The (pro)renin receptor and the mystic HRP -Is there a role in cardiovascular disease?

Ulrike Maschke¹, Dominik N. Muller¹

1Max-Delbruck-Center for Molecular Medicine and Experimental and Clinical Research Center, Berlin-Buch, Germany

TABLE OF CONTENTS

1. Abstract

2. Introduction

2. Introduction

3. Is the function of the (P)RR not only related to the RAS, but also to the vacuolar ATPase?

4. Can we block renin or prorenin binding to the (P)RR with existing compounds?

5. Should we block the (P)RR?

6. Perspectives

7. References

1. ABSTRACT

In 2002, Nguyen et al. cloned the (pro)renin receptor ((P)RR). Two years later, Suzuki, Ichihara and colleagues provided a concept to inhibit the (P)RR through HRP. This decapeptide mimics a sequence of the prorenin prosegment and functions thereby as a decoy peptide. They showed that HRP prevented diabetic nephropathy in rodents and ameliorated renal and cardiac damage in spontaneously hypertensive rats. We tested HRP and the human renin inhibitor aliskiren in transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR). Only aliskiren, but not HRP, was able to ameliorate target organ damage in this model. HRP had also no effect on target organ damage in renovasular hypertensive rats. In vitro studies showed that HRP did not inhibit (pro)renin binding and signaling. More confusing was the fact that HRP bound to cells lacking (P)RR on their surface. We believe that HRP does not act as a competitive antagonist for the (P)RR and promotes its action via an alternative mechanism. Elucidating this mechanism could offer further opportunities, in terms of (pro)renin research.

2. INTRODUCTION

Renin the rate-limiting enzyme in the renin-angiotensin system (RAS) cleaves angiotensinogen into angiotensin I. For decades, researchers hunted for a receptor, which binds renin and its inactive precursor, prorenin (1). In 2002, Nguyen et al. cloned a receptor termed (Pro)Renin Receptor (P)RR, which is able to bind both renin and prorenin. The (P)RR is a 35 kDa single-transmembrane receptor that is able to activate intracellular signaling, and surprisingly, (P)RR-bound prorenin becomes enzymatically active as a result of a conformational change without cleavage of the prosegment (2). The degree of homology between human, rat and, mice (P)RR is about 95% for the nucleotide sequence and over 80% at the amino-acid level, indicating an extremely conserved protein. Unlike other components of the RAS, the (P)RR gene is highly conserved among species, and (P)RR orthologues are found in species as far from mammals as C.elegans and Drosophila, which express some of the RAS components, but not for hemodynamic functions (3,4). The highest homology is in the trans-membrane and cytoplasmic regions pointing to an important function of this fragment of (P)RR (5,6). Therefore, the (P)RR may be involved in other action beside the modulation of the RAS. Furthermore, (P)RR also exists as a soluble receptor s(P)RR. The truncated 28 kDa form occurs after furin cleavage and provides the molecular basis for these additional functions (7).

3. IS THE FUNCTION OF THE (P)RR NOT ONLY RELATED TO THE RAS, BUT ALSO TO THE VACUOLAR ATPASE?

Up today the generation of (P)RR null mice was not successful, possibly because (P)RR-/- embryonic stem (ES) cells do not form chimeras after blastocyst injection (Michael Bader, personal communication). (P)RR deletion in C. elegans and zebrafish yield embryos that die before the end of embryogenesis, thus supporting an essential cellular function for (P)RR (8). Interestingly, a truncated part of the (P)RR composed of the trans-membrane and cytoplasmic domains of (P)RR co-purifies with the vacuolar H⁺-ATPase (V-ATPase) (9). It is tempting to speculate that an interplay between this of part of the (P)RR and v-ATPase is essential for various biological processes. The v-ATPase plays an essential role in controlling cellular and intracellular vesicle pH (10). The gene coding this (P)RR is called ATP6ap2 (ATPase associated protein 2). Zebrafish embryo mutants for v-ATPase subunits, and for (P)RR/ATP6AP2, display similar phenotypes, suggesting a possible link between the two proteins (9). More evidence for a functional link between the two proteins derives from a recent study by Advani et al. who showed that (P)RR co-localized with the v-ATPase in the apical villi of intercalated cells of the distal nephron and that inhibition of v-ATPase with bafilomycin impaired ERK activation induced by (pro)rennin (11).

