renin Receptor Blocker HRP Fails to Prevent (Pro)renin Signaling

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renin Receptor Blocker HRP Fails to Prevent (Pro)renin Signaling

BASIC RESEARCH www.jasn.org

The Putative (Pro)renin Receptor Blocker HRP Fails to

Prevent (Pro)renin Signaling

Sandra Feldt,* Ulrike Maschke, † Ralf Dechend,* Friedrich C. Luft,* † and

Dominik N. Muller* †

*Medical Faculty of the Charité, Experimental and Clinical Research Center, Franz Volhard Clinic, and HELIOS

Klinikum Berlin-Buch, † Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany

ABSTRACT

The prorenin/renin receptor is a recently discovered component of the renin-angiotensin system. The

effects of aliskiren, a direct inhibitor of human renin, were compared with the handle region decoy

peptide (HRP), which blocks the prorenin/renin receptor, in double-transgenic rats overexpressing the

human renin and angiotensinogen genes. After 7 wk, all aliskiren-treated rats were alive, whereas

mortality was 40% in vehicle-treated and 58% in HRP-treated rats. Aliskiren but not the HRP reduced BP

and normalized albuminuria, cystatin C, and neutrophil gelatinase-associated lipocalin, a marker of renal

tubular damage, to the levels of nontransgenic controls. In vitro, human renin and prorenin induced

extracellular signal–regulated kinase 1/2 phosphorylation, independent of angiotensin II (AngII), in

vascular smooth muscle cells. Preincubation with the HRP or aliskiren did not prevent renin- and

prorenin-induced extracellular signal–regulated kinase 1/2 phosphorylation, whereas the MAP kinase

kinase (MEK1/2) inhibitor PD98059 prevented both. In conclusion, renin inhibition but not treatment with

the HRP protects against AngII-induced renal damage in double-transgenic rats. In addition, the in vitro

data do not support the use of the HRP to block AngII-independent prorenin- or renin-mediated effects.

J Am Soc Nephrol 19: 743–748, 2008. doi: 10.1681/ASN.2007091030

A low molecular weight, nonpeptide, direct human

renin inhibitor (DRI), aliskiren, is now available to

treat hypertension. 1 The DRI demonstrated targetorgan

protection in a double-transgenic rat (dTGR)

model of high human renin hypertension. 2–4

Nguyen et al. 5 cloned a novel receptor (prorenin/

renin receptor [(P)RR]) that interacts with both renin

and prorenin. The (P)RR, more correctly

termed RR/ATP6AP2, is a single-transmembranedomain

protein of 350 amino acids with a large unglycosylated

and highly hydrophobic N-terminal

domain and a short cytoplasmic tail of approximately

20 amino acids. The (P)RR is highly conserved

across species. 6 Renin bound to the (P)RR

has a three- to five-fold increased catalytic activity. 5

Furthermore, when the (P)RR binds prorenin, the

latter molecule displays catalytic activity without a

proteolytic conversion to active renin (“nonproteolytic

activation”). 5,7 In contrast to proteolytic activation,

whereby the prosegment is cleaved off, non-

proteolytic activation is characterized by the

unfolding of the prosegment from the enzymatic

cleft, leading to a (P)RR-induced conformational

change in the prorenin molecule. In addition, renin

induces direct (P)RR signaling, leading to extracellular

signal–regulated kinase 1/2 (ERK1/2) mitogen-activated

protein kinase (MAPK) activation

that is independent of angiotensin II (AngII). 5,8,9

Ichihara et al. 10 and Suzuki et al. 11 introduced an

epitope of the prorenin prosegment that they termed

the handle region peptide (HRP). The HRP presum-

Received September 23, 2007. Accepted November 22, 2007.

Published online ahead of print. Publication date available at

www.jasn.org.

