Clinical Science (2011) 120, 169–178 (Printed in Great Britain) doi:10.1042/CS20100432

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Renin, (pro)renin and receptor: an update Genevieve NGUYEN Institut National de la Sant´e et de la Recherche M´edicale (INSERM) and Coll`ege de France ‘Early Development and Pathologies’ Center for Interdisciplinary Research in Biology and Experimental Medicine Unit, 11 place Marcelin Berthelot, 75005, Paris, France

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PRR [(pro)renin receptor] was named after its biological characteristics, namely the binding of renin and of its inactive precursor prorenin, that triggers intracellular signalling involving ERK (extracellular-signal-regulated kinase) 1/2. However the gene encoding for PRR is named ATP6ap2 (ATPase 6 accessory protein 2) because PRR was initially found as a truncated form co-purifying with V-ATPase (vacuolar H+ -ATPase). There are now data showing that this interaction is not only physical, but also functional in the kidney and the heart. However, the newest and most fascinating development of PRR is its involvement in both the canonical Wnt/β-catenin and non-canonical Wnt/PCP (planar cell polarity) pathways, which are essential for adult and embryonic stem cell biology, embryonic development and disease, including cancer. In the Wnt/β-catenin pathway, it has been shown that PRR acts as an adaptor between the Wnt receptor LRP5/6 (low-density lipoprotein receptor-related protein 5/6) and Fz (frizzled) and that the proton gradient generated by the V-ATPase in endosomes is necessary for LRP5/6 phosphorylation and β-catenin activation. In the Wnt/PCP pathway, PRR binds to Fz and controls its asymetrical subcellular distribution and therefore the polarization of the cells in a plane of a tissue. These essential cellular functions of PRR are independent of renin and open new avenues on the pathophysiological role of PRR. The present review will summarize our knowledge of (pro)renin-dependent functions of PRR and will discuss the newly recognized functions of PRR related to the V-ATPase and to Wnt signalling.

INTRODUCTION A receptor for renin and for the inactive proenzyme form of renin prorenin was cloned in 2002 and was called PRR [(pro)renin receptor] [1]. Rapidly, it became clear that PRR was the full-length form of a smaller protein described previously associated with the V-ATPase (vacuolar H+ -ATPase) [2,3] and this is why the PRR gene is named ATP6AP2 (ATPase 6 accessory protein

2)/PRR. In humans, there is a unique ATP6AP2/PRR gene on the X chromosome encoding a unique protein that undergoes intracellular processing, such that PRR exists in three different molecular forms: (i) a fulllength integral TM (transmembrane) protein, (ii) a soluble PRR (sPRR) found in plasma and urine, and (iii) a truncated form composed of the transmembrane and cytoplasmic domains [4,5]. Because PRR was identified as a component of the RAS (renin–angiotensin system) and

Key words: cardiovascular disease, (pro)renin receptor (PRP), renal pathology, Wnt/β-catenin signalling, Wnt/planar cell polarity signalling. Abbreviations: ACE, angiotensin-converting enzyme; AngII, angiotensin II; AT1 R, AngII type 1 receptor; ATP6ap2, ATPase 6 accessory protein 2; BP, blood pressure; CNS, central nervous system; COX2, cyclo-oxygenase 2; EC, extracellular; ERK, extracellular-signal-regulated kinase; Fz, frizzled; HRP, ‘handle region peptide’; IC, intracellular; LRP, low-density lipoprotein receptor-related protein; MAPK, mitogen-activated protein kinase; α-MHC, α-myosin heavy chain; NGF, nerve growth factor; PCP, planar cell polarity; PLZF, promyelocytic zinc finger transcription factor; PRR, (pro)renin receptor; SHR, spontaneously hypertensive rat; SON, supraoptic nucleus; sPRR, soluble PRR; TGF-β, transforming growth factor-β; RAS, renin–angiotensin system; TM, transmembrane; TNF-α, tumour necrosis factor-α; V-ATPase, vacuolar H+ -ATPase. Correspondence: Dr Genevieve Nguyen (email [email protected]).  C

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because of its potential pro-fibrotic effects, it has been primarily studied in cardiovascular and renal diseases. Study of its pathophysiological role has been hampered by the fact that total ablation of PRR was impossible, suggesting an essential role in cell biology, and recent data showing an unexpected involvement of PRR in VATPase function and in Wnt signalling pathways have indeed corroborated this hypothesis (discussed below). The present review will begin with a section on the characteristics of PRR and the experimental data in favour of a role of PRR in relation with (pro)renin in cardiovascular and renal diseases, followed by recent advances in my laboratory on the structure of PRR, and a final section describing the newly discovered functions of PRR related to V-ATPase and to Wnt signalling, which are totally independent of renin and prorenin.