4. CAN WE BLOCK RENIN OR PRORENIN BINDING TO (P)RR WITH EXISTING COMPOUNDS?

Suzuki, Ichihara, and colleagues identified a sequence within the prosegment and proposed that a decapeptide that they called HRP blocks binding of prorenin to the (P)RR (12, 13). The whole concept is based on competitive binding of HRP to the (P)RR. Per definition, a competitive antagonist is a receptor antagonist that binds to a receptor but does not activate the receptor. The antagonist will compete with available agonist for receptor binding sites on the same receptor. Given in a sufficient high dosage, the antagonist will displace the agonist from the binding sites, resulting in a lower frequency of receptor activation.

Here comes the first controversy. Ichihara et al. used in their in vivo studies HRP doses ranging from 7 �g/kg/d up to 1 mg/kg/d (13-16). They used the same doses in rats and mice despite an about 10-fold difference in blood volume. Beside the question why different doses were used in different models, the major issue arises if at all these doses of a peptide are sufficient for competitively blocking a receptor. It is well known that peptides are rapidly degraded by peptidases in the blood.

Nevertheless, several groups including ours have tried to repeat the protective actions of HRP. Recently, Reudelhuber et al. failed to demonstrate an effect of HRP in prorenin-transgenic mice (17). Wenzel et al. tested the concept of (P)RR blockade with HRP in ischemic clipped kidneys. However, in their study HRP also did not ameliorate renal injury (18). We tested HRP and the human renin inhibitor aliskiren in transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) (19). In dTGR, the renin gene is under the control of its own promoter. DTGR rats exhibit high human renin, 10-fold higher human prorenin levels, and 3-5 fold increased circulating and local angiotensin II levels. To our surprise only aliskiren, but not HRP, was able to ameliorate target organ damage in this model. In dTGR rats, we took care to apply both human and rat HRP sequences since presumably in the dTGR, the endogenous rat (P)RR is operative in these animals. We wondered whether the lack of HRP potency might have been caused by species difference between the human ligands (human renin and prorenin) and the rat (P)RR. Therefore, we tested HRP in renovascular hypertensive rats, which solely depend on rat components (20). Nevertheless, also in this model a rat HRP did not ameliorate target organ damage.

We cannot absolutely exclude the possibility that HRP is a blocker for prorenin in situations, where prorenin is elevated, but renin and angiotensin are suppressed. Nevertheless, the data of Ichihara et al. in SHR rats argue against this hypothesis. They showed that HRP treatment reduced local cardiac Ang II levels leading to reduced cardiac hypertrophy and fibrosis (21). Current notions suggest that cardiac Ang II depends to a large extent on renal renin that is released in its active form and is taken up by the heart and thus initiates local Ang II generation (22, 23). Susic et al. also treated SHR rats with HRP. They described an amelioration of left ventricular hypertrophy, an improved left ventricular function, cardiac collagen content and a decrease in serum creatinine levels under high salt condition. Surprisingly, under low salt HRP decreased left ventricular mass only, while all other parameters were not improved (24).

Could (pro)renin be more relevant in the brain or could its interaction with the (P)RR be the key to increased renin activity in the CNS? The Raizada's group showed that renin stimulation via the (P)RR involved MAP kinase phosphorylation and was able to inhibit neuronal activity in vitro. The effect could be reversed with HRP. However, this is rather unexpected since activation of the RAS is usually associated with neuronal excitation, and it has potentially interesting consequences (25).

Taken together the conflicting data, we believe that HRP efficacy in vivo depends on an undefined mechanism, but not on competitive antagonism for the (P)RR.

