

significantly at 6-month biopsy 5.3±2.9 vs. 7.8±3.6 (p


Seron et al. reported a prevalence of CAN in about 42% of 

protocol biopsies at 3 months after transplantation (11,12). 

Recent reports provide evidence in favor of treating biopsyproven 

BC and SR, and that timely therapeutic intervention 

improves clinical outcomes, thereby preventing development 

of CAN. This raises the issue of early protocol biopsies of 

stable allografts and the clinically useful information they 

provide. 


The cohort of forty consecutive living related (LR) transplant 

patients with their first allograft received induction therapy 

with methylprednisolone (500 mg) and Daclizumab 

(Zenapax; 1 mg/kg BW at implantation and thereafter every 2 

weeks x five doses). The post-transplant standard triple 

immunosuppression therapy consisted of: cyclosporine 

(Neoral; 4 to 6 mg/kg/day) to reach target C2 levels (blood 

concentration 2 hours after administration of the drug), 

prednisolone (1 mg/kg/day tapered to 0.1 mg/kg/day after 4 

weeks) and mycophenolate mofetil (Cellcept 1 g bid.). 

During the first postoperative month patients with delayed 

graft function (DGF) who suffered post-transplant acute 

tubular necrosis or experienced a clinical episode of acute 

rejection (AR) were treated with hemodialysis or pulse 

corticosteroids, respectively, whenever an increase in serum 

creatinine >20% or decrease in urine output for 2 consecutive 

days was observed. These cases were included if their graft 

function had been stable (no change in serum creatinine > 

20%) for at least 2 weeks before the first biopsy. Patients 

with histology at 1 month biopsy of BC or AR type I or IIA 

and an increase in serum creatinine between 10 and 20 % 

from baseline (serum creatinine 2 weeks prior the biopsy) 

were assessed as SR and consequently treated with pulse 

corticoid therapy. The patients with histology of BC or AR 

followed by rise in serum creatinine < 10% from baseline 

were not treated. 

Protocol biopsies were performed using ultrasound-guided 

automated biopsy "gun". The formalin fixed biopsies were 

embedded in paraffin, serially sectioned at 3 to 5 µm 

thickness and stained with hematoxylin-eosin (HE), periodic 

acid-Schiff (PAS), Masson's trichrome as well as 

methenamine silver. Biopsies were considered adequate when 

they contained ≥7 glomeruli and at least one artery. The same 

pathologist blindly reviewed the renal lesions for evidence of 

acute and chronic changes by using descriptive morphologic 

criteria according to the Banff 97 scoring schema on a scale 

from 0 to 3 (13). CAN score was calculated as a sum of 

scores for the individual histological markers for chronicity: 

interstitial fibrosis, tubular atrophy, vascular fibrous intimal 

thickening, arterial hyalinosis and chronic glomerulopathy. 

The histological index (HI) was calculated as total sum of 

scores for acute and chronic changes. 

The clinical and biochemical data were recorded at 

the time of transplantation as well as at 1 and 6 months after 

transplantation. Results were expressed as mean values ± SD. 

An unpaired two-tailed Student t test was used to examine 

differences in mean values between the groups. Chi square 

analysis was used to compare the categorical variables. 

Results 

Donors and recipients mean age was 59.3 13 and 34.3 9.8 

years, respectively. Only 7.5% (6/80) of the biopsies showed 

no histopathological lesions. BC was found in 13/40 (32.5%) 

and 12/40 (30%), and SR in 15/40 (37.5%) and 21/40 

(52.2%) of patients, on 1 and 6 months biopsies, respectively. 

The mean HI and CAN score increased significantly at 6- 

month biopsy, 5.3 ± 2.9 vs. 7.8 ± 3.6 (p


first month after transplantation sCr was significantly higher 

in the group with high HI, 134 ± 35.9 vs.113 ± 27.5 (p


Table 4: Comparison of biochemical, clinical data and histological findings and scores at 1 and 6 months post-transplantation 

between the groups according to the treatment of borderline changes (BC) and subclinical rejections (SR) 

CLASSIFICATIONS MEAN SD MEAN SD P value 

1 month 6 months 

TREATMENT OF BC/SR Treatment (Tx) group Non-treatment (NTx) 

Number of patients (n = 9) (n = 19) 

sCr 1 month 130.56 33.09 122.26 29.24 n.s. 

sCr 6 months 151.44 38.36 136.11 44.23 n.s. 

Crcl 1 month 62.15 15.15 66.38 18.34 n.s. 

Crcl 6 months 58.11 21.50 63.35 18.31 n.s. 

DGF 1/9 (11.113%) 5/19 (26.32%) < 0.05 

AR (before 1 month biopsy) 1/9 (11.113%) 3/19 (15.79 %) n.s. 

AR 1 month / / 

BC 1 month 2/9 (22.22%) 11/19 (54.89%) < 0.05 

SR 1 month 7/9 (77.78%) 8/19 (42.11%) < 0.05 

BC 6 months 4/9 (44.44%) 5/19 (26.32%) < 0.05 

SR 6 months 4/9 (44.44%) 12/19 (63.16%) < 0.05 

HI 1 month 7.11 ± 1.90 4.95 ± 1.99 0.013 

HI 6 month 7.11 ± 2.32 8.16 ± 4.30 n.s. 

CAN score 1 month 2.33 ± 1.41 1.89 ± 1.37 n.s. 

CAN score 6 months 4.33 ± 1.73 8.16 ± 4.30 n.s. 

Discussion 

Chronic allograft nephropathy (CAN) causes most kidney 

allograft losses and despite improvements in 

immunosuppression, remains the central clinical challenge 

(14). In protocol biopsy studies, allograft damage is common, 

time-dependent, progressive and underestimated by 

measurement of serum creatinine. Rejection leads to 

allograft damage but the expression depends on its type, 

timing, severity and persistence. Subclinical rejection (SR) is 

defined as histologically-proven acute rejection without 

concurrent renal dysfunction (15), and is influenced by the 

time after transplantation, prior acute rejections, HLA 

mismatches, and immunosuppression (9,15). Untreated SR 

inflicts permanent tubulointerstitial damage and fibrosis (9). 

There is an evidence that protocol biopsy of stable renal 

allografts may be valuable tool to uncover early clinically 

invisible acute histological lesion, such as BC and SR (16). 

Previous reports show the prevalence of SR approximately in 

30-50% of protocol biopsies in the first 3 months after 

transplantation (9). The principal finding of our study is the 

histological evidence of BC and SR in relatively high 

percentage (33 and 38%) at first month, being even higher at 

6-month biopsies (30 and 52%), respectively. A comparable 

experience have been reported by Rush et al. (21 and 33- 

50%), and by Schweitzer et al. (23 and 33%), for the 

prevalence of BC and SR, respectively (4, 9, 17). 

Our study demonstrates that corticosteroid treatment of early 

BC and SR found at 1-month biopsies, is associated with 

improved histology of acute and chronic lesions at 6 months 

biopsies. Additionally, 4 patients in Tx group initially 

diagnosed as SR (classified as type IIA), showed an 

improvement in histology towards type I A, while 7 patients 

in NTx group diagnosed as BC at 1-month biopsies, 

worsened their histology towards SR type IA, at 6-month 

biopsies. Despite the progression of renal scarring in NTx 

group (significant increase of mean CAN and HI at 6-month 

biopsies), both groups presented with similar serum 

creatinine at the same time point. However, treatment of BC 

and SR did not result in improved allograft function at 6 

months. In contrast, Rush et al. reported that the treatment of 

early SR decreases chronic pathology (CAN), late clinical 

rejection episodes and improves long-term graft function (9). 

In addition, Shweitzer et al. reported a complete therapeutic 

response to steroid tretament in 58%, and Gaber et al. in 

100% of patients with BC (17,18). 

Conclusions 

A protocol 1-month biopsy may uncover a high prevalence of 

BC or SR in stable allografts. The presence of untreated BC 

or SR showed a greater susceptibility for histological acute 

and chronic deterioration on 6-month biopsy, accelerating the 

process of CAN. The beneficial effect of the treatment of 

BC/SR should be confirmed at follow-up of the graft function 

at 1 and 2 years. 

