The Toxicologist - Society of Toxicology

sodium citrate. Citrates (0.2 mmol/kg, saline for controls) were given IV at 6, 12,
24, 36, 48, and 60 h. Urines and plasmas were analyzed for markers of kidney injury
and levels of aluminum, citrate, oxalate and calcium. Blood urea nitrogen
(BUN) levels in EG + aluminum citrate rats were significantly lower than those in
EG + sodium citrate rats, but not different from either EG or control rats. Citrate
was decreased in all groups treated with EG compared to the control group, due to
the EG-induced metabolic acidosis which promotes citrate reabsorption in the
proximal tubules. Urine samples were centrifuged to separate soluble urinary components
from insoluble moieties like COM. Urinary aluminum was primarily
found in the supernatant and not in the pellet. In contrast, calcium levels were
highest in the pellets of rats treated with EG and treatment with aluminum citrate
increased calcium excretion in the pellet. Similarly, treatment with EG + aluminum
citrate appeared to increase urinary oxalate excretion compared to EG and EG +
sodium citrate rats. Taken together, these results suggest that aluminum citrate
could decrease COM-induced renal injury by enhancing the excretion of both calcium
and oxalate thus decreasing their retention in the kidney.
759 SUB-CHRONIC ETHANOL EXPOSURE INDUCES
ACUTE RENAL INJURY AND FIBROSIS IN RATS.
C. Latchoumycandane 1 , J. Liu 1 , L. E. Nagy 2 , A. E. Feldstein 1 and T. M.
McIntyre 1 . 1 Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH
and 2 Pathobiology and Gastroenterology, Lerner Research Institute, Cleveland Clinic,
Cleveland, OH.
Ethanol exposure induces hepato-renal injury in human and rodent models, and recently
we have reported that sub-chronic ethanol exposure increases circulating oxidized
phospholipids in rats as well as in alcoholic steatohepatitis patients.
However, the biological role of circulating oxidized phospholipids in extra-hepatic
tissues remains unclear. Interestingly, exogenously administered fluorescent Platelet
activating factor (PAF, a bioactive phospholipid) is predominantly eliminated by
kidney, and some oxidized phospholipids have PAF-receptor agonist activity. Based
on this, we hypothesized that ethanol induces acute renal injury. In this study we
find by LC/MS/MS analysis that rats exposed to a Lieber-DeCarli ethanol diet for
4 weeks significantly increased circulating PAF, peroxidized phophatidylcholine
(HpODE-PC), and the pro-apoptotic oxidatively truncated phospholipid azelaoyl-
PC derived from HpODE-PC. The expression of inflammatory cytokine Tumor
necrosis factor-α and apoptotic active-caspase-3 as well as the number of TUNEL
positive cells (apoptotic cells) increased significantly in kidney of ethanol-fed rats as
compared with pair-fed. Most strikingly, the renal fibrosis markers, Transforming
Growth Factor β-1 and α-smooth muscle actin are increased in ethanol-fed rats,
along with collagen IV. Masson trichrome staining of ethanol-fed rat kidney also
confirms the presence of collagen fibers in inter tubular space. Kidney injury molecule-1
(KIM-1), a marker for proximal tubular injury has been reported to increase
during acute renal injury and we find higher expression of KIM-1 in the proximal
tubules of ethanol fed-rats kidneys. The increase in circulating oxidized phospholipids
in rats is concordant with all renal injury markers. These results clearly suggest
that circulating bioactive oxidized phospholipids correlate with acute renal injury,
and development of renal fibrosis. This is the first demonstration of
ethanol-induced renal fibrosis.
760 BODY ADIPOSITY AND DIETARY FATTY ACIDS
INDUCE CYTOKINE EXPRESSION AND ACTIVATION
OF TUMOR PROMOTING PATHWAYS IN THE RAT
KIDNEY.
A. Bort 1, 2 , G. Ananthakrishnan 1 , D. Perez-Tilve 1 , D. R. Dietrich 2 , M. H.
Tschöp 1 , P. T. Pfluger 1 and K. Stemmer 1, 2 . 1 Department of Internal Medicine,
University of Cincinnati, Cincinnati, OH and 2 Human and Environmental
Toxicology, University of Konstanz, Konstanz, Germany.
Obesity is a major risk factor for numerous co-morbidities, including renal cancer.
However, the mechanisms by which obesity increases renal cancer risk are still unclear.
Here, we aim to compare the impacts of body adiposity vs. increased dietary
fatty acids on renal inflammatory and carcinogenic pathways in rats.
To delineate renal consequences of body adiposity and dietary fatty acids, we selected
two different phenotypes from high fat diet fed rats: high susceptibility to
diet induced obesity (DIOsens) and partial resistance to diet induced obesity
(DIOres) with 56% and 18% overweight compared to a lean chow fed group, respectively.
Circulating cytokines, renal cytokine expression and overall pathology
were investigated in vivo. Activation of corresponding downstream pathways like
tumor promoting mTOR/S6 kinase pathway, were analyzed in vivo and in fatty
acid-exposed NRK-52E rat kidney cells.