PRR, (PRO)RENIN-DEPENDENT FUNCTIONS, AND CARDIOVASCULAR AND RENAL DISEASES PRR binds renin and prorenin [collectively named (pro)renin] with an affinity in the nanomolar range [1,6– 10], and their binding triggers a range of cellular events depending on the cell type. Commonly, PRR activation triggers the phosphorylation of the MAPKs (mitogenactivated protein kinases) ERK (extracellular-signalregulated kinase) 1/2, inducing the up-regulation of the pro-fibrotic genes such as TGF-β1 (transforming growth factor-β1), PAI-1 (plasminogen activator inhibitor-1), collagens and fibronectin [1,7,8,11,12]. In addition, PRR also up-regulates COX2 (cyclo-oxygenase 2) [13] and activates the p38 MAPK/Hsp27 (heat-shock protein 27) pathway [14], PI3K (phosphoinositide 3-kinase) p85 and PLZF (promyelocytic zinc finger transcription factor) that represses the expression of PRR itself [15,16]. Moreover, PRR-bound prorenin undergoes a conformational change that opens its pro-segment and uncovers the active site, so that, on a cell surface, prorenin becomes enzymatically active [1,10,17,18]. Because PRR was characterized as a new component of the RAS, the first studies aimed to address its pathophysiological role in hypertension and organ damage in situations known to be associated with RAS activation. In order to do so, as with other components of the RAS, several groups tried to generate PRR − / − mice, which represents the most powerful tool to study the role of a new receptor. Against all odds, PRR-knockout mice could not be generated and therefore research groups have turned to transgenic animals overexpressing PRR or have tried to design PRR antagonists. To date, evidence that PRR is linked to cardiovascular and renal diseases relies exclusively on studies in animals overexpressing PRR and on the use of a putative PRR blocker. Indeed, ubiquitous overexpression of human PRR in rats was  C

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associated with proteinuria, nephropathy and COX2 up-regulation in spite of normal renal levels of AngII (angiotensin II) and BP (blood pressure) [13,19], whereas overexpression of the human PRR gene in VSMCs (vascular smooth-muscle cells) induced high BP and increased heart rate but resulted in normal kidney function [20], showing that increased PRR synthesis could be linked in some way to altered cardiovascular and renal functions. In an attempt to identify a compound able to block renin and/or prorenin binding to PRR, Suzuki et al. [21] observed that an antibody against a sequence of the pro-segment of human prorenin (I11 PFLKRP15 ) was able to open the pro-fragment, yielding a ‘nonproteolytically’ activated prorenin, in a manner similar to PRR-binding-induced prorenin activation. They also tested the ability of a peptide mimicking this part of the pro-segment called the HRP (‘handle region peptide’) to block (pro)renin binding to PRR. HRP was infused in several experimental models and, in particular, in diabetic rodents that have increased renin and prorenin synthesis. HRP was shown to be able to prevent or even reverse diabetic nephropathy [22–25], and to block ocular inflammation in endotoxin-induced uveitis [26], ischaemia-induced retinal neovascularization [27] and diabetic retinopathy [28–30]. Moreover, HRP could decrease cardiac and renal fibrosis in stroke-prone SHRs (spontaneously hypertensive rats) [31–33] and reduce insulin resistance [34]. The great interest in PRR in diabetic nephropathy and retinopathy is explained by the fact that diabetic patients have low active renin levels, but very high prorenin levels, which are reported to be associated with and even to be predictive of the occurrence of microvascular complications as evidenced in microalbuminuria and retinopathy [35–38], but, in spite of the low renin levels, RAS blockers have a tissueprotective effect, suggesting local RAS activation. In addition, it has been shown that prorenin synthesis was locally enhanced in the collecting duct of diabetic rats [39], and many studies have also shown an up-regulation of PRR in the kidney of diabetic humans [40] and rats [41,42], so that the combination of increased prorenin and PRR may create a local environment favourable for PRR activation and for AngII generation. The increased PRR synthesis was attributed to the activation of AT1 R (AngII type 1 receptor) and NADPH oxidase activity, but also to high glucose by a mechanism involving NF-κB (nuclear factor κB) and AP1 (activator protein 1) [41,43]. In return, PRR activation enhances renal synthesis of the inflammatory molecules TNF-α (tumour necrosis factorα) and IL-1β (interleukin-1β), maintaining the vicious pro-inflammatory pro-fibrotic circle. Taken together, the hypothesis that the non-proteolytic activation of prorenin by PRR could be responsible for increased RAS activation in diabetes was tempting and blocking prorenin binding to PRR and prorenin enzymatic activation with HRP, claiming it to be a PRR blocker, was a logical move.

