The prorenin receptor in the cardiovascular system and beyond

Abstract

Since the prorenin receptor (PRR) was first reported, its physiological role in many cellular processes has been under intense scrutiny. The PRR is currently recognized as a multifunctional receptor with major roles as an accessory protein of the vacuolar-type H⁺-ATPase and as an intermediary in the Wnt signaling pathway. As a member of the renin-angiotensin system (RAS), the PRR has demonstrated to be of relevance in cardiovascular diseases (CVD) because it can activate prorenin and enhance the enzymatic activity of renin, thus promoting angiotensin II formation. Indeed, there is an association between PRR gene polymorphisms and CVD. Independent of angiotensin II, the activation of the PRR further stimulates intracellular signals linked to fibrosis. Studies using tissues and cells from a variety of organs and systems have supported its roles in multiple functions, although some remain controversial. In the brain, the PRR appears to be involved in the central regulation of blood pressure via activation of RAS- and non-RAS-dependent mechanisms. In the heart, the PRR promotes atrial structural and electrical remodeling. Nonetheless, animals overexpressing the PRR do not exhibit cardiac injury. In the kidney, the PRR is involved in the development of ureteric bud branching, urine concentration, and regulation of blood pressure. There is great interest in the PRR contributions to T cell homeostasis and to the development of visceral and brown fat. In this mini-review, we discuss the evidence for the pathophysiological roles of the PRR with emphasis in CVD.

THE PRORENIN RECEPTOR: A FUNCTIONAL COMPONENT OF THE RENIN-ANGIOTENSIN SYSTEM

The renin-angiotensin system (RAS) regulates blood pressure (BP) and Na⁺ and electrolyte balance (64). The long-term use of RAS blockers in antihypertensive therapies documents the clinical relevance of the RAS, and new discoveries of components and novel mechanisms keep this system a focus of active investigation. Renin, the rate-limiting step of the RAS, is synthesized as prorenin and activated in the granular cells of the juxtaglomerular apparatus (11). The discovery of a receptor for prorenin, the prorenin receptor (PRR) (52, 53), has clarified many issues but has also prompted questions about the roles of the PRR in cardiovascular diseases (CVD) (37).

The PRR (Atp6ap2) gene is located in chromosome X and encodes an extracellular domain (≈310 amino acids), a signal peptide at the NH₂-terminal region, and a transmembrane region followed by the COOH-terminus at the intracellular domain (90). The intracellular domain of PRR is the mediator for signal transduction, whereas the COOH-terminal fragment is involved in the assembly of the vacuolar H⁺-ATPase (v-ATPase) (53, 90). Seminal reports have demonstrated that transfection of PRR cDNA into non-PRR-expressing cells resulted in the expression of a 350-amino acid membrane protein able to bind renin and prorenin (53). Later evidence suggested that PRR binding to prorenin causes a conformational change of prorenin, enabling it to full enzymatic activity without proteolytical removal of the pro segment (90). The binding of renin to PRR promotes an increased catalytic efficiency of circulating and tissue angiotensinogen (AGT) conversion to angiotensin (ANG) I promoting ANG II formation if an adequate source of angiotensin converting enzyme (ACE) is available. Stimulation of PRR also activates intracellular pathways related to fibrosis, independent of ANG II. The binding of PRR to its natural agonists causes its phosphorylation on serine and tyrosine residues, thus activating MAPKs ERK1/2 (53). The PRR is part of v-ATPase responsible for intracellular and extracellular pH regulation (35), acting as an accessory protein v-ATPase H⁺-transporting lysosomal accessory protein 2 giving the name Atp6ap2 to the protein. The implications of the PRR in cardiac pathology, activation of the RAS in the brain, and regulation of BP and Na⁺ homeostasis in the kidney emphasize the importance of the interactions among renin, prorenin, and the PRR in CVD (23, 30, 49). Efforts directed toward understanding how the PRR is regulated in pathological conditions are the current focus of attention.