Our preliminary data support the hypothesis that treatment of 

the subclinical conditions (BC and SR) may counteract the 

development of CAN, aiming to provide a strong impetus to 

incorporate early allograft protocol biopsies into a standard 

clinical practice. Understanding the causes and the 

mechanisms of chronic allograft nephropathy may suggest 

targeted therapies to prevent the initiation or progression of 

CAN. 

References 

1. Paul LC. Chronic renal transplant loss. Kidney Int 1995; 47: 

1491-1499 

2. Isoniemi HM, Krogerus L, von Willebrand E, Taskienen E et 

al. Histological findings in well-functioning, long term renal 

allografts. Kidney Int 1992; 41: 155-160 

3. Rush DN, Henry SH, Jeffery JR, Schroeder TJ, Gough J. 

Histological findings in early routine biopsies of stable renal 

allograft recipients. Transplatation 1994; 57: 208-211 

4. Rush D, Nickerson P, Gough J et al. Beneficial effects of 

treatment of early subclinical rejection: a randomized study. J 

Am Soc Nephrol 1998; 9: 2129-2134 

5. Nickerson P, Jeffery JR, Gough J et al. Effect of increasing 

baseline immunosuppression on the prevalence of clinical and 

subclinical rejection: a pilot study. J Am Soc Nephrol 1999; 

10: 1801-1805 

6. Shapiro R, Randhawa P, Jordan M et al. An analysis of early 

renal transplant protocol biopsies- the high incidence of 

subclinical tubulitis. Am J Transpl 2001; 1: 47-50


7. Morath C, Ritz E and Zeier M. Protocol biopsy: what is the 

rationale and what is the evidence? Nephrol Dial Transplant 

2003; 18: 644-647 

8. Rush D, Karpinski M, Nickerson P, Dancea S et al. Does 

subclinical rejection contribute to chronic rejection in renal 

transplant patients? Clin Transplant 1999; 13: 441-446 

9. Rush DN, Jeffery J, Trpkov K et al. Effect of subclinical 

rejection on renal allograft histology and function at 6 months. 

Transpl Proc 1996; 28: 494-495 

10. Shapiro R, Randhawa P, Jordan ML et al. An analysis of early 

renal transplant protocol biopsies-the high incidence of 

subclinical tubilitis. Am J Transplant 2001; 1: 47-50 

11. Nankivell BJ, Fenton-Lee CA, Kuypers DJ et al. Effect of 

histological damage on long-term kidney transplant outcome. 

Transplantation 2001; 71: 515-523 

12. Seron D, Moreso F, Bover J, Condom E et al. Early protocol 

renal allograft biopsies and graft outcome. Kidney Int 1997; 

51: 310-316 

13. Racusen LC, Solez K, Colvin RB et al. The Banff 97 working 

classification of renal allograft pathology. Kidney Int 1999; 55: 

713-723 

14. Meier-Kriesche HU, Schold JD, Kaplan B. Long-term renal 

allograft survival: have we made significant progress or is it 

time to rethink out analytic and therapeutic strategies? Am J 

Transplant 2004; 4: 1289-1295 

15. Kuypers DR, Chapman JR, O`Connell PJ et al. Predictors of 

renal transplant histology at three months. Transplantation 

1999; 75: 1222-1230 

16. Gough J, Rush D, Jeffery J et al. Reproducibility of the Banff 

schema in reporting protocol biopsies of stable renal allografts. 

Nephrol Dial Transplant 2002; 17: 1081-1084 

17. Schweitzer EJ, Drachenberg CB, Anderson L, Papadimerou 

HC et al. Significance of the Banff borderline biopsy. Am J 

Kidney Dis 1996; 28: 585-588 

18. Gaber LW, Moore LW, Alloway RR, Flax S, gaber AO. 

Correlation between Banff classification, acute renal rejection 

scores, and reversal of rejection. Transplant Proc 1995; 27: 

1019


Factors associated with carotid and femoral atherosclerosis in non-diabetic 

hemodialysis patients 

S. Gelev, S. Dzikova, A. Sikole, Gj. Selim, P. Dzekova, V. Amitov and G. Spasovski 

Department of Nephrology, University of Skopje, Skopje, Republic of Macedonia 

Abstract 

Information on different stages of atherosclerotic changes has 

been obtained by combining information from B-mode 

ultrasonography recordings of the common carotid (CCA) 

and femoral arteries (FA). The aim of our study was to 

analyze the potential risk factors associated with different B- 

mode ultrasonography measurements on CCA and FA in 

non-diabetic hemodialysis (HD) patients. 

In a cross-sectional study we examined 67 non-diabetic, 

asymptomatic HD patients. All patients underwent high 

resolution B-mode ultrasonography of the CCA and FA. We 

compared the ultrasonographic findings in patients stratified 

according to various cut off levels for each clinical and 

biochemical parameter (mean value of one year laboratory 

data). 

The intima media thickness (IMT) exceeded the upper limit 

of normal range in 97.5% on CCA and in 100% of patients 

on FA. Atherosclerotic plaques (AP) were detected in 43.3 

and 55.2%, and were calcified in 20.9 and 13.4% of the 

patients on CCA and FA, respectively. An increased CCA- 

IMT was found in older patients (> 50 years and > 45 years at 

start of HD), with lower (< 1.2) kt/V, higher (> 1.5 mmol/L) 

phosphate and LDL (> 2,8 mmol/L) levels. An increased FA- 

IMT was only associated with HD duration > 72 months. 

Male gender, kt/V < 1.2, systolic blood pressure (SBP) > 150 

mmHg and pulse pressure (PP) > 70 mmHg were associated 

with increased CCA and FA internal diameter (ID). An 

increased CCA-ID was revealed in older patients (>50 years 

and > 45 years at the start of HD) and those with body mass 

index (BMI) > 23 kg/m 2 . Mean arterial pressure (MAP) > 

100 mmHg was associated with an increased FA-ID. 

Higher frequency of CCA and FA-AP were detected in older 

patients (> 50 years and > 45 years at start of HD), and higher 

total serum calcium (Ca) > 2,4 mmol/L. The finding of CCA- 

AP was associated with male gender and lower (< 150 pg/ml) 

serum intact parathyroid hormone (iPTH), while the presence 

of FA-AP was associated with higher (> 70 mmHg) PP and 

serum C-reactive protein (CRP) levels (> 3 mg/L). The 

gender, age (> 50 years and > 45 years at start of HD), PP > 

70 mmHg and iPTH < 150 pg/ml were associated with higher 

presence of calcified CCA and FA-AP. The calcified CCA 

and FA-AP were associated with lower (< 0.9 mmol/L) HDL 

and higher (> 3.5 mmol/L) Ca x phosphate product, 

respectively. 

The atherosclerotic changes on CCA and FA were frequently 

found in our cohort of asymptomatic, non-diabetic HD 

risk for cardiovascular disease. Age, gender, blood pressure, 

dialysis adequacy, bone markers and serum lipid disorders 

were associated with the atherosclerotic lesions in our 

patients. Timely management of the associated factors may 

be beneficial in preventing of atherosclerosis. 

Key words: dialysis, atherosclerosis, ultrasonography 

Introduction 

The association between uremia and increased risk of 

atherosclerotic disease has been documented in many 

investigations (1). Atherosclerotic damage of large arteries is 

major contributory factor to the high cardiovascular 

morbidity and mortality of hemodialysis (HD) patients (2). 

Since cardiovascular complications are a common cause of 

death in HD patients, the early detection and prevention of 

atherosclerosis are desirable. Arterial intima calcification 

represents an advanced stage of atherosclerosis and is 

associated with development of plaques and occlusive lesions 

(3). Moreover, the vascular calcification in advanced 

atherosclerosis is a common feature in HD patients (4). 