Correspondence: Dr. Dominik N. Muller, Experimental and Clinical

Research Center, Max-Delbruck-Center, Lindenberger Weg

80, 13125 Berlin, Germany. Phone: �49-30-9406-4581; Fax: �49-

30-9406-4220; E-mail: dominik.mueller@mdc-berlin.de

Copyright © 2008 by the American Society of Nephrology

J Am Soc Nephrol 19: 743–748, 2008 ISSN : 1046-6673/1904-743 743


BASIC RESEARCH www.jasn.org

ably blocks binding of prorenin to the (P)RR, thereby functioning

as a decoy peptide. Satofuka et al. 12 published a schematic of how

they envision the HRP to work. The model relies on the handle

region and would predict to be confined to prorenin. The group

demonstrated that long-term HRP treatment in diabetic mice and

rats improved nephropathy without affecting blood glucose levels.

10,13 The group also showed that HRP ameliorated renal and

cardiac damage in hypertensive spontaneously hypertensive rats

(SHR). 14,15 We investigated the role of DRI treatment and (P)RR

blockade in a transgenic rat model with human high renin and

prorenin hypertension. We also conducted studies in human coronary

vascular smooth muscle cells (VSMC) to test the hypothesis

that renin and prorenin induce signaling independent of AngII

and to evaluate DRI and HRP treatment on this signaling.

RESULTS

Aliskiren but not the (P)RR Blocker Ameliorates AngII-

Induced Renal Damage

At week 7, no aliskiren-treated dTGR died, whereas mortal-

ity was 58% in HRP-treated and 40% in vehicle-treated

dTGR (Figure 1A). All nontransgenic Sprague-Dawley (SD)

controls survived until the end of the study. Aliskiren normalized

systolic BP (106 � 3 for aliskiren and 111 � 1

mmHg for SD; Figure 1B). In contrast to aliskiren treatment,

HRP-treated rats developed high systolic BP similar

to vehicle-treated dTGR (196 � 9 for HRP versus 200 � 5

mmHg for dTGR; Figure 1B). Whereas aliskiren normalized

albuminuria (Figure 2A) and cystatin C, a marker for GFR

(Figure 2B), and renal neutrophil gelatinase-associated lipocalin

(NGAL) mRNA expression, a marker for tubular

damage (Figure 2C), to SD levels, HRP-treated dTGR were

not different from vehicle-treated dTGR. The 30-fold

higher dosage of rat and human HRP also showed no protective

effect (data not shown). These results demonstrate

that the DRI protected fully, whereas the putative competitive

(P)RR blocker HRP was ineffective in our dTGR

model. We also investigated whether renal (P)RR expression

was altered in our study. Vehicle-treated dTGR showed

a lower (P)RR expression compared with aliskiren-treated

dTGR and nontransgenic SD rats (Figure 3). The (P)RR

Figure 1. Effect of aliskiren and HRP on mortality (A) and systolic BP (B). Data are means � SEM. *P � 0.05 versus vehicle-treated dTGR

and HRP-treated dTGR.

Figure 2. Effect of aliskiren and HRP on albuminuria (A), cystatin C (B), and NGAL (C). All three markers demonstrated that aliskiren

but not HRP improved renal damage. Data are means � SEM. *P � 0.05 versus vehicle-treated dTGR and HRP-treated dTGR.

744 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 743–748, 2008


expression of dTGR and dTGR�HRP treatment were not

different. In any event, the (P)RR expression remained robust

in all groups throughout the study.

Figure 3. (P)RR mRNA expression in the kidney. Vehicle-treated

dTGR showed lower renal (P)RR expression compared with

aliskiren-treated dTGR and SD rats. HRP and SD rat (P)RR expressions

were not different. Data are means � SEM. *P � 0.05 versus

aliskiren-treated dTGR and SD rats. AU, arbitrary units.

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BASIC RESEARCH

Neither Aliskiren nor HRP Affects Renin and Prorenin-

Induced ERK1/2 Phosphorylation in Human Coronary

VSMC

To exclude any AngII-mediated signaling effect, we performed

all of our signaling experiments in the presence of the AngII

type 1 (AT1) blocker losartan and AT2 blocker PD123319.

Time-course analysis revealed that human recombinant renin

induced ERK1/2 phosphorylation in a time-dependent manner

starting at 5 min with a strong signal up to 15 min and

slightly elevated phosphorylation up to 45 min (Figure 4A).