Renin, (pro)renin and receptor: an update

However, if the first results with HRP created a lot of excitation because they identified PRR as a new valuable therapeutical target in organ damage, alas HRP did not meet all of the expectations as a PRR blocker as it has since failed to show any effects in many other studies or even had adverse effects [30]. Furthermore, its ability to inhibit (pro)renin binding to PRR was even questioned in vitro, and the fact that HRP cannot block prorenin binding and ERK activation by native PRR expressed on the surface of a cell [10,12,44,45] must definitely rule out its name of ‘PRR blocker’. If the non-proteolytic activation of prorenin by PRR is important in diabetes, then increased prorenin levels in pregnancy [46] or in patients treated with an ACE (angiotensin-converting enzyme) inhibitor [47] should also be a real concern. To study whether increased prorenin could be responsible for fibrosis, two independent groups have generated transgenic rats [48] and mice [49] respectively with inducible or constitutive overexpression of prorenin. Their results clearly show that increased prorenin up to 200 times the normal concentrations and over a period of 6 months is not associated with any cardiac or kidney fibrosis, as assessed by histological examination and by PCR analysis for TGF-β and collagens. Yet, the animals had a severe hypertension attributed to the increased generation of AngII, since it was controlled by ACE treatment and because mice overexpressing active-sitemutated prorenin had BPs comparable with controls [48,49]. In summary, on the basis of the absence of an established PRR antagonist, the weak phenotype of animals overexpressing PRR and the absence of fibrosis in prorenin-overexpressing animals, we must admit that the evidence for a role of PRR in cardiovascular and renal diseases is not very strong at the moment and we are still waiting for tissue-specific knockout mice to establish the real pathophysiological role of PRR. Apart from these experimental models focusing on cardiovascular and renal diseases, data have been published suggesting a role for PRR in the central control of BP. Indeed, we have shown that PRR is expressed in nuclei important in the central control of BP, including the SON (supraoptic nucleus) [50], a finding confirmed by Shan et al. [51]. Moreover, these authors have shown that the level of PRR expression in the SON was higher in SHRs compared with Wistar–Kyoto control animals, and that neuronal cells of SHRs have a higher susceptibility to prorenin stimulation and displayed a 50 % higher ERK1/2 phosphorylation in response to prorenin. A down-regulation of PRR expression in the SON of SHRs by local injection of an adenovirus coding for PRR shRNA (small-hairpin RNA) reduced the agedependent increase in BP in SHRs [51]. Importantly, Hirose et al. [52] provided the first human data supporting the role of PRR in the control of BP by showing

that the polymorphism IVS5+169T in the PRR gene was associated with significantly higher systolic and diastolic BP in a cohort of Japanese males, a result recently confirmed by another study in Caucasians [53]. When aliskiren, a renin inhibitor, was made available in clinics, it became important to determine its effect on the binding and activation of PRR by (pro)renin. All of the studies concluded that renin and prorenin with the active site blocked by aliskiren still bound to PRR and activated intracellular signalling [12,54] and, conversely, aliskiren was able to inhibit PRR-bound renin and prorenin [10,55]. In other words, aliskiren has no effect on the binding and activation of PRR by (pro)renin. Nevertheless, hypertensive diabetic rats treated with aliskiren had a significant down-regulation in PRR expression in the kidney [56]. However, the mechanisms underlying this effect of aliskiren are still not clear. This could be due to the (pro)renin repression of PRR expression via the PLZF pathway or to the inhibition of AngII generation by aliskiren, as a study has shown that treatment of diabetic rats with valsartan was also able to reduce PRR expression [41]. It would be interesting to compare the effects of a renin inhibitor and an ARB (AT1 R blocker) on the expression of PRR in vivo.

THE MANY MOLECULAR FORMS OF PRR All of the results and conclusions of studies in cells in culture or in animals were based on PRR being considered exclusively as an integral membrane protein and acting as the receptor of renin and prorenin. However, recent data have now shown that these assumptions were wrong, because PRR exists in three different molecular forms that may have different functions and that PRR is also essential for a process completely independent of renin and prorenin, namely the Wnt signalling pathway (Figure 1).

The soluble form of PRR PRR is a small protein of an apparent molecular mass of 35–39 kDa organized into a large EC (extracellular) domain, a single TM domain and a short IC (intracellular) domain (Figure 2). The truncated form of PRR initially described to be associated with the V-ATPase in bovine chromaffin granules [57] is composed of the TM–IC domains and a short portion of the EC domain, cleaved at a close vicinity of a putative furin-cleavage site (RKTR). The existence of the truncated TM–IC form of PRR suggested that another truncated form composed of the EC must exist. By analysing the conditioned medium of cultured cells, we have demonstrated the existence of a low-molecular-mass 28 kDa PRR [4]. Sitedirected mutagenesis of the furin-cleavage site and the use of a furin-specific protease inhibitor abolished the  C

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Figure 1 Processing of PRR

(a) Furin cleavage in the trans -Golgi. (b) Vesicles containing the three molecular forms of PRR: (i) an integral membrane protein associated with the V-ATPase, (ii) a truncated form associated with the V-ATPase and (iii) the sPRR. (c) The PRR–V-ATPase–LRP–Fz–Wnt complex. C-ter, C-terminus; N-ter, N-terminus.

generation of the 28 kDa PRR by cells in culture, whereas inhibitors of metalloproteases, ADAM17 (a disintegrin and metalloproteinase 17) and TNF-α protease had no effect, confirming furin as the enzyme responsible for the generation of the 28 kDa PRR. As this molecular form of PRR was found in the conditioned medium, it was named sPRR. Most importantly, sPRR could be found in plasma, where it is able to bind renin [4], and also in urine [58]. The role of sPRR has not been elucidated yet and we  C