It is assumed that the atherosclerotic changes in carotid artery 

may reflect the general atherosclerosis (5). High resolution B- 

mode carotid ultrasonography is a fundamental technique for 

the noninvasive investigation of atherosclerosis in HD 

patients, combining the data from measurements of the 

carotid and femoral arteries (5,6). The investigation of the 

carotid artery with this technique is important not only for the 

assessment of its structural alterations but also because the 

extent of atherosclerosis of this vessel reflects the severity of 

arterial damage in other districts (6). Increased common 

carotid artery intima-media thickness (IMT) is a marker of 

early atherosclerosis (7). 

Traditional risk factors for atherosclerotic disease can not 

completely explain the excess of cardiovascular disease 

(CVD) in HD patients (5). Among a variety of 

pathophysiological conditions, age, gender, HD duration (5), 

hypertension (8,9), diabetes (10), chronic inflammation 

(11,12), malnutrition (13), abnormalities in lipid composition 

(14), serum calcium (Ca), hyperphosphatemia, calciumphosphorus 

product (Ca x P) (15-17) and intact parathyroid 

hormone (iPTH) concentrations (18), have been closely 

associated with atherosclerotic changes in HD populations. 

However, further information about the possible factors 

associated with atherosclerosis is needed in order to better 

prevent and control development of CVD. 

patients, implying the need for examination of this group at 

______________________ 

Correspodence to: G. B. Spasovski, Department of Nephrology, University Clinical Center, 

Vodnjanska 17, 1000 Skopje, Macedonia; Telephone: + 389 70 268 232; Fax: + 389 2 3231 501; 

E-mail: gspas@sonet.com.mk


The primary endpoint of present study was to analyze the 

association between B-mode ultrasonography findings in the 

carotid and femoral arteries and the possible risk factors for 

atherosclerosis in our non-diabetic HD population. Secondary 

endpoint was to assess the presence of atherosclerotic 

changes in the carotid and femoral arteries in our nondiabetic, 

asymptomatic HD patients. 


Patients 

In a cross-sectional study we examined 67 patients (at least 

12 months on HD), with no records and symptoms of CVD 

(19), infection or diabetes. The patients were dialyzed with 

low-flux synthetic membranes, bicarbonate dialysate (1,75 

mmol/l calcium), on erythropoietin therapy to maintain predialysis 

hemoglobin between 100 and 120 g/l, regular 

supplementation with iron, and calcium carbonate (CaCO 3 ) 

use to maintain pre-dialysis serum phosphate levels < 1,8 

mmol/l. The duration of HD was individualized (4-5 hours, 

thrice weekly) to control body fluids and blood chemistries. 

Systolic (SBP) and diastolic blood pressure (DBP) was 

recorded monthly before HD, at the same day for blood 

sampling, and averaged for the last 12 months prior to the B- 

mode ultrasonography for statistical analysis. Brachial pulse 

pressure (PP) and mean arterial pressure (MAP) were 

calculated by the formula PP = SBP-DBP; MAP = DBP + 

(SBP-DBP) / 3. Body mass index (BMI), prescription for 

vitamin D (µg/weekly) and CaCO 3 (g of elemental Ca/day) 

prescribed to each patient were recorded from the patients' 

files. The demographic data on age at inclusion and start of 

HD, gender, HD duration, smoking, and dialysis adequacy 

were also recorded in the database. 

Blood chemistries 

Pre-dialysis hemoglobin, leukocyte, serum Ca, phosphate, 

albumin, triglycerids, total, HDL and LDL cholesterol and C- 

reactive protein (CRP) were determined once monthly. 

Serum iPTH and ferritin were measured every 4 months. The 

values of biochemical data considered in the study were 

averaged over the last 12 months preceding the B-mode 

ultrasonography measurements. 

Ultrasonography 

All patients underwent bilateral B-mode ultrasonography 

(Toshiba HDI 3000 with 7,5 MHz transducer) of the common 

carotid arteries (CCA) and femoral arteries (FA) for the 

evaluation of IMT, internal diameter (ID), atherosclerotic 

plaque (AP) detection and the presence of calcified AP. CCA 

measurements were made 2 cm beneath the bifurcation (20) 

and included approximately 4 cm of the CCA, and FA was 

examined approximately 4 cm below the inguinal ligament at 

the site where the artery divides into the superficial and the 

profound FA (5). IMT measurements were made on the far 

walls at the same level as the ID measurements (20). IMT 

was defined as the distance between the leading edge of the 

lumen-intima interface to the leading edge of the mediaadventitia 

interface of the far walls (5). Abnormal IMT is 

defined as IMT > 0,8 mm (6). A localized echo-structure 

encroaching into the vessel lumen was considered to be AP if 

the IMT was > 50% thicker than neighboring sites (20). 

Measurements of IMT and ID were always made in AP-free 

arterial segments (20). 


Variables were expressed as frequencies, percentages for 

assessed factors and mean values ± SD for normally 

distributed continuous parameters. 

Patients were divided into 2 groups as a result of different cut 

off levels of each parametric variable and different code of 

nonparametric variables, respectively. Fifty years at 

inclusion, 45 years at the start of HD, 72 months HD 

duration, Kt/V ≥ 1.2, SBP ≥ 150mmHg, DBP ≥ 90mmHg, 

MAP ≥ 100mmHg, PP ≥ 70mmHg, BMI ≥ 23 kg/m2, CaCO 3 

≥ 3g Ca/day, hemoglobin ≥ 110g/L, leukocyte ≥ 6x10 9 /L, 

serum albumin ≤ 40 g/L, total serum Ca ≥ 2.4mmol/L, serum 

PO 4 ≥ 1.5mmol/L, Ca x P product ≥ 3.5mmol/L, iPTH ≥ 150 

pg/ml, TG ≥ 1.9 mmol/L, cholesterol ≥ 4.8mmol/L, HDL ≥ 

0.9mmol/L, LDL ≥ 2.8mmol/L, ferritin ≥ 500mg/L and CRP 

≥ 3.0mg/L were determined for cut off values. Gender (0- 

male, 1-female), vitamin D treatment, smoking, AP detection 

and presence of calcified AP (0-no, 1-yes) were used as 

categorical variables. Ultrasonography findings (IMT, ID, 

plaque detection and presence of calcified plaques) were 

compared among the groups. 

Statistical comparison between the groups was performed 

with Student t-test for continuous variables, and chi-square 

analysis for the categorical variables. 

Statistical analysis was performed with standard statistical 

package (SPSS for Windows version 9.0). For all 

comparisons, a P-value < 0.05 was considered statistically 

significant. 

Results 

This is the first study performed in our dialysis patients using 

high-resolution B-mode ultrasound for evaluation of the 

atherosclerotic changes in CCA and FA. 

The intima media thickness (IMT) exceeded the upper limit 

of normal range in 66 (97.5%) on CCA and in 100% of 

patients on FA. Atherosclerotic plaques (AP) were detected 

in 29 (43.3) and 37 (55.2%), and were calcified in 14 (20.9) 

and 9 (13.4%) of the patients on CCA and FA, respectively. 

Ultrasonographic findings on CCA and FA of the whole 

study group are presented in Table 1. 

Table 1. Ultrasonograpic characteristics of all patients 

CCA-IMT (mm) 1.46±0.29 FA-IMT (mm) 1.43±0.23 

frequency of CCA-AP (%) 43,28 Frequency of FA-AP (%) 55.22 

Freq. of calcified CCA-AP (%) 20.9 freq. of calcified FA-AP (%) 13.43 

CCA-ID (mm) 7.36±0.99 FA-ID (mm) 7.43±1.05 

Age: When divided according to age (>50< years) the older 

patients (n=37) had mean age of 60.9±7.2 years (range 51-74) 

as compared with 41.4±6.3 years (range 26-49) in the 

younger group, while patients (> 45 years) at start of HD


(n=35) had mean age of 55.3±7.0 years (range 45-67) as 

compared with 33.7±7.7 years (range 19-44) in the younger 

patients. Both older groups of patients showed significantly 

increased CCA-IMT, CCA-ID, higher frequency of CCA and 

FA-AP, calcified CCA and FA-AP (the last being at 

borderline of significance for age at start of HD) (Table 2). 