In vitro results have even increased the confusion. Several groups using different methods such as inhibition of binding of radiolabeled prorenin and ERK 1/2 activation in the presence of HRP, found no inhibitory effects, even at HRP concentrations as high as 10 �M (19, 26, 27). Others have reported that HRP can inhibit not only prorenin binding but also renin binding to recombinant PRR and have also reported that HRP stimulates ERK1/2 by itself, which pinpoints to a partial agonist concept (28, 29). Altogether, it is difficult to understand how HRP could totally inhibit ERK1/2 phosphorylation in some in vivo studies when it could itself induce ERK 1/2 phosphorylation. Another unsolved conceptual issue is why HRP would not be effective in high renin models when it could inhibit renin binding in vitro.

At the moment, there remains skepticism concerning the mode of action of HRP, and the discrepancy between the in vitro and the in vivo data cannot be explained at this time.

5. SHOULD WE BLOCK THE (P)RR?

In the light of (P)RR gene deletion experiments in mice and zebrafish leading to an embryonic phenotype, it is quite questionable whether a complete (P)RR blockade might be favorable. From the cardiovascular point of view, one could imagine that blockade of the (P)RR could ameliorate target organ damage. Given that the (P)RR might also be involved in basic cell biological processes, the risk for side-effects seems to be rather high. The only known mutation in the (P)RR gene in humans leads (P)RR protein lacking amino acid 100 until 132. Patients with this the (P)RR mutation develop epilepsia with mental retardation without obvious cardiovascular problems (30).

6. PERSPECTIVES

The (P)RR is more complex than expected in several ways. It is a multifunctional protein existing in different molecular forms, and even its cellular localization is intriguing. Currently, we still know very little about the structure of this protein because recombinant PRR is very difficult to generate in native form and about its other cell functions beyond serving as a (pro)renin receptor. It may well be that the soluble 28 kDa (P)RR has an own function, while the transmembrane cytoplasmic part of the (P)RR has a completely different one rather linked to v-ATPase function. Therefore, studies with tissue-specific (P)RR knock-out mice are desperately needed to elucidate the role of the (P)RR in development, physiology and pathophysiology.

7. REFERENCES

1. Sealey JE, Glorioso N, Itskovitz J, Atlas SA, Pitarresi TM, Preibisz JJ, Troffa C & Laragh JH. Ovarian prorenin. Clin Exp Hypertens A9: 1435-1454, 1987.
doi:10.3109/10641968709158994

2. Nguyen G, Delarue F, Burcklé C, Bouzhir L, Giller T & Sraer J-D Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J Clin Invest 109: 1417-1427, 2002.
PMid:12045255    PMCid:150992

3. L'Huillier N, Sharp MGF, Dunbar DR & Mullins JJ. On the relationship between the renin receptor and the vacuolar proton ATPase membrane sector associated protein (M8-9). The Local Cardiac Renin Angiotensin-Aldosterone System. Editors Edward D. Frolich and Richard N. Re. Springer. Chapter 3, pp 17-34

4. Bader M. The second life of the (pro)renin receptor. J Renin Angiotensin Aldosterone Syst 8: 205-208, 2007.
doi:10.3317/jraas.2007.031
PMid:18205100

5. Burckle C & Bader M. Prorenin and its ancient receptor. Hypertension 48: 549-551, 2006.
doi:10.1161/01.HYP.0000241132.48495.df
PMid:16940209

6. Wakeel A, Kuriakose JA, McBride JW. An Erhrlichia chaffeensis tandem repeat protein interacts with multiple host targets involved in cell signaling, transcriptiona regulation and vesicle trafficking. Infection and Immunity 77; 1734-1745, 2009
doi:10.1128/IAI.00027-09
PMid:19273555    PMCid:2681728

7. Cousin C, Bracquart D, Contrepas A, Corvol P, Muller L & Nguyen G. Soluble form of the (Pro)renin receptor generated by intracellular cleavage by furin is secreted in plasma. Hypertension 53: 1077-1082, 2009.
doi:10.1161/HYPERTENSIONAHA.108.127258
PMid:19380613

8. Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S & Hopkins N. Identification of 315 genes essential for early zebrafish development. Proc Natl Acad Sci USA 101: 12792-12797, 2004.
doi:10.1073/pnas.0403929101
PMid:15256591    PMCid:516474

9. Ludwig J, Kerscher S, Brandt U, Pfeiffer K, Getlawi F, Apps DK & Schägger H. Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules. J Biol Chem 273: 10939-10947, 1998.
doi:10.1074/jbc.273.18.10939
PMid:9556572

10. Nishi T & Forgac M. The vacuolar (H+)-ATPases--nature's most versatile proton pumps. Nat Rev Mol Cell Biol 3: 94-103, 2002.
doi:10.1038/nrm729
PMid:11836511

11. Advani A, Kelly DJ, Cox AJ, White KE, Advani SL, Thai K, Conelly KA, Yuen D, Trogadis J, Herzenberg AM, Kuliszewski MA, Leong-Pio H & Gilbert RE. The (pro)renin receptor:site-specific and functional linkage to the vacuolar H+-ATPase in the kidney. Hypertension 2009 54:261-269.
doi:10.1161/HYPERTENSIONAHA.109.128645
PMid:19546380

12. Suzuki F, Hayakawa M, Nakagawa T, Nasir UM, Ebihara A, Iwasawa A, Ishida Y, Nakamura Y, Murakami K. Human prorenin has "gate and handle" regions for its non-proteolytic activation. J Biol Chem 278: 22217-22222, 2003
doi:10.1074/jbc.M302579200
PMid:12684512

13. Ichihara A, Hayashi M, Kaneshiro Y, Suzuki F, Nakagawa T, Tada Y, Koura Y, Nishiyama A, Okada H, Uddin MN, Nabi AH, Ishida Y, Inagami T & Saruta T. Inhibition of diabetic nephropathy by a decoy peptide corresponding to the "handle" region for nonproteolytic activation of prorenin. J Clin Invest 114: 1128-1135, 2004.
PMid:15489960    PMCid:522242

14. Ichihara A, Suzuki F, Nakagawa T, Kaneshiro Y, Takemitsu T, Sakoda M, Nabi AH, Nishiyama A, Sugaya T, Hayashi M & Inagami T. Prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin II type 1a receptor-deficient mice. J Am Soc Nephrol 17: 1950-1961, 2006.
doi:10.1681/ASN.2006010029
PMid:16738017

15. Ichihara A, Kaneshiro Y, Takemitsu T, Sakoda M, Suzuki F, Nakagawa T, Nishiyama A, Inagami T & Hayashi M. Nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension. Hypertension 47:894-900, 2006.
doi:10.1161/01.HYP.0000215838.48170.0b
PMid:16585419

16. Satofuka S, Ichihara A, Nagai N, Noda K, Ozawa Y, Fukamizu A, Tsubota K, Itoh H, Oike Y, Ishida S. (Pro)renin receptor-mediated signal transduction and tissue renin-angiotensin system contribute to diabetes-induced retinal inflammation. Diabetes 58: 1625-33, 2009.
doi:10.2337/db08-0254
PMid:19389828

17. Mercure C, Prescott G, Lacombe MJ, Silversides DW & Reudelhuber TL. Chronic Increases in Circulating Prorenin Are not Associated With Renal or Cardiac Pathologies. Hypertension 53: 1062-1069, 2009.
doi:10.1161/HYPERTENSIONAHA.108.115444
PMid:19364992

18. Krebs C, Weber M, Steinmetz O, Meyer-Schwesinger C, Stahl R, Danser AH, Garrelds I, van Goor H, Nguyen G, Müller DN & Wenzel U. Effect of (pro)renin receptor inhibition by a decoy peptide on renal damage in the clipped kidney of Goldblatt rats. Kidney Int. 74: 823-4, 2008.
doi:10.1038/ki.2008.315
PMid:18756297

19. Feldt S, Maschke U, Dechend R, Luft FC & Müller DN. Role of the (pro)renin receptor in a transgenic model of high human renin hypertension J Am Soc Nephrol.19: 743-8, 2008.
doi:10.1681/ASN.2007091030
PMid:18235083    PMCid:2390971