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do not know whether it can increase renin activity and activate prorenin as is the case for membrane PRR, or if sPRR behaves as a circulating antagonist of membrane PRR. An ELISA for sPRR will be available soon and this will allow us to measure sPRR concentrations in the plasma and urine of patients. The study of the variations in sPRR in different types of pathologies might help us to better understand the pathophysiology of PRR and, for example, it would be of utmost interest to see

Renin, (pro)renin and receptor: an update

Figure 2 Organization of the multi-complex PRR–V-ATPase–LRP–Fz on the plasma membrane (a), and a schematic structure of the three molecular forms of PRR (b)

C-ter, C-terminus; N-ter, N-terminus.

whether IVS5+169T in the PRR gene, associated with higher ambulatory BP, is also associated with increased levels of sPRR.

PRR associated with the V-ATPase A multi-species protein sequence comparison revealed homologues with the human PRR in most, if not all, species including rat and mouse, and also chicken, frog, zebrafish, mosquito and Drosophila, and species remotely related to humans such as Caenorhabditis elegans (WormBase gene ID WBGene00010993; www.wormbase.org) and the bacterium Ehrlichia chaffeensis [60], with the highest homology being in the TM–IC region [2,3]. The truncated TM–IC form of PRR was identified by chance when Ludwig et al. [57] were looking for proteins associated with the V-ATPase (Figure 1). V-ATPases are ATP-dependent proton pumps that acidify intracellular compartments and transport protons across plasma membranes in intercalated cells, osteoclasts, macrophages and tumour cells. The structure of the V-ATPase is well established: they are multi-subunit proteins composed of a V1 domain containing eight different subunits (A–H), which is responsible for ATP

hydrolysis, and a V0 domain composed of six different subunits (a, c, c , d, e and the accessory protein subnit Ac45 in mammals), which is responsible for proton translocation (Figure 2). Some subunits in the V1 and V0 domains may be present in multiple copies and most subunits have tissue-specific isoforms [61,62]. In mammals, there are two accessory subunits binding to the V0 sector that have been described to regulate V-ATPase activity: Ac45 encoded by the gene ATP6ap1 and the truncated form of PRR encoded by the gene APT6ap2. If Ac45 is a genuine accessory protein, because it is found as an integral component of the V0 sector, and therefore deserves to be named a V-ATPase subunit [63], PRR is mistakenly considered and named a V-ATPase subunit because it has never been found as an integral component of the V0 sector. However, Ac45 and PRR share some similarities. First, they are processed by furin. This furin processing appears to be a prerequisite for Ac45 function, as reduced proteolytic processing of Ac45 impairs acidic vesicle formation and is responsible for defective insulin secretion in pancreatic β-cells [64]. This could explain why the majority of PRR is also cleaved by furin [4] to generate the truncated TM–IC necessary for V-ATPase  C

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assembly. Secondly, ATP6ap1/Ac45 knockout in mice is embryonic-lethal [65] and PRR-knockout mice could not be generated. Thirdly, mutation of ATP6ap1/Ac45 and ATP6ap2/PRR in zebrafish gives a similar phenotype: oculocutaneous albinism, small head and eyes, CNS (central nervous system) necrosis and embryonic lethality [66,67]. There is only one known human mutation for the accessory proteins Ac45 and PRR, and it is in the ATP6ap2/ PRR gene encoding PRR and is responsible for X-linked mental retardation, epilepsy and ataxia without cardiovascular or renal dysfunction [68]. The silent mutation is in an exon splicing enhancer site, resulting in the inefficient inclusion of exon 4 in 50 % of the mRNA and hence the synthesis of full-length and of exon 4-deleted PRR. Mutant PRR behaves as a dominantnegative, impairing ERK1/2 activation induced by renin and inhibiting neuronal differentiation of PC12 cells stimulated by NGF (nerve growth factor) [50]. We attributed the X-linked mental retardation to impaired NGF-induced neuronal maturation, but in light of the recent data on the role of PRR in Wnt signalling and CNS patterning in Xenopus embryos [69], the possibility of a defective interaction of exon 4-deleted PRR with Fz (frizzled) and impaired Wnt signalling is highly conceivable. Consistent with an important role in the CNS, we have shown that PRR is highly expressed in neurons and on plasma membranes, where it binds renin and mediates ERK1/2 phosphorylation [50,70], but it is also present in synaptic vesicles, where V-ATPase is essential for neurotransmitter concentration and maturation, suggesting again a link between PRR and V-ATPase. A functional link between PRR and V-ATPase has been suggested for the first time in renal cells. The membrane V-ATPase found at the luminal side of intercalated cells of the late distal tubule and collecting duct is responsible for acid secretion into the urine. To no surprise, intercalated cells are also rich in PRR that co-localizes with the V-ATPase, and Advani et al. [71] have shown that blocking V-ATPase function with bafilomycin in MDCK (Madin–Darby canine kidney) cells of a collecting duct/distal lineage also inhibited PRR activation and ERK phosphorylation induced by (pro)renin, thus establishing a possible link between PRR and V-ATPase. Recently, Kinouchi et al. [72] have generated mice with specific ATP6ap2/PRR-knockout in cardiomyocytes by crossing ATP6ap2/PRR-floxed mice with mice expressing Cre recombinase under the control of the α-MHC (αmyosin heavy chain) promoter. The affected males ATP6ap2/PRRflox/Y ×α − MHC-Cre+/0 are born, but the mice die within 3 weeks from severe heart failure. The histological alterations included large fibrotic areas and degenerative cardiomyocytes. These cardiomyocytes had disorganized fibrillar structures, excessive vacuoles and were congested by autophagic vacuoles, a phenotype that  C