Table 2. Ultrasonograpic characteristics of patients divided according to age (>50< years) 

age at inclusion > 50 years < 50 years P 

CCA-IMT (mm) 1.53±0.27 1.37±0.29 0.02 

CCA-ID (mm) 7.62±1.02 7.04±0.88 0.01 

frequency of CCA-AP (%) 22/37 (59.46) 7/30 (23.33) 0.000 

freq. of calcified CCA-AP (%) 13/37 (35.13) 1/30 (3.33) 0.000 

frequency of FA-AP (%) 25/37 (67.57) 12/30 (40.0) 0.03 

freq. of calcified FA-AP (%) 8/37 (21.62) 1/30 (3.33) 0.04 

age at start of HD 45 years < 45 years P 

CCA-IMT (mm) 1.55±0.27 1.37±0.28 0.009 

CCA-ID (mm) 7.68±1.01 7.01±0.87 0.005 





Gender: Male patients (n=43) had significantly increased 

CCA-ID, FA-ID, higher frequency of CCA-AP, calcified 

CCA-AP and calcified FA-AP when compared with female 

patients (n=24) (Table 3). 

Table 3. Ultrasonograpic characteristics of patients divided according to the gender 

Gender Male Female P 

CCA-ID (mm) 7.60±0.96 6.92±0.93 0.006 

FA-ID (mm) 7.84±0.98 6.69±0.72 0.000 




Dialysis adequacy and HD duration: CCA-IMT, CCA-ID and 

FA-ID were significantly lower in patients (n=37) with 

higher (> 1.2) kt/V (mean 1.37±0.14, range 1.21-1.76) as 

compared to the patients with kt/V 1.2 (mean 1.08±0.1, 

range 0.81-1.2). Patients (n=33) with longer (> 72 months) 

HD treatment (mean 130±47, range 72-236) in comparison 

with patients being shorter on HD (mean 47±15, range 14-68) 

showed significantly increased FA-IMT (Table 4). 

Table 4. Ultrasonograpic characteristics of patients according to dialysis adequacy and HD duration 

dialysis adequacy kt/V 1.2 kt/V > 1.2 P 

CCA-IMT (mm) 1.54±0.26 1.39±0.29 0.03 

CCA-ID (mm) 7.74±0.87 7.04±0.99 0.003 

FA-ID (mm) 7.91±0.96 7.05±0.97 0.000 

HD duration 72 months < 72 months P 

FA-IMT (mm) 1.49±0.19 1.37±0.24 0.028 

Blood pressure: The patients (n=33) with higher 

(150mmHg) SBP (mean 162±11, range 151-190) presented 

with significantly increased CCA and FA-ID as compared 

with patients with lower SBP (mean 124±14, range 91-145). 

Significantly higher FA-ID 

Table 5. Ultrasonograpic characteristics of patients divided according to the blood pressure 

SBP 150 mmHg < 150 mmHg P 

CCA-ID (mm) 7.60±1.03 7.12±0.92 0.049 

FA-ID (mm) 7.74±1.06 7.13±0.96 0.01 

MAP > 100 mmHg < 100 mmHg P 

FA-ID (mm) 7.69±1.08 7.18±0.98 0.049 

PP 70 mmHg < 70 mmHg P 

CCA-ID (mm) 7.65±0.92 7.19±1.01 0.06 


FA-ID (mm) 7.85±0.98 7.19±1.03 0.012 


freq. of calcified FA-AP (%) 6/24 (25.0) 3/43 (6.98) 0.028


measurements were found in patients (n=33) with MAP > 

100mmHg (mean 115±7, range 103-130) as compared with 

those with lower MAP (mean 89±9, range 63-100). Patients 

(n=24) with higher ( 70 mmHg) PP (mean 77±8, range 70- 

100) tended to have increased CCA-ID, while FA-ID, FA- 

AP, calcified CCA and FA-AP were significantly higher 

when compared to the patients with lower PP (mean 52±9, 

range 31-61) (Table 5). 

BMI: Significantly higher CCA-ID was shown in patients 

(n=33) with an increased (> 23 kg/m 2 ) BMI (mean 27.1±3.5, 

range 23.0-35.7) as compared with patients having lower 

BMI (mean 20.5±1.5, range 16.4-22.7) (Table 6). 

Table 6. Ultrasonograpic characteristics of patients divided according to BMI 

BMI > 23 kg/m 2 < 23 kg/m 2 P 

CCA-ID (mm) 7.69±1.11 7.17±1.11 0.04 

Bone mineral metabolism parameters: Patients (n=31) with 

an increased ( 2.4 mmol/L) serum Ca (mean 2.49±0.08, 

range 2.4-2.8) had significantly higher frequency of CCA-AP 

and FA-AP being at borderline of significance in comparison 

with patients with lower Ca (mean 2.25±0.12, range 1.84- 

2.39). CCA-IMT was significantly increased in patients 

(n=34) with serum phosphate 1.5 mmol/L (mean 1.8±0.22, 

range 1.5-2.32) when compared with patients with lower 

phosphate levels (mean 1.23±0.18, range 0.86-1.47). 

Calcified FA-AP tended to be more frequent in patients 

(n=35) with serum Ca x P > 3.5 mmol/L (mean 4.27±0.62, 

range 3.51-5.84) as compared with patients with lower Ca x P 

product (mean 2.89±0.49, range 1.71-3.46). Patients (n=36) 

with lower serum iPTH < 150 pg/ml (mean 64.5±33.2, range 

11.5-140) showed significantly higher frequency of CCA-AP, 

calcified CCA and FA-AP in comparison with patients with 

higher iPTH (mean 298.1±130, range 150-599) (Table 7). 

Table 7. Ultrasonograpic characteristics of patients divided according to the bone mineral metabolism parameters 

total serum Ca 2.4 mmol/L < 2.4 mmol/L P 



serum phosphate 1.5 mmol/L < 1.5 mmol/L P 

CCA-IMT (mm) 1.53±0.29 1.39±0.27 0.04 

Ca x P product > 3.5 mmol/L < 3.5 mmol/L P 


serum iPTH > 150 pg/ml < 150 pg/ml P 



frequency of calcified FA-AP (%) 1/31 (3.23) 8/36 (22.22) 0.03 

Serum HDL and LDL cholesterol: Significantly lower 

frequency of calcified CCA-AP was observed in patients 

(n=36) with higher (> 0.9mmol/L) HDL cholesterol (mean 

1.17±0.25, range 0.92-1.94) as compared with patients lower 

HDL (mean 0.77±0.08, range 0.57-0.88); while significantly 

increased CCA-IMT was found in patients (n=33) with 

higher ( 2.8mmol/L) serum LDL cholesterol (mean 

3.42±0.57, range 2.8-4.92) in comparison with patients with 

lower LDL (mean 2.11±0.38, range 1.43-2.77) (Table 8). 

Table 8. Ultrasonograpic characteristics of patients divided according to the lipid parameters 

serum HDL cholesterol < 0.9 mmol/L > 0.9 mmol/L P 

frequency of calcified CCA-AP (%) 11/31 (35.48) 3/36 (8.33) 0.04 

serum LDL cholesterol 2.8 mmol/L < 2.8 mmol/L P 

CCA-IMT (mm) 1.56±0.34 1.39±0.32 0.04 

Serum CRP: Patients (n=33) with low ( 3.0 mg/L) serum 

CRP (mean 1.63±0.84, range 0.25-3.0) had significantly 

lower frequency of calcified FA-AP as compared with 

patients with high CRP (mean 7.02±4.89, range 3.07-30.0) 

(Table 9). 