20. Müller DN, Klanke B, Feldt S, Cordasic N, Hartner A, Schmieder RE, Luft FC & Hilgers KF. (Pro)renin receptor peptide inhibitor "handle-region" peptide does not affect hypertensive nephrosclerosis in Goldblatt rats. Hypertension 51: 676-681, 2008.
doi:10.1161/HYPERTENSIONAHA.107.101493
PMid:18212268

21. Ichihara, A, Kaneshiro, Y, Takemitsu, T, Sakoda, M, Suzuki, F, Nakagawa, T, Nishiyama, A, Inagami, T & Hayashi, M: Nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension. Hypertension, 47:894-900, 2006.
doi:10.1161/01.HYP.0000215838.48170.0b
PMid:16585419

22. Muller, DN, Fischli, W, Clozel, JP, Hilgers, KF, Bohlender, J, Menard, J, Busjahn, A, Ganten, D & Luft, FC: Local angiotensin II generation in the rat heart: role of renin uptake. Circ Res, 82:13-20, 1998.
PMid:9440700

23. van Kesteren CA, Danser AH, Derkx FH, Dekkers DH, Lamers JM, Saxena PR & Schalekamp MA: Mannose 6-phosphate receptor-mediated internalization and activation of prorenin by cardiac cells. Hypertension, 30:1389-96, 1997.
PMid:9403558

24. Susic D, Zhou X, Frohlich ED, Lippton H & Knight M. Cardiovascular effects of prorenin blockade in genetically spontaneously hypertensive rats on normal and high-salt diet. Am J Physiol Heart Circ Physiol 295: H1117-H1121, 2008.
doi:10.1152/ajpheart.00055.2008
PMid:18621861

25. Shan Z, Cuadra AE, Sumners C & Raizada MK : Characterization of a functional (pro)renin receptor in rat brain neurons Exp Physiol, 93.5:701-708, 2008.
doi:10.1113/expphysiol.2008.041988
PMid:18326551

26. Batenburg WW, Krop M, Garrelds IM, de Vries R, de Bruin RJA, Burcklé C, Müller DN, Bader M, Nguyen G, Danser AHJ. Prorenin is the endogenous agonist of the (pro)renin receptor. Binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor. J Hypertens 25: 2441-2453, 2007.
doi:10.1097/HJH.0b013e3282f05bae
PMid:17984666

27. Feldt S, Batenburg WW, Mazak I, Maschke U, Wellner M, Kvakan H, Dechend R, Fiebeler A, Burckle C, Contrepas A, Danser AHJ, Bader M, Nguyen G, Luft FC & Muller DN. Prorenin and Renin-Induced Extracellular Signal-RegulatedKinase 1/2 Activation in Monocytes Is Not Blocked by Aliskiren or the Handle-Region Peptide. Hypertension 51: 682-688, 2008.
doi:10.1161/HYPERTENSIONAHA.107.101444
PMid:18212269

28. Nabi AHMN et al. 'Decoy peptide' region (RIFLKRMPSI) of prorenin prosegment plays a crucial role in prorenin binding to the (pro)renin receptor. Int J Mol Med 24: 83-89, 2009.
PMid:19513539

29. Ichihara A, Kaneshiro Y & Suzuki F. Prorenin receptor blockers: effects on cardiovascular complications of diabetes and hypertension. Expert Opin Investig Drugs 15:1137-1139, 2006.
doi:10.1517/13543784.15.10.1137
PMid:16989590

30. Ramser J, Abidi FE, Burckle CA, Lenski C, Toriello H, Wen G, Lubs HA, Engert S, Stevenson RE, Meindl A, Schwartz CE & Nguyen G. A unique exonic splice enhancer mutation in a family with X-linked mental retardation and epilepsy points to a novel role of the renin receptor. Hum Mol Genet 14: 1019-1027, 2005.
doi:10.1093/hmg/ddi094
PMid:15746149

Key Words : (Pro)renin receptor, HRP, cardiovascular disease, v-ATPase, Review