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could be reproduced by treating normal cardiomyocytes with bafilomycin. These authors also showed that deletion of ATP6ap2/PRRflox/Y ×α − MHC-Cre+/0 in fibroblasts isolated from ATP6ap2/PRRflox/Y mice resulted in the down-regulation of all subunits of the V0 sector, whereas the subunits of the V1 sector were unaffected [72]. These in vitro data confirm the importance of ATP6ap2/PRR in the assembly and function of the V-ATPase, as do the phenotype of PRR-knockout mice. Whether the phenotype of PRR-knockout mice is solely attributable to V-ATPase dysfunction is certainly a hasty conclusion, as it is well known that Wnt signalling is essential for cardiac development and differentiation [73] and that altering ATP6ap2/PRR must have affected Wnt signalling and function independent of V-ATPase.

PRR associated with LRP (low-density lipoprotein receptor-related protein)/Fz and involved in Wnt signalling As is so often the case, the most unexpected data on the function of PRR were reported by a group not working in the cardiovascular or renal field, but in embryonic development, who showed an absolute requirement for PRR in the Wnt/β-catenin signalling pathway [69]. The Wnt proteins are growth factors and their signalling is fundamental for a normal patterned embryo. In the adult, Wnt signalling is involved in cell proliferation, migration and polarity, and abnormal Wnt signalling is known to promote human degenerative diseases including cancer. The Wnt proteins refer to a family of structurally related secreted, although highly hydrophobic, proteins. Wnt proteins act on target cells by binding to a complex formed of Fz, their primary receptor containing a seven-transmembrane region, and LRP, a single-pass transmembrane protein (Figure 2). The members of the Wnt family can activate different highly interconnected signalling pathways, and these signalling pathways are subdivided into the canonical Wnt/βcatenin-dependent pathway and several non-canonical Wnt signalling pathways not involving β-catenin. Among the non-canonical pathways, one is called the Wnt/PCP (planar cell polarity) signalling pathway with regard to a mechanism similar to the Drosophila PCP, a process that allows cell to polarize in the plane of a tissue and which is complementary to their apical–basal orientation. As the present review is not intended to cover Wnt signalling, we direct readers to some excellent reviews by specialists in the Wnt signalling field [73–77]. The first relationship between PRR and Wnt was published by Cruciat et al. [69], who were looking for proteins involved in the regulation of canonical Wnt/β-catenin signalling. The authors took a genomewide siRNA (small inhibitory RNA) screening approach in HEK-293T cells (human embryonic kidney cells

Renin, (pro)renin and receptor: an update

expressing the large T-antigen of simian virus 40) and used a Wnt-responsive luciferase reporter assay as a readout. PRR was then identified as an adaptor between the Wnt receptor complex and the V-ATPase, and the results indicated that endocytosis of LRP5/6 was necessary for its phosphorylation and downstream β-catenin activation [69]. This function of PRR requires the EC and TM, but not the cytoplasmic domain, and the authors showed that the EC domain of PRR bound to LRP5/6 and Fz (Figure 1). When PRR antisense morpholinos were injected into Xenopus embryos, the tadpoles had smaller heads and a defect in melanocyte and eye pigmentation, a phenotype very similar to that of ATP6ap2/PRR-mutant zebrafish embryos [66,67]. Because the expression of PRR was prominent in the CNS of Xenopus, the authors performed a detailed study of the CNS in Xenopus embryos injected with PRR antisense morpholinos and found a defect in the anterio–posterior neural patterning. Shortly after, Hermie et al. [78] and Buechling et al. [79] simultaneously confirmed that the Drosophila homologue of PRR binds to the Fz receptor in not only in the Wnt/β-catenin pathway, but also in the Wnt/PCP pathway, thus regulating two major processes: planar polarity in Drosophila and convergent-extension movements in Xenopus gastrulae. The implication of these observations is huge, because PCP is an absolute requirement for most if not all cell types to function properly. In adult tissue, the typical illustrations of the role of PCP in vertebrates are feathers in birds and hair in mammals. During embryonic development, PCP signalling is necessary for the convergent-extension of mesenchymal cells during gastrulation and neural tube cells during neurulation, a process where cells move towards the midline and intercalate, allowing extension of the body axis. In the renal field and during kidney development, PCP signalling is tightly linked to ciliopathies and therefore to polycystic kidney disease [80]. It is important to stress that the function of PRR in the Wnt/β-catenin and Wnt/PCP signalling pathways is totally independent of renin, as renin is not expressed in Xenopus embryos at the early phase of development where the study was performed, and Drosophila and Hydra do not have renin. These studies showing the involvement of PRR in Wnt signalling during embryonic development are extremely exciting and they probably explain why no PRRknockout mice have ever been generated. However, we should also keep in mind that Wnt signalling is important in adult stem cell biology, especially in the field of degenerative diseases and cancer. We have reported a high level of expression of PRR in human glioblastoma and have shown that inhibition of renin activity in glioblastoma cells reduced their proliferation rate and induced apoptosis [81]. Now, with this new development in PRR biochemistry, it would be interesting to correlate