Table 9. Ultrasonograpic characteristics of patients divided according to CRP levels 

serum CRP > 3 mg/L 3.0 mg/L P 

frequency of FA-AP (%) 19/34 ( 55.88 ) 10/33 ( 30.3 ) 0.03 

Other variables: When divided according to various cut off 

levels of parameters like DBP, daily dose of CaCO 3, blood 

hemoglobin and leukocyte, serum albumin, ferritin, 

triglycerides and total cholesterol, there was no significant 

difference between the groups of patients, neither for 

comparison of the use of vitamin D or smoking. 

Discussion 

The results of this study confirm that atherosclerotic changes 

on CCA and FA in asymptomatic, non-diabetic HD patients 

are rather frequent, implying the need for examination of


systemic atherosclerosis in HD patients even they are free of 

symptoms. Moreover, the higher risk of cardiovascular 

mortality was reported to be associated with IMT of CCA 

obtained by ultrasonographic measurement (6). The present 

data has confirmed previous reports that age, gender, blood 

pressure, dialysis adequacy, bone mineral markers and serum 

HDL/LDL cholesterol disorders in HD patients are 

predominantly associated with their atherosclerotic lesions 

(8,9,14-17). 

Increased CCA-IMT in our patients was associated with 

traditional risk factors such as older (> 50 years) age at 

inclusion, and (> 45 years) at start of HD, worse dialysis 

adequacy (kt/V levels < 1.2), higher (> 1.5 mmol/L) 

phosphate and LDL (> 2.8 mmol/L) levels. In many studies 

no relationship was found between CCA and FA-IMT and 

the duration of HD (5). However, we found that increased 

FA-IMT was associated with longer (> 72 months) HD 

duration, although for better explanation we need a further 

statistical evaluation with multiple regression analysis. Male 

gender, lower kt/V (< 1.2), higher (> 150 mmHg) SBP and 

PP (> 70 mmHg) were associated with increased CCA and 

FA-ID. Increased CCA-ID was also associated with an older 

age at inclusion and at start of HD, and increased (> 23 

kg/m 2 ) BMI. Higher (> 100 mmHg) MAP was also 

associated with an increased FA-ID. 

Higher frequency of CCA and FA-AP in our patients was 

associated with an older age at inclusion and at start of HD, 

as well as an increased total serum Ca levels (> 2.4 mmol/L). 

The last could be possibly explained by the high (1.75 

mmol/L) concentration of calcium in the dialysis fluid, and 

calcium influx during each of the sessions. In addition, a 

higher frequency of CCA-AP was also associated with male 

gender and lower (< 150 pg/ml) iPTH, and a higher presence 

of FA-AP was found in patients with higher (> 70 mmHg) PP 

and serum CRP levels (> 3 mg/L). Calcified CCA and FA- 

AP were most frequently observed in males, older patients, 

with higher PP and lower iPTH levels. A recent 

epidemiological survey on bone markers in our HD 

population has shown adynamic bone disease (ABD) as most 

frequent renal bone disease (21). It was concluded that the 

use of high (1.75 mM) dialysate Ca concentration, calcium 

carbonate and vitamin D treatment might be associated with 

development of ABD. Hence, the over-suppression of PTH 

and ABD as predominant type of renal osteodystrophy 

(ROD) contribute to high levels of calcium and phosphorus 

in the blood, which are strongly associated with 

cardiovascular disease, a major cause of mortality and 

morbidity (22). This is also in line with our study data that 

higher presence of calcified CCA and FA-AP was associated 

with lower HDL and an increased Ca x P product, 

respectively. 

DBP, serum albumin, ferritin, triglycerides, cholesterol and 

the amount of ingested CaCO 3, vitamin D intake and smoking 

did not show significant associations with CCA and FA 

ultrasonographic findings in our patients. Here, the possible 

explanation could be a regular supplementation with 

parenteral iron therapy if needed, prescription of 

hypolipemics upon strict indication and precautious treatment 

with CaCO 3 and vitamin D. 

In line with the previous reports that IMT is a strong 

predictor for cardiovascular events in the HD population (23), 

and that CCA-ID, but not CCA-IMT predict overall mortality 

and cardiovascular outcomes in HD patients (6), we need 

further keep trying to get under control the atherogenic 

factors like disorders of mineral metabolism (abnormalities in 

plasma Ca, phosphorus and iPTH concentration), blood 

pressure, HDL/LDL cholesterol, inflammation (CRP), and to 

improve the dialysis adequacy. The case if we can't establish 

any association between ultrasonographic findings and other 

atherogenic factors do not release us to keep up controlling 

further nutritional and inflammation markers, serum 

triglycerides and cholesterol, and the amount of ingested Ca 

from CaCO 3, vitamin D intake and smoking. 

Conclusions 

The measurement of IMT on CCA and FA in the present 

study demonstrated that atherosclerosis in asymptomatic nondiabetic 

HD patients is frequent. Our results suggest a few 

emerging risk factors for atherosclerosis such as older age, 

male gender, impaired dialysis adequacy, longer HD 

duration, increased BMI, SBP, MAP and PP, increased serum 

Ca, phosphate and Ca x P product, lower iPTH and HDL and 

higher LDL. We need to examine atherosclerotic conditions 

of CCA and FA in HD patients even if they are 

asymptomatic. The management of these associated factors 

may be beneficial in preventing of atherosclerosis and CVD 

development in this population at risk. 

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Acid-base disorders in patients with hypoproteinemia 

P. Mavromatidou 1 , N. Sotirakopoulos 2 , T. Tsitsios 2 , I. Skandalos 3 , M. Peiou 2 , and K. Mavromatidis 2 

1 Evosmos, Thessaloniki, 2 Renal Unit of Gen. Hosp. of Komotini, 3 Second Surgical Department, Gen. Hosp. Papageorgiou, 

Thessaloniki, Greece 

Abstract 

Hypoproteinemia is a common disturbance in patients 

suffering from various diseases. It can be accompanied by 

hypovolemia and hypotension that influences the volume 

space of drugs in the body as well as the acid-base balance. 

In the present study, 22 patients with hypoproteinemia (8F, 

14M) constituted group A. In this group the acid-base 

disorders as well as their importance were evaluated. From 

group A, 10 patients suffered from nephrotic syndrome, 

while 17/22 from chronic renal failure (CRF). The level of 

serum proteins varied from 46 gr/L to 64 gr/L, while the 

levels of albumin ranged from 13 to 32 gr/L. Furthermore, 15 

normal individuals were used as controls (group B) aging 

from 24 to 72 years old with normal levels of serum proteins. 

The results suggested that in group A, 21/22 patients had 

acid-base disorders. Moreover, 11/22 patients had a mixed 

disturbance (metabolic alkalosis combined with respiratory 

alkalosis). Only 4/22 patients had metabolic alkalosis, 4/22 

had metabolic acidosis (mixed) and 2/22 respiratory 

alkalosis. In particular, the blood’s pH appeared to be slightly 

alkaline. 

In group A the patients’ anion gap ranged from 5,7 to 18,5 

mmol/L. Based on this fact, group A patients were divided in 

two sub-groups. The first one (sub-group A1) consisted of 12 

patients measuring an anion gap higher than 10 mmol/L, 

while the second group (sub-group A2) consisted of 10 

patients with an anion gap lower than 10 mmol/L. The 

comparison of the pH between the two sub-groups suggested 

that A1 had respiratory alkalosis, while A2 had metabolic 

alkalosis. 

It can be concluded, that patients with hypoproteinemia 

appear to have disturbances of acid-base balance (mainly 

metabolic alkalosis), because of the reduction in serum 

proteins, which improves the pH levels in patients with CRF. 

The second most common acid-base disorder that appears in 

hypoproteinemic patients is respiratory alkalosis. 

Key words: hypoproteinemia, acid-base disorders, metabolic 

alkalosis, respiratory alkalosis, anion gap 

Introduction 

The electrochemical balance that exists in the body’s fluid 

compartments (also in plasma) suggests that the anions are 

equal to the cations. Nevertheless, it is not possible, in routine 

blood tests, to estimate most of the anions (non-counted 

anions) in comparison to the cations. For this reason, an 

anion gap appears which is expressed by a number that 

represents the non-counted negative ions (which, when added 

to the negatives’ ions sum, result to a number that is a equal 

to the positives ions sum). 