THE PRR AND ITS POTENTIAL ROLE IN THE BRAIN

The brain lacks the machinery necessary to cleave prorenin to renin (27); therefore, the synthesis of renin in the brain remains controversial. Li et al. (38–40) suggested that the PRR may have important roles in the activation of prorenin in the brain. The PRR transcript and protein have been described in cardiovascular regulatory nuclei in the brain such as the subfornical organ (SFO), paraventricular nucleus of the hypothalamus, rostral ventral lateral medulla, nucleus tractus solitarius, and area postrema. The PRR has been detected in neurons, astrocytes, and microglial cells (38–40, 86). In microglial cells, its activation promotes the release of proinflammatory factors (71). The importance of brain PRR in the central control of BP was evidenced by knockdown of the PRR in the SFO of human renin and AGT transgenic hypertensive mice, leading to decreased BP, cardiac, and vasomotor sympathetic tone (39). Li et al. (40) further demonstrated that chronic intracerebral infusion of the PRR peptide antagonist PRO20 attenuated the development of DOCA-salt induced hypertension and decreased brain ANG II formation. Another study (69) revealed that the PRR mediates antihypertensive effects in the, nucleus tractus solitarius in spontaneously hypertensive rats. Furthermore, prorenin treatment increases firing activity of magnocellular neurosecretory cells and paraventricular nucleus hypothalamic neurons (57). Despite strong evidence demonstrating expression of the PRR in the brain, the presence of renin in the brain is still controversial (49). Additionally, brain prorenin levels are the lowest among other tissues, suggesting that its effect in the activation of the PRR, if any, may be explained by plasma prorenin (15). The body of evidence suggests that brain PRR may play a role in the regulation of BP, but the intrinsic mechanisms remain debatable.

THE PRR IN THE HEART AND KIDNEY DURING PATHOPHYSIOLOGICAL STATES

Activation of the PRR stimulates intracellular pathways related to cardiac and kidney tissue damage (31, 55). Mahmud et al. (44) quantified the expression of the PRR in several animal models of heart failure. In postmyocardial infarcted hearts, they found increased PRR mRNA levels. Moreover, they observed significant increases in PRR levels in hearts of patients with dilated cardiomyopathy (44). The role of the PRR in congestive heart failure is unclear; however, it may be related to ANG II-independent effects. Lian et al. (42) reported that heart-specific overexpression of the PRR causes atrial fibrillation in mice associated with the activation of ERK1/2. This remains controversial. Rosendahl et al. (66) demonstrated that overexpression of the PRR in mice does not cause hypertension, renal fibrosis, or cardiac damage.

In the kidney, the PRR is expressed in the macula densa, mesangial cells, podocytes, proximal tubule, distal convoluted tubule, interstitial cells, and intercalated cells of the collecting duct (2, 21, 22). During kidney development, PRR expression is observed during gestation and then declines after postnatal development (73, 74). While the kidney is developing, cap mesenchyme signals induce elongation of ureteric bud (UB), which promotes consecutive branching (72, 74). The PRR in the UB is important for nephrogenesis (73, 74). In mice with PRR deletion in Six2⁺ progenitors (Six2^PRR−/−), mutant kidneys lacked a defined nephrogenic zone and showed abnormalities in the medulla and decreased nephron number (75). Additionally, lack of the PRR in nephron progenitor cells resulted in podocyte abnormalities and proteinuria (72, 74). The exact mechanism by which PRR intervenes in nephron progenitors during kidney development has not been fully explained, but Song et al. (72) hypothesized that PRR may control nephrogenesis through canonical Wnt/β-catenin signaling. It is also likely that the conditional deletion of PRR from the collecting duct cells causes functional alterations of H⁺-ATPase, resulting in intracellular pH imbalance (56). These alterations may produce cytotoxicity in collecting duct progenitor cells and disturbances in kidney development (35). In addition, the PRR has been involved in antidiuretic actions by regulating aquaporin (AQP)2 expression and water homeostasis in the collecting duct (82). Lu et al. (42a) demonstrated that recombinant soluble PRR (sPRR) in the nanomolar range increased AQP2 protein in renal medullary collecting duct cells via a sequential activation of the frizzled 8/β-catenin pathway and activation of cAMP-PKA.