PRR expression and the degree of Wnt/β-catenin signalling activity in these glioblastoma cells.

CONCLUSIONS AND PERSPECTIVES In 2006, at the Gordon Conference on angiotensins, 4 years after its first description, we predicted that PRR was a multi-functional protein on the basis of the observation that its wide tissue distribution in adult and enormous expression in embryonic mouse did not fit with a function restricted to the RAS. There is now accumulating evidence that this is indeed true and that PRR is involved in V-ATPase function and in the Wnt receptor complex signalling. The newly discovered function of PRR in the Wnt signalling pathways adds a new dimension to the study of the pathological relevance of PRR and is taking us from organ damage and fibrosis in hypertension and diabetes to the biology of embryonic stem cell and development, and of adult stem cell and tissue repair and cancer [82]. Although the new aspects of PRR as an accessory subunit of the V-ATPase and a cofactor of the Wnt receptor complex are really exciting, we must keep in mind that the human data available so far establish a link between ATP6ap2/PRR and higher BP in the case of the IVS5+169T polymorphism and to altered neuronal function due to the silent 321C > T mutation. Two major advances have been made that will help us understand the functions of PRR: (i) the generation of ATP6ap2/PRR-floxed mice that allow the embryonic lethality due to total ablation during development to be overcome to enable the study of PRR in adult mice in disease models, and (ii) an ELISA to measure sPRR. We predicted in a recent review [5] that tissue-specific knockout of PRR would give surprising results and we were indeed right. The phenotype of animals lacking PRR in cardiomyocytes is totally unexpected, with massive cardiac defects attributed to impaired V-ATPase assembly and defective autophagy, once again totally ignoring the complexity of PRR functions and its role of PRR in Wnt signalling and the importance of Wnt signalling in cardiac development. In 2010, recombinant full-length PRR or sPRR still remains to be produced, which would allow the crystal structure to be solved and be of use in studying the function of the soluble receptor. Nevertheless, we believe that animal studies, if carefully examined and interpreted in light of the many functions of PRR, will be able to give us clues as to the role of PRR in pathology. Then, the fact that PRR is a receptor with a soluble counterpart might make it a valuable therapeutic target not only in cardiovascular and renal disease, but also in degenerative diseases.

ACKNOWLEDGEMENT I thank France Maloumian for her expert assistance in preparing the illustrations.  C