The anions, that are included usually to the anion gap, are 

serum proteins as well as organic acid, phosphate and sulfate 

ions (1). The acidosis with normal anion gap is caused by 

either bicarbonate loss or addition of chloride salts to the 

body. Greater than normal anion gap suggests the presence of 

retained acid anions other than chloride. 

The anion gap is actually low in the conditions stated, where 

an increase to the non-counted cations is present (acute 

toxicity from lithium, paraproteinemias-such as multiple 

myeloma, because of their positive charge-hypercalcaemia, 

hypermagnesemia). Extremely low anion gap occurs because 

of the reduction of the counted anions in hypoproteinemia 

and, in addition, in this case the anion gap is technically 

reduced (1). Understanding the anion gap in the above 

situations is very important since all these are life-threatening 

(2). 

The protein contributes to the anion gap approximately 2-2,8 

mmol/L for every 10 gr/L protein that exists in the serum (3). 

Since the human proteins are non-volative acids, their 

increase causes acidosis and their reduction causes alkalosis 

(4,5) and for this reason their estimation is necessary for the 

evaluation of the patients’ acid-base balance. In conclusion, it 

is estimated that normally about 8 mmol/L of the anion gap is 

due to serum proteins (2x4=8) and this is the reason 

responsible for the reduction of the anion gap in 

hypoproteinemia. In the present study the influence of 

hypoproteinemia in the acid-base balance of patients was 

evaluated. 


In the study were included patients with hypoproteinemia 

(serum albumin10 mmol/L) as well as those with hyperlipidemia 

(serum total cholesterol>3,4 mmol/L or triglycerides>3,4 

mmol/L). Furthermore, patients with hypernatremia, 

hypomagnesemia, hyperkalemia, paraproteinemia and 

bromism were excluded. Patients who received diuretics were 

also excluded. The study included 22 patients (8F, 14M), 

aging from 37 to 88 years old (median=63 years old). The 

control group (group B), consisted of 15 healthy individuals 

______________________ 


K. Mavromatidis, Ant. Rossidi 11 – 69100 , N. Mosinoupoli, Komotini – Greece; 

Tel. (+30) 25310 57149; Fax (+30) 25310 30706; E-mail: mavromatidis_k@yahoo.com


(7F, 8M), aging from 24 to 72 years old (median =40 years 

old) with normal levels of serum proteins. 

Blood for the gas determination was taken from the radical 

artery, while for the determination of serum electrolytes was 

taken from a peripheral vein. Blood gases were determined as 

well as haematocrite, creatinine and serum electrolytes 

(potassium, sodium, calcium, chloride), while the anion gap 

was calculated by using the formula: anion gap= (sodium) – 

(chloride + bicarbonate). The cause responsible for 

hypoproteinemia was marked as well as the presence or 

absence of nephrotic syndrome. 

The existence or not of hypovolemia in our patients was 

estimated based on medical history, clinical findings (arterial 

pressure in sitting and trendelenburg position, pulse wave, 

jugular vein condition) and laboratory findings (proportional 

increase of ratio Na + /CI - ). The diagnosis of acid-base 

disorders was based on medical history, clinical findings, 

blood gases, serum electrolytes and lactates, urine 

examination and serum anion gap. 

Serum electrolyte levels were determined by the use of an 

iontoepilectic analyzer (Dimension A.R., Acsess, USA) and 

blood gases were determined by the use of an automatic 

analyzer (GemPrimer 3000, USA). The statistical analysis 

was carried out by an unpaired student t-test. Changes with a 

level of significance p


(normal values for the serum total proteins: 65-80 gr/L and 

for albumin: 35-50 gr/L) (Table 1). 

It was found that, in group A, 21 patients showed 

disturbances of the acid-base balance. Specifically, 11 

patients appeared to have a mixed disturbance (metabolic 

alkalosis with respiratory alkalosis), 4 had metabolic 

alkalosis, 4 had metabolic acidosis (mixed), two had 

respiratory alkalosis and one had normal values. The mean 

average of the blood pH was slightly alkaline (7,44±0,06). 

Bicarbonates appeared to be at a mean average increased 

(24,1±5,2) and were ranging from 10,6 to 35,3 mmol/L. 

The comparison of the results of group A to those of group B 

(Table 1, 2) revealed a significant difference to the serum 

proteins and albumin (p=0,00001 in both cases), while in 

group B no acid-base disturbance was noted. 

n (7F, age pH PaC2 HCO3- Hct Cr Pro Alb + a+ CI- Ca++ AG 

8M) 

1 35 7.413 33.8 21.1 42.5 0.9 7.9 5 4.2 143 110 10.2 12.1 

2 60 7.434 36.7 24 41.5 0.9 7.4 4.3 4.4 142 108 10.8 10 

3 43 7.427 35.5 22.9 48.3 1 9.1 5.6 4.6 141 105 10.5 13.1 

4 24 7.423 36.9 23.6 38 1 8.9 5.4 4.2 141 104 10.7 9 

5 45 7.438 34.2 22.6 38.2 0.7 8.3 5.2 4.3 139 106 10.9 10.4 

6 30 7.414 41.3 25.8 38.2 0.8 8.3 5.2 4.4 140 102 10.8 12.2 

7 37 7.441 32.8 21.9 38.7 0.7 8.7 5.1 3.9 145 109 10.2 14.1 

8 67 7.42 38 24.1 39.5 0.8 8.7 6.7 4.2 141 105 9.9 11 

9 35 7.41 33.7 20.9 40 0.7 8 5.3 4.3 142 104 10.3 17.1 

10 40 7.446 32.1 21.6 38.2 0.7 7 4.4 4.4 142 106 9.8 14.4 

11 72 7.364 40.4 22.5 36.8 1.1 7.1 4.5 4.6 148 104 10 11.5 

12 50 7.437 39.3 25.9 43.5 0.9 7.3 4.5 4.7 143 103 9.6 14.1 

13 26 7.407 40.9 25.2 41.5 1.1 7.4 4.9 4.1 144 104 9.6 14.8 

14 30 7.419 40.2 25.2 36.8 0.9 6.8 4.2 4.5 139 102 9.7 11.8 

15 54 7.376 41.6 23.9 38.6 0.8 7.3 4.3 4.3 141 106 8.3 11.1 

Table 2: Blood gases, serum electrolytes, creatinine, proteins and haematocrite of normal individuals 

(Hct=haematocrite, Cr=creatinine, Pro=proteins, Alb=albulin, K+=potassium, Na+=sodium, CI- 

=chloride, Ca++=calcium, AG=anion gap) 

mean+/- 

sd 

43,2+/ 

-14,7 

7,42+/- 

0,02 

range 24-72 7,36- 

7,475 

37,2+/- 

3,3 

32,1- 

41,6 

23,4+/- 

0,4 

20,9- 

25,9 

40+/- 

3,1 

36,8- 

48,3 

0,9+/- 

0,1 

0,7- 

1,1 

7,9+/- 

0.8 

6,8- 

9,1 

4,8+/- 

0,7 

4,2- 

6,7 

4,3+/- 

0,2 

3,9- 

4,7 

142+/- 

2,3 

139- 

148 

105+/- 

2,4 

102- 

110 

10,1+/ 

-0,7 

8,3- 

10,9 

12,8+/ 

-1,9 

10- 

17,1 

The anion gap in group A was ranging from 5,7 to 18,5 

mmol/L (11,5±3,7). Because of this wide range, the patients 

were divided in two further sub-groups (Table 3). These were 

sub-group A1 with 12 patients that had an anion gap higher 

than 10 mmol/L and sub-group A2 with 10 patients that had 

an anion gap lower than 10 mmol/L. Evaluating the pH of the 

two sub-groups revealed the first (A1) had respiratory 

alkalosis (mean±SD, pH=7,42±0,06, PaCO 2 =32,0±4,2 and 

HCO 3 - =21,6±4,6), while the second (A2) had metabolic 

alkalosis (mean±SD, pH=7,46±0,04, HCO 3 - =27,3±5,2 and 

PaCO 2 =38,3±7,5). 