Similar circulating levels of interleukin 1β and monocyte chemo-attractant protein
1 (MCP1) in DIOres, DIOsens and chow rats did not suggest systemic inflammation.
However, a local renal inflammatory reaction, paralleled by an increased ex-
164 SOT 2011 ANNUAL MEETING
pression of interleukin 1β and TNFα was detected in the renal cortex of DIOsens
rats, and to lesser extent DIOres rats. Using immunohistochemistry, we detected
active mTOR signaling in cortex areas with increased monocyte infiltration and regenerative
cell proliferation, which was more pronounced in DIOsens than in
DIOres rats. mTOR activation was verified in fatty acid-exposed NRK-52E cells.
In summary, our data suggests that the degree of adiposity in diet-induced obesity,
and to lesser extent the excess of dietary fatty acids, are major determinants for a
pro-inflammatory and pro-carcinogenic condition in rat kidneys, which might
provide an ideal microenvironment for the development of obesity-induced renal
cancer.
761 TRANSPORT OF MERCURIC SPECIES IN RABBIT
RENAL BRUSH-BORDER MEMBRANE VESICLES.
C. Bridges, L. Joshee and R. K. Zalups. Division of Basic Medical Sciences, Mercer
University School of Medicine, Macon, GA.
Cysteine- (Cys) and homocysteine- (Hcy) S-conjugates of inorganic mercury (Hg),
have been shown to be taken up at the luminal membrane of renal proximal tubular
cells. Although one mechanism for this uptake (i.e., amino acid transporter, system
b 0,+ ) has been identified, it is likely that other carriers are also involved in this
process. Therefore, the purpose of the current study was not only to characterize the
uptake of Cys- and Hcy-S-conjugates of Hg at the brush-border membrane of
proximal tubular cells, but also to identify other possible mechanisms that may be
involved in the uptake of these conjugates. Brush-border membrane vesicles were
isolated from the cortex and outer stripe of the outer medulla from rabbit kidneys
as described previously. LLC-PK 1 cells were also used as a control model system.
Analyses of saturation kinetics for arginine and phenylalanine indicate that the isolated
vesicles are viable models for studies of proximal tubular transport processes.
The uptake of each mercuric conjugate was found to be time-dependent and saturable.
Addition of Cys or Hcy significantly reduced the uptake of Cys- and Hcy-
S-conjugates of Hg, respectively. Analyses of substrate specificity using LLC-PK 1
cells showed that various amino acids were able to block the uptake of each conjugate.
We conclude that Cys- and Hcy-S-conjugates of Hg are taken up at the brushborder
membrane of proximal tubular cells by multiple amino acid transporters, including
system b 0,+ .
762 KIDNEY PROXIMAL TUBULE-ON-A-CHIP FOR DRUG
TRANSPORTER STUDIES AND NEPHROTOXICITY
ASSESSMENT.
K. Jang 1, 2 , D. Huh 1, 3 , G. A. Hamilton 1 , A. Bahinski 1 , K. Suh 2 and D. E.
Ingber 1, 3, 4 . 1 Wyss Institute for Biologically Inspired Engineering at Harvard
University, Boston, MA, 2 Interdisciplinary Program in Nano-Science and Technology,
Seoul National University, Seoul, Republic of Korea, 3 Vascular Biology Program,
Departments of Pathology and Surgery, Children’s Hospital, Boston, MA and 4 School
of Engineering and Applied Sciences, Harvard University, Cambridge, MA.
There is a great need for more predictive in vitro human kidney models for investigating
absorption, distribution, metabolism, excretion, and toxicological properties
of new chemical entities during the drug development process. Here we describe a
biomimetic microsystem that reconstitutes critical functional aspects of renal proximal
tubule of the human kidney. In this system, kidney tubular epithelial cells are
cultured on a porous flexible matrix-coated membrane within a microfluidic system
and exposed to cyclic mechanical strain and fluid dynamic forces that mimic the in
vivo microenvironment. Culture of primary human proximal tubule cells under a
fluid shear stress similar to that of normal human proximal tubule (~ 0.2 to 5
dyn/cm2) resulted in enhanced cell polarization, cytoskeletal reorganization, and
molecular transport in response to hormonal stimulation. Renal drug transporter
expression, specific markers of proximal tubule function, and effects of known
nephrotoxic compounds were also analyzed using this microdevice, which reproduces
complex physiological responses to hormonal stimulation, molecular transport,
and metabolic activities of the renal tubule. The results obtained in this investigation
suggest that this novel system may provide a useful and cost-effective tool
for studying biotransformation profiles, renal pharmacology, renal drug transport
and toxicity relevant to the human kidney, and hence help to facilitate the drug development
process.
763 ATTENUATION OF OCHRATOXIN A TOXICITY BY
INHIBITION OF ORGANIC ANION TRANSPORT.
A. Sarma and S. M. Ford. Toxicology Program, St. Johns University, Queens, NY.
Ochratoxin A (OTxA) is a nephrotoxic mycotoxin which is transported in the renal
proximal tubule by organic anion transporters (OATs). OAT1 and OAT3 on the
basolateral membrane contribute to OTxA secretion into tubular fluid. The re-