Human recombinant prorenin also induced a long-lasting

ERK1/2 phosphorylation with a signal up to 45 min (Figure

4B). The final proof that renin- and prorenin-induced ERK1/2

phosphorylation is not AT1 A receptor dependent comes from

experiments in VSMC deficient for the receptor (Figure 4C),

where both stimuli were still active. We subsequently addressed

the question of whether aliskiren might also interfere

with renin- and prorenin-mediated (P)RR signaling. Our data

indicate that aliskiren affected neither renin- or nor prorenininduced

ERK1/2 phosphorylation (Figure 4D). We next investigated

the effect of the HRP. To our surprise, we found no

evidence that HRP blocked either renin- or prorenin-induced

ERK1/2 phosphorylation (Figure 4E). In contrast, the MAP

kinase kinase (MEK1/2) inhibitor PD98059, which blocks an

upstream kinase of ERK1/2, significantly reduced renin- and

prorenin-mediated ERK1/2 phosphorylation (Figure 4F).

Figure 4. (A and B) Time-course analysis of renin-induced (10 nM; A) and prorenin-induces (2 nM; B) ERK1/2 phosphorylation (p-ERK1/2) in

human VSMC (top) with unphosphorylated ERK as loading control (bottom). (C) Renin and prorenin (10 min) also induced p-ERK1/2 in AT1 A

receptor–deficient VSMC. (D through F) Effects of 10 �M aliskiren (D), 1 �M HRP (E), and MEK1/2 inhibitor (PD98059; 100 nM; F) on reninand

prorenin-induced (both 10 min) p-ERK1/2 in human VSMC (top) with unphosphorylated ERK as loading control (bottom). All experiments

were performed after 24-h serum starvation in the presence of losartan (Los) and the AT2 receptor blocker (PD123319).

J Am Soc Nephrol 19: 743–748, 2008 Renin and Prorenin Signaling 745


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DISCUSSION

We provide the first evidence that both prorenin and renin

rapidly induce cellular signals in human VSMC and mouse

AT1 A receptor–deficient mouse VSMC that lead to MAPK

ERK1/2 phosphorylation, completely independent of AngII.

Prorenin- and renin-induced ERK1/2 phosphorylation was inhibited

by MEK1/2 inhibition. Furthermore, we demonstrated

that aliskiren affected neither prorenin- nor renin-induced

ERK1/2 activation; therefore, aliskiren is a pure DRI that has

no (P)RR-blocking potency. Presumably, the enzymatic renin

cleft or any conformational changes resulting from occupancy

are not involved with any (P)RR interactions. Our results also

provided no evidence of a specific (P)RR blockade by the HRP,

despite its putative in vivo potency in various experimental

diabetic and nondiabetic nephropathy models. 10,12–16 HRP

also did not improve target-organ damage in dTGR. In contrast,

aliskiren completely prevented hypertension, renal damage,

and mortality.

In humans, aliskiren exhibits a adverse effect profile no

different from placebo, 17 while decreasing systolic and diastolic

BP alone or in combination with ramipril, valsartan,

amlodipine, and hydrochlorothiazide. 18–20 In rat models,

aliskiren potentiated the BP-lowering effects of angiotensin-converting

enzyme inhibitors and AT1 receptor blockers.

21 We recently demonstrated that aliskiren improved renal

and cardiac damage in a rat model of high human renin

hypertension. 2–4 Aliskiren offers a novel opportunity to reduce

plasma renin activity (PRA) that increases in response

to most other antihypertensive drugs. We speculated that

the binding of aliskiren in the active cleft of the renin molecule

could lead to a conformational change and thus prevent

the renin-aliskiren complex from binding to the

(P)RR; however, our in vitro signaling experiments suggest

that this is not the case.