Seven patients in sub-group A2 suffered from CRF, while 3 

of these patients had a serum creatinine greater than 264 

mmol/L (advanced stage) (Table 3). In total, 6/22 patients 

had serum creatinine>264 mmol/L and from these patients, 3 

had clearly metabolic acidosis combined with some other 

disturbance (metabolic or respiratory alkalosis). 

Discussion 

In the extracellular fluids, the total concentration of weak 

acids cannot be regulated in order to achieve acid-base 

balance. However, since the total concentration of weak acids 

is an independent variable, a change to their levels would 

cause a change to the acid-base balance. Proteins are weak 

acids, and when they are reduced in the plasma, the 

concentration of bicarbonates is increased (causing metabolic 

alkalosis), in order to retain electric balance, a fact that was 

noted in most of the patients in this study (21/22). The 

metabolic alkalosis in 15/22 patients was caused by 

hypoproteinemia. If alkalosis was due to an acid deficiency, 

the serum chloride levels would be much more reduced, 

while the calculated bicarbonates would be increased (over 

42 mmol/L). However, in the patients of this study, the mean 

value of serum bicarbonates and chloride was approximately 

in the normal limits (small increase of bicarbonates), as it 

happens in the case of a primary hypoproteinemic alkalosis. 

The above information is of particular importance 

considering that 6/22 patients suffered from CRF with serum 

creatinine over 264 mmol/L. While this situation is 

accompanied by metabolic acidosis (usually with an 

increased anion gap), this was a fact only in 3 of our patients.


n age pH PaC2 HCO3- Hct Cr Pro Alb + a+ CI- Ca++ AG 

Subgroup 

A2 

1 60 7.48 35 27 34 2.8 5 2.1 3.5 142 101 7.2 14 

2 65 7.426 40.3 25.8 37.3 1.3 6 3.1 4.1 135 100 9.4 9.2 

3 74 7.427 38.7 24.9 27.6 2.2 6 3.2 4 132 98 9.5 9.1 

4 68 7.5 35.2 25.5 35 3 6 2.6 4.3 138 95 8.8 18.5 

5 50 7.4 32.2 23.3 30.8 0.6 4.6 2.5 4.5 143 108 8.5 11.7 

6 45 7.44 30.7 21.2 27.1 2.9 5.1 2.2 3.2 145 107 8.1 17 

7 88 7.4 34 22.1 40.7 1.1 5.3 2.5 4.7 148 112 9 10.9 

8 62 7.465 36.4 25.6 34.5 2.9 5.7 2.5 3.7 134 97 8 11 

9 37 7.5 33.6 28.3 43.4 0.7 6 2.5 3.8 144 104 11.7 9.7 

10 76 7.43 36.5 24 28 5.2 5 1.8 3.6 142 102 6.5 16 

11 59 7.289 22.6 10.6 25.1 6.9 4.9 1.9 3.8 140 116 8.4 13.4 

12 54 7.4 25.5 18.9 30.3 2.8 5 1.7 6.2 150 119 8.2 12 

13 64 7.304 32.4 15.7 29.7 4.7 6 3.2 4.5 139 108 8.3 15.3 

14 65 7.484 31.8 23.4 39 2.6 6.4 2.9 4.3 139 103 8.5 12 

15 74 7.459 32.1 22.3 28.5 2.4 6.1 2 5.1 136 96 8.1 17.7 

(mean+/-sd) 

62,7+/- 

12,9 

7,43+/- 

0,06 33,1+/-4,6 22,6+/-4,6 

range 37-88 7,29-7,5 22,6-40,3 10,6-28,3 

13,2+/- 

3,1 

9,1- 

18,5 

32,7+/- 

5,5 

2,8+/- 

1,7 

5,5+/- 

0,6 

2,5+/- 

0,5 

4,2+/- 

0,7 

140+/- 

4,9 

104,4+/- 

7,2 

8,6+/- 

1,2 

25,1- 

43,4 0,6-6,9 4,6-6,4 1,7-3,2 3,2-6,2 132-150 95-119 6,5-11,7 

Subgroup 

A1 

1 58 7.455 46.7 32 34 1.7 5.4 2.3 4.4 143 103 8 8 

2 70 7.491 34.9 25.4 34.8 2.9 5.8 2.5 2.9 138 94 8.1 8.6 

3 62 7.489 34.8 25.9 33.4 3.1 4.7 1.9 4 139 107 7.3 6.1 

4 38 7.397 41.5 25 37.5 3.2 6.2 3.2 4.7 135 103 9.1 7 

5 73 7.529 43.3 35.3 31.6 3.1 6.2 2.4 2.4 131 86 8.3 9 

6 53 7.433 44.2 28 44.4 0.8 5.7 1.3 3.7 137 100 8.1 9 

7 84 7.493 25 19.3 31.7 0.9 6.1 2.7 4 137 112 9 5.7 

(mean+/-sd) 

62,6+/- 

14,9 

7,47+/- 

0,04 38,6+/-7,5 27,3+/-5,2 

range 38-84 7,4-7,53 25-46,7 19,3-35,3 

35,3+/- 

4,5 

2,2+/- 

1,1 

5,6+/- 

0,5 

2,3+/- 

0,6 

3,7+/- 

0,8 

137+/- 

3,7 

100,7+/- 

8,6 

8,3+/- 

0,6 

7,6+/- 

1,4 

31,6- 

44,4 0,8-3,2 4,7-6,2 1,3-3,2 2,4-4,7 31-143 86-112 7,3-9,1 5,7-9 

Table 3: Patients with hypoproteinemia in subgroups depends on their anion gap (AG) 

(Hct=haematocrite, Cr=creatinine, Pro=proteins, Alb=albumin, K+=potassium, Na+=sodium, 

CI-=chloride, Ca++=calcium) 

Normal serum proteins are polyanionic molecules and their 

contribution to the electrochemical balance cannot be easily 

specified with accuracy. They contribute to the change of the 

charge, a change that depends on the kind of proteins, their 

concentration as well as the serum pH. In particular, albumin 

contributes at 2,0-2,8 mmol/L for every 10 gr/L of change in 

the serum level, while the globulins 1,3-1,9 mmol/L for every 

10 gr/L of change. Hence, this explanation constitutes the 

possible mechanism according to which the reduced anion 

gap in patients with nephrotic syndrome or severe stage of 

liver cirrhosis may arise (2). In sub-groups A1 and A2 it is 

obvious that at a mean average in the first one (A1) there was 

respiratory alkalosis due to tachypnea because they were 

hypovolemic, while in the second one (A2), there was at a 

mean average metabolic alkalosis, apparently due to the 

relative hypoproteinemia. 

In addition, patients of group A showed to have at a mean 

average a mild increase of the pH (7,44±0,06), approximately 

normal bicarbonates (24,1±5,2) and reduced PaCO 2 

(34,9±6,1), all suggesting the presence of respiratory 

alkalosis, apparently due tachypnea because the presence of 

hypovolemia. Rossing et al., studied 23 patients with 

hypoproteinemia due to liver cirrhosis or other diseases and 

ascertained that alkalemia (metabolic alkalosis of 

hypoproteinemia) was accompanied by hypocapnea 

(compensatory hypercapnea would be expected) for which 

hypoxemia was not responsible. In addition, the PaCO 2 levels 

were proportional to the reduction of albumin as well as the 

total serum proteins (6). All these facts were established in 

the present study in most of the patients, as mentioned above. 

Taking in consideration that 12 patients of sub-group A2 had 

CRF, while in 3 of these patients serum creatinine was over 

264 mmol/L, it is obvious that the importance of the reduced 

gap is greater, since in cases of an advanced stage of CRF an 

increased anion gap is expected (Table 3). Furthermore, the 

fact that 6/22 patients had serum creatinine>264 mmol/L


suggests that there should be, at least, an evident metabolic 

acidosis, with some patients showing an increased anion gap. 