During physiological intrarenal RAS activation, as occurs during low-salt diet, expression of the PRR is augmented in tubular epithelial cells (24) via stimulation of the cGMP/PKC pathway (28). Indeed, it has been shown that the PRR is regulated in kidney inner medullary collecting duct (IMCD) cells by glycogen synthase kinase-3β (GSK-3β)-nuclear factor of activated T cells 5 (NFAT5)-sirtuin-1 (SIRT-1) signaling pathway (62). Shao et al. (70) showed that male Sprague-Dawley rats subjected to a low-salt diet exhibited high intrarenal ANG II content and augmented collagen deposition, without increases in other major kidney injury markers. Conceivably, profibrotic factors are evoked by the PRR because of local activation of the PRR (24). Although there is consensus that a moderate reduction in salt consumption helps to decrease BP in both hypertensive and nonhypertensive subjects (13), a chronic low-salt diet may promote renal fibrosis via enhanced PRR activation. Additionally, water deprivation induces antidiuresis mediated by sequential activation of prostaglandin E (EP) receptor type 4 and the PRR through the regulation of AQP2 (82).

The PRR is augmented in experimental models of ANG II-dependent hypertension, including the ANG II-infused rat and mouse, the two-kidney one-clip Goldblatt rat, and the Ren2 gene overexpression transgenic rat (Ren2-TGR) (21, 36, 59, 60). The mechanisms by which ANG II upregulates the PRR have been previously described (22, 62, 63, 81). ANG II upregulates PRR expression in rat IMCD cells independent of osmolality (22, 24). In addition, PGE₂ mediates the ANG II-dependent regulation of PRR via EP₄ receptors (81). We showed that, during early stages of chronic ANG II infusions in rats, there is upregulation of the PRR with augmentation of cyclooxygenase (COX)-2 and PGE₂ in the renal inner medulla (20). Furthermore, PGE₂ treatment of collecting duct (M-1) cells induces a biphasic stimulation of renin-dependent PRR activation via EP₁ and EP₄ receptors. These findings support the notion that, in the distal nephron, the PRR exerts a positive feedback stimulation of COX-2/PGE₂ to buffer the development of hypertension during ANG II-dependent hypertension (20, 89).

Functional studies performed in mice with PRR deficiency either along the nephron or in the collecting duct have demonstrated the relevance of this receptor in the development of hypertension. Ramkumar et al. (63) showed that mice with inducible nephron-wide PRR deletion exhibited attenuated hypertension and Na⁺ retention responses to chronic ANG II infusion. We further showed that mice with conditional PRR-specific deletion in the collecting duct exhibited an attenuated BP response to chronic ANG II infusion along with alterations in renal function (58). Both studies independently demonstrated that PRR in the collecting duct facilitates the activation of prorenin locally produced to increase intratubular ANG II content and stimulates epithelial Na⁺ channel-mediated Na⁺ reabsorption. Overexpression of the PRR leads to kidney injury (34). PRR blockade with the 20-amino acid PRR antagonist PRO20 (41) decreases kidney inflammation and injury during chronic ANG II-dependent hypertension (80) and slows down mesangial cell proliferation and the progression of diabetic nephropathy (31). The use of the PRR blocker called handle region peptide (HRP) to target cardiovascular and renal diseases remains controversial (36, 47). The HRP is a 10-amino acid sequence (76) that competitively inhibits the binding of prorenin to PRR (48, 78). Using streptozotocin-induced diabetes, Ichihara et al. (29) demonstrated that treatment with the HRP decreased ANG I and ANG II in the kidney and completely suppressed diabetic nephropathy, even in the presence of hyperglycemia. Later on, Muller et al. (47) challenged previous findings, demonstrating that the HRP did not improve BP, cardiac hypertrophy, or renal damage in rats with renovascular hypertension. Moreover, it was shown that concomitant treatment with HRP and aliskiren in spontaneously hypertensive rats did not improve the effects of renin inhibition on BP. Indeed, it counteracted the beneficial effects of aliskiren in the kidney, increasing plasminogen activator-inhibitor 1, COX-2, and cardiac collagen, thus suggesting that HRP might act as a PRR agonist (77). Furthermore, in diabetic hypertensive rats, renin inhibition improved vascular dysfunction, but HRP did not (6). The same observations were described in spontaneously hypertensive rats (79). Taken together, further evidence is needed to demonstrate the beneficial effects of HRP alone or accompanied by renin inhibition.