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30 Wilkinson-Berka, J. L., Heine, R., Tan, G., Cooper, M. E., Hatzopoulos, K. M., Fletcher, E. L., Binger, K. J., Campbell, D. J. J. and Miller, A. G. (2010) RILLKKMPSV influences the vasculature, neurons and glia, and (pro)renin receptor expression in the retina. Hypertension 55, 1454–1460 31 Susic, D., Zhou, X., Frohlich, E. D., Lippton, H. and Knight, M. (2008) 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 32 Ichihara, A., Kaneshiro, Y., Takemitsu, T., Sakoda, M., Suzuki, F., Nakagawa, T., Nishiyama, A., Inagami, T. and Hayashi, M. (2006) Nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension. Hypertension 47, 894–900 33 Ichihara, A., Kaneshiro, Y., Takemitsu, T., Sakoda, M., Nakagawa, T., Nishiyama, A., Kawachi, H., Shimizu, F. and Inagami, T. (2006) Contribution of nonproteolytically activated prorenin in glomeruli to hypertensive renal damage. J. Am. Soc. Nephrol. 17, 2495–2503 34 Nagai, Y., Ichihara, A., Nakano, D., Kimura, S., Pelisch, N., Fujisawa, Y., Hitomi, H., Hosomi, N., Kiyomoto, H., Kohno, M. et al. (2009) Possible contribution of the non-proteolytic activation of prorenin to the development of insulin resistance in fructose-fed rats. Exp. Physiol. 94, 1016–1023 35 Luetscher, J. A., Kraemer, F. B., Wilson, D. M., Schwartz, H. C. and Bryer-Ash, M. (1985) Increased plasma inactive renin in diabetes mellitusA marker of microvascular complications. N. Engl. J. Med. 312, 1412–1417 36 Wilson, D. M. and Luetscher, J. A. (1990) Plasma prorenin activity and complications in children with insulindependent diabetes mellitus. N. Engl. J. Med. 323, 1101–1106 37 Deinum, J., Rønn, B., Mathiesen, E., Derkx, F. H., Hop, W. C. and Schalekamp, M. A. (1999) Increase in serum prorenin precedes onset of microalbuminuria in patients with insulin-dependent diabetes mellitus. Diabetologia 42, 1006–1010 38 Chiarelli, F., Pomilio, M., De Luca, F. A., Vecchiet, J. and Verrotti, A. (2001) Plasma prorenin levels may predict persistent microalbuminuria in children with diabetes. Pediatr. Nephrol. 16, 116–120 39 Kang, J. J., Toma, I., Sipos, A., Meer, E. J., Vargas, S. L. and Peti-Peterdi, J. (2008) The collecting duct is the major source of prorenin in diabetes. Hypertension 51, 1597–1604 40 Takahashi, K., Yamamoto, H., Hirose, T., Hiraishi, K., Shoji, I., Shibasaki, A., Kato, I., Kaneko, K., Sasano, H., Satoh, F. and Totsune, K. (2010) Expression of (pro)renin receptor in human kidneys with end-stage kidney disease due to diabetic nephropathy. Peptides 231, 1405–1408 41 Siragy, H. M. and Huang, J. (2008) Renal (pro)renin receptor upregulation in diabetic rats through enhanced angiotensin AT1 receptor and NADPH oxidase activity. Exp. Physiol. 93, 709–714 42 Matavelli, L. C., Huang, J. and Siragy, H. M. (2010) (pro)renin receptor contributes to diabetic nephropathy by enhancing renal inflammation. Clin. Exp. Pharmacol. Physiol. 37, 277–282 43 Huang, J. and Siragy, H. M. (2009) Glucose promotes the production of interleukin-1β and cyclooxygenase-2 in mesangial cells via enhanced (pro)renin receptor expression. Endocrinology 150, 5557–5565 ¨ 44 Muller, D. N., Klanke, B., Feldt, S., Cordasic, N., Hartner, A., Schmieder, R. E., Luft, F. C. and Hilgers, K. F. (2008) (pro)renin receptor peptide inhibitor ‘handle-region’ peptide does not affect hypertensive nephrosclerosis in Goldblatt rats. Hypertension 51, 676–681 45 Krebs, C., Weber, M., Steinmetz, O., Meyer-Schwesinger, C., Stahl, R., Danser, A. H., Garrelds, I., van Goor, H., ¨ Nguyen, G., Muller, D. and Wenzel, U. (2008) Effect of (pro)renin receptor inhibition by a decoy peptide on renal damage in the clipped kidney of Goldblatt rats. Kidney Int. 74, 823–824