Indeed, 3 of these patients had metabolic acidosis combined 

with some other disturbance (metabolic or respiratory 

alkalosis). These facts clearly show the influences of 

hypoproteinemia in the acid-base balance of those patients. 

Conclusions 

It can be concluded that: a) patients with hypoproteinemia 

have disturbances of the acid-base balance (mainly metabolic 

and respiratory alkalosis), which are due to the reduction of 

the serum proteins and b) the influence of hypoproteinemia in 

patients with CRF contributes to the improvement of the 

blood’s pH level. 

References 

1. Gabow PA. Disorders associated with an altered anion gap. 

Kidney Int 1985; 27: 472-83 

2. Emmett M & Narins RG. Clinical use of the anion gap. N Engl 

J Med 1977; 56: 38-54 

3. McAuliffe JJ, Lind LJ, Leith DE, Fenci V. Hypoproteinemic 

alkalosis. Am J Med 1986; 8: 86-90 

4. Rossing TH, Maffeo N, Fenci V. Acid-base effects of altering 

plasma protein concentration in human blood in vitro. J Appl 

Physiol 1986; 61: 2260-5 

5. Schlichtig R. Base excess vs strong ion difference. Which is 

more helpful? Adv. Exp Med Biol 1997; 411: 91-5 

6. Rossing TH, Boixeda D, Maffeo N, Fenci V. Hyperventilation 

with hypopropteinemia. J Lab Clin Med 1988; 112: 553-9

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Report, Technical Report or Brief Report, and 1 page for a Letter. Excess page charges will be 

charged 10 EURO per page. 

Case Reports must be 3 pages or less, and the standard length of a Letter to the Editor is 200 

words. A printed page is ~850 words, but pro rata reductions in the length of the text must be 

made for Tables, Figures and illustrations. 

In the proof email there is a reminder of the excess page charges levied on manuscripts that are 

over length. It is the authors responsibility to check their proof page extent and act according if 

they are do not wish to be charge for excess pages. 

AUTHORS 

Each author should have participated sufficiently in the work to take public responsibility for the 

content. This participation must include: 

Conception or design, or analysis and interpretation of data, or both. 

Drafting the article or revising it for critically important intellectual content. 

Final approval of the version to be published. (See Br Med J 1985; 291: 722-723.) 

TEXT 

NB - Please include your abstract and title page in the main body of your manuscript, as 

well as submitting it separately. 

The order of original articles should be as follows: 

Title page giving details of all authors, including first or given name (see above). 

On a separate page an abstract of ~250 words. It should consist of four paragraphs labelled, 

`Background', `Methods', `Results'and `Conclusions'. They should briefly describe, respectively, 

the problems being addressed in this study, how the study was performed, the salient results and 

what the authors conclude from the results. 

Keywords not more than 6, in alphabetical order, characterizing the scope of the paper, the 

principal materials, and main subject of work. Authors are requested to supply an additional 10- 

15 keywords for electronic publication purposes. 

Running title of not more than 75 letters and spaces. 

On a new page: Introduction, Subjects and Methods, Results, Discussion, Acknowledgements, 

References (see below), Tables, Legends to Figures and Figures. All pages should be numbered 

consecutively commencing with the title page. Headings (Introduction; Subjects and Methods, 

etc) should be placed on separate lines. It is most important that authors number their pages 

prior to submission as reviewers will refer to particular pages when providing their 

comments on the manuscript. 

Please ensure that your abstract and title page are inlcuded in the main body of your manuscript, 

as well as submitting it separately.

Any statistical method must be detailed in the Subjects and methods section, and any not in 

common use should be described fully or supported by references. 

Authors should not use abbreviations in headings, and Figure legends should be comprehensive 

without extensive perusal of the Subjects and methods section. Authors are advised to refrain 

from excessive use of uncommon abbreviations, particularly to describe groups of patients or 

experimental animals. 

Images in Nephrology aims to publish 1 or 2 high quality pictures of great clinical interest 

accompanying by a minimal amount of text and 2-3 references. The piece can not exceed 2 

printed pages. 

TABLES 

Tables must be typed on separate pages and should follow the reference list. All Tables must be 

numbered consecutively and each must have a brief heading describing its contents. Any 

footnotes to Tables should be indicated by superscript characters. Tables must be referred to in 

the main text in order. All Tables must be simple and not duplicate information given in the text. 

FIGURE PREPARATION 

Please be aware that the requirements for online submission and for reproduction in the journal 

are different: (i) for online submission and peer review, please upload your Figures either 

embedded in the word processing file or separately as low-resolution images (.jpg, .tif, .gif or. 

eps); (ii) for reproduction in the journal, you will be required after acceptance to supply highresolution 

.tif files (1200 d.p.i. for line drawings and 300 d.p.i. for colour and half-tone artwork) 

or high-quality printouts on glossy paper. We advise that you create your high-resolution images 

first as these can be easily converted into low-resolution images for online submission. 

For useful information on preparing your Figures for publication, go to 

http://cpc.cadmus.com/da. Figures will not be relettered by the publisher. The journal reserves 

the right to reduce the size of illustrative material. All micrographs must carry a magnification 

bar. Any photomicrographs, electron micrographs or radiographs must be of high quality. 

Wherever possible photographs should fit within the print area of 169 x 235 mm (full page) or 

within the column width of 82 mm. Photomicrographs should provide details of staining 

technique and a scale bar. Patients shown in photographs should have their identity concealed or 

should have given their written consent to publication. Normally no more than six illustrations 

will be accepted without charge. 

TRADE NAMES 

Non-proprietary (generic) names of products should be used. If a brand name for a drug is used, 

the British or International non-proprietary (approved) name should be given. The source of any 

new or experimental preparation should also be given. 

REFERENCES 

The references should be numbered in the order in which they appear in the text. At the end of 

the article the full list of references should give the name and initials of all authors unless there 

are more than six, when only the first three should be given followed by et al. The authors' 

names should be followed by the title of the article, the title of the Journal abbreviated according 

to the style of Index Medicus, the year of publication, the volume number and the first and last 

page numbers. References to books should give the title of the book, which should be followed 

by the place of publication, the publisher, the year and the relevant pages.

EXAMPLES 

1. Madaio MP. Renal biopsy. Kidney Int 1990; 38: 529-543 

Books: 

2. Roberts NK. The cardiac conducting system and the His bundle electrogram. Appleton- 

Century-Crofts, New York, NY: 1981; 49-56 

Chapters: 

3. Rycroft RJG, Calnan CD. Facial rashes among visual display unit (VDU) operators. In: Pearce 

BG, ed. Health hazards of VDUs. Wiley, London, UK: 1984; 13-15 

References should be limited to those relating directly to the content of the paper and should not 

exceed 20. Case Reports should contain no more than 10 references, and letters to the editor no 

more than five. 

Note: It is the responsibility of the author to ensure the accuracy of the references in the 

submitted article. Downloading references direct from Medline is highly recommended. 

COPYRIGHT 

It is a condition of publication in the Journal that authors grant an exclusive licence to the 

Journal. 

EDITORIAL ENQUIRIES: 

A Basci 

Ege University Medical School Nephrology Dept. 

Bornova, Universite Caddesi 

IZMIR, 35100 

TURKEY 

Tel/Fax: + 90 232 388 9231 

E-mail: alibasci@med.ege.edu.tr 

Deputy editor: Clinical Nephrology Goce Spasovski 

Email: gspas@sonet.com.mk (for original articles - clinical research) 

Deputy editor: Basic research Adrian Covic 

Email: acovic@xnet.ro (for original articles - basic research) 

COLOUR ILLUSTRATIONS 

Colour illustrations are accepted, but the authors will be required to contribute to the cost of the 

reproduction. Colour figures will incur a printing charge of 50 euros. Illustrations for which 

colour is not essential can be reproduced as black and white images in the print journal. 

BANTAO Journal 

Published on behalf of 

Balkan Cities Association 

of Nephrology, Dialysis, 

Transplantation and Artificial Organs

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