THE sPRR

Circulating levels of prorenin and renin may give insights about possible interactions with the sPRR (14). Plasma sPRR levels correlate with renal function in essential hypertension (46), preeclampsia (50, 84), and chronic kidney disease (25); however, the mechanisms involved remain unclear. The binding affinity of the PRR for prorenin occurs at nanomolar concentrations but in vivo occurs in the picomolar range (5, 15, 52). Regardless, great interest has been given to the presence of the sPRR in the plasma because diabetic patients display low to normal levels of renin but increased levels of prorenin, which are associated with microvascular complications (17, 18, 32). The urine of type 1 diabetic patients also contains high levels of renin and the sPRR (3). We demonstrated that the sPRR in urine of ANG II-dependent hypertensive rats functionally activates prorenin secreted by the collecting ducts (21). Clinically, this is important in light of recent studies demonstrating that serum levels of the sPRR have a positive relationship with the extent of tubulointerstitial fibrosis (25) and correlate with the stage of chronic kidney disease (54). These data further support the hypothesis that increased levels of the sPRR contribute to the activation of prorenin in the extracellular space. Nonetheless, further studies are necessary to examine the role of the sPRR as a potential biomarker of renal damage and RAS activation.

THE PRR IN ADIPOSE TISSUE

Adipose tissue synthesizes all of the components of the RAS, including the ANG II type 1 receptor (AT₁R), ACE, renin, and the PRR (10). The PRR is produced in stromal adipose tissue and is likely regulated posttranscriptionally because PRR protein levels drop during differentiation but mRNA levels remain constant (1). Renin binding to the PRR in human adipose cells augments renin enzymatic efficiency, thus contributing to the local generation of ANG II (1). Furthermore, the administration of renin to human cultured 3T3-L1 preadipocytes induced phosphorylation of ERK1/2 (1). Activation of ERK1/2 signaling regulates adipogenic transcription factors (61) linked to the regulation of adipocyte differentiation, adiposity, and high-fat diet-induced obesity (7); thus, it is likely that the PRR is implicated in the metabolism of visceral adipose tissue by inducing adipocyte accumulation and obesity. The phenotype evoked by the lack of PRR in adipocytes has been previously described. Mice with PRR deletion in adipose tissues have reduced body weight gain and improved insulin sensitivity (68) along with reduced adipose tissue mass, increased liver lipid deposition, and steatosis (85). In addition to these phenotypes, these mice exhibit increases in systolic BP and plasma sPRRs (85). The mechanism by which deletion of the PRR in adipocytes causes high BP is not clear, but it is likely that this response is directly dependent of the sPRR. High levels of the sPRR in plasma may activate circulating prorenin, leading to ANG I formation and further conversion to ANG II, resulting in hypertension (21). Constitutive deletion of the PRR in adipocytes (84) leads to alterations in adipocyte development; thus, it is a mouse model of lipodystrophy with limitations to assess physiological or functional roles of the PRR in adipocytes. The role of the PRR in the development of brown adipose tissue may be of clinical relevance. Sustained PRR activation suppresses brown adipogenesis in multipotent mesodermal cells and brown preadipocytes, perhaps by peroxisome proliferator-activated receptor (PPAR)-γ transactivation (4). Thus, anti-inflammatory therapies targeting PPARs may have beneficial effects through impairment of brown adipose tissue development. The role of the adipocyte-PRR beyond its potential role in local RAS activation needs further investigation to determine the PRR mechanism that regulates adipose cell formation, lipid homeostasis, and BP.