46 Derkx, F. H. M., Alberda, A. T., de Jong, F. H., Zeilmaker, F. H., Makovitz, J. W. and Schalekamp, M. A. D. H. (1987) Source of plasma prorenin in early and late pregnancy: observations in a patient with primary ovarian failure. J. Clin. Endocrinol. Metab. 65, 349–354 47 Azizi, M., Webb, R., Nussberger, J. and Hollenberg, N. K. (2006) Renin inhibition with aliskiren: where are we now, and where are we going? J. Hypertens. 24, 243–256 48 Peters, B., Grisk, O., Becher, B., Wanka, H., Kuttler, B., Ludemann, J., Lorenz, G., Rettig, R., Mullins, J. J. and Peters, J. (2008) Dose-dependent titration of prorenin and blood pressure in Cyp1a1ren-2 transgenic rats: absence of prorenin-induced glomerulosclerosis. J. Hypertens 26, 102–109 49 Mercure, C., Prescott, G., Lacombe, M. J., Silversides, D. W. and Reudelhuber, T. L. (2009) Chronic increases in circulating prorenin are not associated with renal or cardiac pathologies. Hypertension 53, 1062–1069 50 Contrepas, A., Walker, J., Koulakoff, A., Franek, K. J., Qadri, F., Giaume, C., Corvol, P., Schwartz, C. E. and Nguyen, G. (2009) A role of the (pro)renin receptor in neuronal cell differentiation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 297, R250–R257 51 Shan, Z., Shi, P., Cuadra, A. E., Dong, Y., Lamont, G. J., Li, Q., Seth, D. M, Navar, L. G., Katovich, M. J., Sumners, C. and Raizada, M. K. (2010) Involvement of the brain (pro)renin receptor in cardiovascular homeostasis. Circ. Res. 107, 934–938 52 Hirose, T., Hashimoto, M., Totsune, K., Metoki, H., Asayama, K., Kikuya, M., Sugimoto, K., Katsuya, T., Ohkubo, T., Hashimoto, J. et al. (2009) Association of (pro)renin receptor gene polymorphism with blood pressure in Japanese men: the Ohasama study. Am. J. Hypertens. 22, 294–299 53 Ott, C., Schneider, M. P, Hilgers, K. F. and Schmieder, R. E. (2010) Association of (pro)renin receptor gene polymorphism with blood pressure in men. J. Hypertens. 28 (esuppl. A), e346 54 Sakoda, M., Ichihara, A., Kurauchi-Mito, A., Narita, T., Kinouchi, K., Murohashi-Bokuda, K., Saleem, M. A., Nishiyama, A., Suzuki, F. and Itoh, H. (2010) Aliskiren inhibits intracellular angiotensin II levels without affecting (pro)renin receptor signals in human podocytes. Am. J. Hypertens. 23, 575–580 55 Biswas, K. B., Nabi, A. N., Arai, Y., Nakagawa, T., Ebihara, A., Ichihara, A., Watanabe, T., Inagami, T. and Suzuki, F. (2010) Aliskiren binds to renin and prorenin bound to (pro)renin receptor in vitro. Hypertens. Res. 33, 1053–1059 56 Feldman, D. L., Jin, L., Xuan, H., Contrepas, A., Zhou, Y., Webb, R. L., Mueller, D. N., Feldt, S., Cumin, F., Maniara, W. et al. (2008) Effects of aliskiren on blood pressure, albuminuria, and (pro)renin receptor expression in diabetic TG(mRen-2)27 rats. Hypertension 52, 130–136 57 Ludwig, J., Kerscher, S., Brandt, U., Pfeiffer, K., Getlawi, F., Apps, D. K. and Sch¨agger, H. (1998) 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 58 Ichihara, A., Sakoda, M., Kurauchi-Mito, A., Narita, T., Kinouchi, K., Murohashi- Bokuda, K. and Itoh, H. (2010) Possible roles of human (pro)renin receptor suggested by recent clinical and experimental findings. Hypertens Res. 33, 177–180 59 Reference deleted 60 Wakeel, A., Kuriakose, J. A. and McBride, J. W. (2009) An Ehrlichia chaffeensis tandem repeat protein interacts with multiple host targets involved in cell signaling, transcriptional regulation, and vesicle trafficking. Infect. Immun. 77, 1734–1745 61 Nishi, T. and Forgac, M. (2002) The vacuolar [H+ ]-ATPases: nature’s most versatile proton pumps. Nat. Rev. Mol. Cell Biol. 3, 94–103 62 Cipriano, D. J., Wang, Y., Bond, S., Hinton, A., Jefferies, K. C., Qi, J. and Forgac, M. (2008) Structure and regulation of the vacuolar ATPases. Biochim. Biophys. Acta 1777, 599–604  C

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72 Kinouchi, K., Ichihara, A., Sano, M., Sun-Wada, G. H., Wada, Y., Kurauchi-Mito, A., Bokuda, K. and Narita, T. Oshima, Y., Sakoda, M. et al. (2010) The (pro)renin receptor/ATP6AP2 is essential for vacuolar H+ -ATPase assembly in murine cardiomyocytes. Circ. Res. 107, 30–34 ¨ M. (2010) The multiple phases and 73 Gessert, S. and Kuhl, faces of Wnt signaling during cardiac differentiation and development. Circ. Res. 107, 186–199 74 Logan, C. Y. and Nusse, R. (2004) The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20, 781–810 75 van Amerongen, R. and Nusse, R. (2009) Towards an integrated view of Wnt signaling in development. Development 136, 3205–3214 76 van Amerongen, R., Mikels, A. and Nusse, R. (2008) Alternative Wnt signaling is initiated by distinct receptors. Sci. Signaling 1, re9 ¨ M. (2010) An updated overview on 77 Rao, T. P. and Kuhl, Wnt signaling pathways: a prelude for more. Circ. Res. 106, 1798–1806 ¨ 78 Hermle, T., Saltukoglu, D., Grunewald, J., Walz, G. and Simons, M. (2010) Regulation of Frizzled-dependent planar polarity signaling by a V-ATPase subunit. Curr. Biol. 20, 1269–1276 79 Buechling, T., Bartscherer, K., Ohkawara, B., Chaudhary, V., Spirohn, K., Niehrs, C. and Boutros, M. (2010) Wnt/Frizzled signaling requires dPRR, the Drosophila homolog of the prorenin receptor. Curr. Biol. 20, 1263–1268 80 Wu, J. and Mlodzik, M. (2009) A quest for the mechanism regulating global planar cell polarity of tissues. Trends Cell Biol. 19, 295–305 81 Juillerat-Jeanneret, L., Celerier, J., Chapuis Bernasconi, C., Nguyen, G., Wostl, W., Maerki, H. P., Janzer, R. C., Corvol, P. and Gasc, J. M. (2004) Renin and angiotensinogen expression and functions in growth and apoptosis of human glioblastoma. Br. J. Cancer 90, 1059–1068 82 Lucero, O. M., Dawson, D. W., Moon, R. T. and Chien, A. J. (2010) A re-evaluation of the ‘oncogenic’ nature of Wnt/β-catenin signaling in melanoma and other cancers. Curr. Oncol. Rep. 12, 314–318

Received 18 August 2010/7 October 2010; accepted 25 October 2010 Published on the Internet 19 November 2010, doi:10.1042/CS20100432

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