Our results showed that prorenin, as well as renin, can induce

ERK1/2 activation in human primary VSMC. Huang et

al. 8,9 studied rodent mesangial cells and showed that renininduced

(P)RR stimulation activates ERK1/2 and stimulates

TGF-�, collagen, and fibronectin expression. Recently, Saris et

al. 22 reported that prorenin induced p38 MAPK but not

ERK1/2 signaling in rat neonatal cardiomyocytes. Taken together,

the results serve to underscore the emphasis that the

Ishihara group placed on a possible role of the (P)RR in targetorgan

damage. 10,12–15 The findings give prorenin a substantially

greater dimension than provided by early acid activation

and cryoactivation studies, which pointed to prorenin’s potential

but were not relevant in vivo because neither acid activation

nor cryoactivation can occur within the body. 23–26 Luetscher

and colleagues 24,27 drew attention to the fact that patients with

diabetes and target-organ damage had much higher prorenin

levels than healthy individuals. High prorenin levels and not

PRA were closely associated with the severity of diabetic complications

in these patients.

Ichihara and colleagues 10–12,28 offered an intriguing new

concept with their idea of (P)RR blockade. They proposed a

10–amino acid sequence as an effective (P)RR blocker that

was efficacious in streptozotocin-induced diabetes. 10,11

They showed that the nephroprotective effect of the HRP

was independent of AngII in AT1 A receptor knockout

mice. 13 HRP infusion also ameliorated target-organ damage

in SHR 14,15 ; however, Susic et al. 29 were unable to substantiate

these findings. Our dTGR have three- to five-fold increased

circulating and tissue AngII levels; have high circulating

human renin and prorenin; develop hypertension,

cardiac, and renal damage; and die at the age of 7 wk. 30 We

found no evidence that HRP had any salubrious effects in

this model. We took care to apply both human and rat HRP

sequences because presumably in the dTGR, the endogenous

rat (P)RR is operative. We used the same sequences

published by Ishihara and colleagues. 14–16 Ishihara et al. 15

reported that HRP-treated SHR had normal cardiac AngII

like WKY rats, whereas untreated SHR had increased levels.

Current notions suggest that cardiac AngII depends on renal

renin that is released in its active form. 31,32 We took the

precaution of studying a dosage of HRP that was 30-fold

higher than that used by Ishihara et al. but still could not

find any target-organ protection. To address the role of

HRP in a simpler experimental system, we performed in

vitro signal transduction experiments. These experiments

also did not support the notion that HRP blocked proreninand

renin-induced (P)RR signaling. Our in vitro and in vivo

results challenge the notion that HRP acts as a specific

(P)RR antagonist. Possibly, HRP efficacy in vivo depends on

an undefined mechanism but not on competitive antagonism

for the (P)RR.

Ichihara et al. 10,13 showed impressive findings in a diabetic

model of nephropathy that presumably had high prorenin

levels and low PRA. Our rat model is substantially

different. We characterized the model earlier and reported

AngII concentrations of 100 pg/ml, human PRA values of 60

ng AngI/ml per h, and human angiotensinogen values of

approximately 40 �g AngI/ml. 30 Kidney AngII concentrations

were almost 800 pg/g tissue, approximately four-fold

higher than in SD controls. The human prorenin-to-renin

ratio in the model is approximately 10:1. Recently, Schefe et

al. 33 described a novel signal transduction cascade involving

direct physical interaction of the (P)RR and the promyelocytic

zinc finger protein transcription factor. Their data

suggest the existence of a pathway involving ligand, (P)RR,

and promyelocytic zinc finger protein, which downregulates

(P)RR. We did not investigate this issue; however,

746 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 743–748, 2008


wedid find that untreated dTGR had lower (P)RR expression,

compared with aliskiren-treated dTGR or SD rats. The

expression differences were relatively modest. The (P)RR

should have had ample ligand in our study considering the

renin and prorenin concentrations in dTGR, compared

with SD rats. The issue requires further direct investigation.

The cloning of the (P)RR contributed a new dimension to

the renin-angiotensin system. The (P)RR clearly is important

in activating prorenin and enhancing renin in terms of angiotensinogen

cleavage. This plasma renin activity can be blocked

by DRI administration; however, the direct (P)RR signaling

resulting in ERK1/2 activation cannot be inhibited by DRI. The

possibility remains that the (P)RR contributes to target-organ

damage directly. Inhibitors of the (P)RR above and beyond

HRP are needed to test this hypothesis.