ROLE OF THE PRR IN THE DEVELOPMENT OF IMMUNE CELLS

Evidence demonstrating that prorenin has angiotensin-independent proinflammatory effects supports the concept that the PRR exerts an active role in immune responses (91). The PRR is expressed in monocytes, T cells, and immature dendritic cells (33). Interestingly, PRR deletion affects thymocyte survival and development at multiple stages (19). However, this study needs to be interpreted with caution because of the use of Lck-Cre technology. PRR deletion with Lck-Cre in T cells has demonstrated severe phenotypes, especially compared with conditional deletion of β-catenin (87). The extent to which the Lck-Cre transgene contributes to T cell development and survival might be hampered by the absence of Lck-Cre in control mice (51). Carow et al. (9) showed a 65% reduction in cellularity and increased expression of IL-7 receptors in the thymus of mice expressing Cre under the proximal lck promoter (Lck-Cre⁺ mice). Naïve T cells were reduced in the spleen and lymph in Lck-Cre⁺ mice (9). The PRR also seems to play a role on infiltrated mononuclear cells in kidneys from humans undergoing heminephrectomy because of primary or metastatic renal carcinoma, which exhibit PRR upregulation, without changes in PRR abundance in the distal nephron and blood vessels (54). Cellular responses evoked by immune system PRR activation are currently under active investigation.

POTENTIAL IMPACT OF SEX HORMONES ON THE PRR

Plasma prorenin and renin activity, ANG II, and AT₁R expressions are sexually dimorphic by hormone influence (43, 65, 88), and the causes of these differences are poorly understood. The counterregulatory effects of estrogen on AGT and AT₁R and on the processing enzymes renin, ACE, and ACE2 are complex. RAS activation is more pronounced in the kidney of male subjects, since androgens increase plasma and renal renin activity, whereas they are decreased by estrogen (12, 45). Conversely, AGT is upregulated by oral administration of estrogen, whereas renin, ACE, and AT₁R are downregulated by the hormone (16). Therefore, in the presence of estrogen, the actions of AT₁R may not be fully evoked by sufficient ANG II levels, whereas prorenin might lead directly to tissue damage via activation of the PRR and downstream pathways.

Polymorphisms located in the PRR gene IVS5+169C>T and 1513A>G have been associated with increased ambulatory BP in Japanese men and with lacunar infarction and left ventricular hypertrophy in Japanese women but not men (26). Likewise, polymorphisms in the PRR gene have been associated with hypertension in two cohorts with coronary artery disease and cerebrovascular disease (8).

It is becoming apparent that pregnancy outcomes, i.e., health of the fetus and survival of the neonate, are influenced by the sex of the fetus. It has been suggested that the placenta regulates fetal growth and survival in a sex-specific manner (67). The sex of the fetus affects the expression of the RAS components. Wang et al. (83) demonstrated that high levels of renin mRNA are associated with stimulation of prorenin secretion and upregulation of PRR in cultured decidua collected from women with a female fetus. To date, whether the expression and actions of the PRR are modulated by sex hormones remain open ended. Further studies on the interactions of the PRR during pregnancy and the influence of sex on the outcome of pregnancy also deserve special attention.

CONCLUSIONS

Accumulated evidence indicates that the PRR exerts crucial functions as a RAS component and beyond the RAS. As part of the RAS, the PRR contributes to ANG II formation in plasma and urine. In addition, the PRR appears to be an essential component of v-ATPase and Wnt/β-catenin signaling. Studies suggesting PRR overexpression have yielded conflicting results and raise concerns about whether PRR effects depend on ANG II generation, prorenin binding, or both mechanisms. On the other hand, deletion of the PRR, even in specific tissues such as podocytes, results in phenotype alterations, including cell death. Further studies using inducible cell-specific PRR knockout mouse models might be necessary to define whether PRR actions are related to RAS activation or cell membrane-mediated activation of the intracellular pathway and local injury. Because evidence from experiments using PRR blockers is also debatable, we must think comprehensively concerning the PRR and its interactions with other proteins and pathways to explore possible therapeutic benefits in CVD.

GRANTS

This work was supported by National Institutes of Health Grants DK-104375 and 1-U54-GM-104940 (LACaTS Center; to M. C. Prieto) and by FONDECYT-Chile Grant 11121217 (to A. A. Gonzalez). M. T. Hennrikus was a recipient of the 2017 American Heart Summer Fellowship Program Award.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.T.H. and A.A.G. performed experiments; M.T.H. and A.A.G. analyzed data; M.T.H., A.A.G., and M.C.P. drafted manuscript; M.T.H., A.A.G., and M.C.P. approved final version of manuscript; A.A.G. and M.C.P. conceived and designed research; A.A.G. and M.C.P. interpreted results of experiments; A.A.G. and M.C.P. edited and revised manuscript.