CONCISE METHODS

Experimental Design

We studied male dTGR (RCC Ltd., Basel, Switzerland) and agematched

nontransgenic SD rats (MDC, Berlin, Germany). 2,3,4,30,34

Local authorities approved the studies, which followed American

Physiologic Society guidelines for animal care. We studied vehicletreated

dTGR (n � 15), dTGR�aliskiren (3 mg/kg per d subcutaneously

by minipump infusion; n � 11), dTGR�HRP (n � 11), and SD

rats (n � 8). Because dTGR produce both rat and human renin, we

co-infused rat (NH2-RILLKKMPSV-COOH; Biosynthan, Berlin,

Germany) and human (NH2-RIFLKRMPSI-COOH; Biosynthan)

HRP (3.6 �g/kg per d each, subcutaneously by minipump infusion) to

block the (P)RR. The dosage was selected on the basis of previous

reports. 14–16 We also treated dTGR with a 30-fold higher human and

rat HRP dosage. Systolic BP was measured weekly by tail cuff. Twentyfour-hour

urine samples were collected in metabolic cages from

weeks 5 through 7 for the determination of rat albumin (CellTrend,

Lukenwalde, Germany). Rats were killed at age 7 wk by decapitation.

Serum was collected for cystatin C analysis. Cystatin C was measured

by routine clinical analysis. The kidneys were harvested for RNA isolation

and TaqMan reverse transcription–PCR as described previously.

2,3 We analyzed NGAL and rat (P)RR in the kidney. For quantification,

the target sequences were normalized in relation to the 36B4

product. Biotez (Berlin, Germany) synthesized the primers; all sequences

are available upon request. Data are presented as means �

SEM. Statistically significant differences in mean values were tested by

ANOVA followed by Scheffé test. Mortality was examined using a

Kaplan-Meier analysis. A value of P � 0.05 was considered statistically

significant.

Cell Culture and Western Blot

Human VSMC (Cambrex, Rockland, USA) were cultured in SmBM

medium (Lonza, Basel, Germany) with human fibroblast growth factor,

EGF (both Biochrome, Berlin, Germany), insulin (Sigma, Deisen-

hofen, Germany), 5% FBS, and Pen/Strep. All stimulation experiments

were performed under 24-h serum-free conditions. To exclude

AngII signaling effects, we also investigated AT1 A receptor–deficient

mouse VSMC. For stimulation experiments, cells were preincubated

with the AT1 receptor blocker losartan (10 �M) and AT2 receptor

blocker PD123319 (10 �M) for 30 min. Thereafter, cells were treated

with human recombinant renin (10 nM) or human recombinant prorenin

(2 nM) as indicated in the legend to Figure 4. In all inhibitor

protocols, cells were preincubated with the respective blocker for 30

min. The following blockers were used: Aliskiren (10 �M; Novartis,

Basel, Switzerland), the HRP (1 �M; NH2-RIFLKRMPSI-COOH;

Biosynthan), and the MEK1/2 inhibitor PD98059 (100 nM; Calbiochem,

Darmstadt, Germany). After stimulation, cells were lysed with

lysis buffer containing protease inhibitor Complete (Roche, Mannheim,

Germany) and phosphatase inhibitor cocktail (Sigma). For

Western blotting, we used ERK1/2 and phosphorylated ERK1/2 (both

Cell Signaling, Frankfurt, Germany), which were detected by standard

technique. At least three different cell stimulation experiments were

performed for each protocol.

ACKNOWLEDGMENTS

Grants-in-aid from the European Union (EuReGene), the Novartis

Foundation, and the Deutsche Forschungsgemeinschaft to D.N.M.

and F.C.L. supported the studies.

We thank Astrid Schiche, Jutta Meisel, and Gabriele N�diaye for

excellent technical assistance.

DISCLOSURES

F.C.L. and D.N.M. have served as advisors for Novartis and have lectured on

aliskiren. F.C.L. is a member of the renin academy.

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