Prorenin receptor regulates more than the renin-angiotensin system

Abstract

The (pro)renin receptor (PRR) was initially believed to be a contributor to the pathogenesis of cardiovascular diseases via the amplification of renin- or prorenin-induced angiotensin (Ang) formation. However, a recent paradigm shift suggests a new role for PRR, separate from the renin-angiotensin system (RAS), in contributing to cellular homeostasis. Specifically, PRR is thought to be essential for vacuolar H⁺ -ATPase (V-ATPase) activity and acts as an adaptor between the V-ATPase and the Wnt signalling pathway. Recent PRR conditional knock-out studies have confirmed this link between V-ATPase and PRR, with deletion resulting in the accumulation of autophagic vacuoles and animal lethality. The molecular mechanism by which PRR contributes to V-ATPase activity, and whether multiple signalling pathways are affected by PRR loss, is currently unknown. Additionally, cleavage by furin at a single site within full-length PRR results in the production of a soluble form of the receptor, which is detectable in plasma. Soluble PRR is hypothesized to bind to specific ligands and receptors and mediate signal transduction pathways. Understanding the physiological function of full-length and soluble PRR will be important for establishing its role in pathology.

Key words::

• Prorenin receptor
• renin-angiotensin system
• V-ATPase
• Wnt signalling

Key messages

• The biological function of the prorenin receptor is unknown; however, recent reports point to it having an important role in V-ATPase activity and signal transduction.

• Understanding the physiology of the prorenin receptor will be essential for determining its role in pathology.

The (pro)renin receptor (PRR/ATP6ap2) was first discovered and cloned by Ngyuen et al. in 2002 and was initially believed to be a contributor to the pathogenesis of cardiovascular diseases by assisting activation of the local renin-angiotensin system (RAS) (1). Renin activates the RAS cascade by cleaving angiotensinogen, resulting in the production of Ang I, which is subsequently cleaved by the angiotensin-converting enzyme to produce Ang II. Current treatment of cardiovascular and renal diseases with RAS-blocking agents does not completely abolish end-organ damage, suggesting additional pathological mechanisms exist (2,3). Upon (pro)renin binding to the PRR, the catalytic activity of prorenin and renin is increased, resulting in increased RAS activation (1). Additionally, signalling cascades are triggered resulting in the expression of inflammatory and fibrotic molecules such as VEGF and PAI-1 (4,5)(Figure 1, left panel). Importantly, these signalling events were shown to occur independently of the generation of Ang II, leading to the hypothesis that the PRR may directly promote tissue damage, particularly in diabetes-related microvascular complications, such as diabetic nephropathy and retinopathy, where 7-fold elevations of serum prorenin can occur (6). Therefore, with the discovery of PRR there was hope that pharmacological blockage of the receptor would prevent RAS-associated organ damage.

Figure 1. The (pro)renin receptor regulates the renin-angiotensin system by increasing the catalytic activity of (pro)renin and increasing angiotensin formation. (Pro)renin binding to the PRR also results in the activation of signalling cascades, resulting in the expression of inflammatory and fibrotic molecules (left panel). Additionally, PRR interacts with members of the Wnt receptor complex and contributes to the activity of the V-ATPase, thereby regulating the Wnt signal activation (right panel). See text for further details.

To establish if PRR has a direct role in mediating pathology, transgenic animals overexpressing PRR were generated. Rats constitutively overexpressing human PRR were found to have normal blood pressure and Ang II levels, but developed renal nephropathy (7). However, this phenotype differed to rats overexpressing PRR only in smooth muscle cells. In contrast, these rats had an elevated systolic blood pressure and heart rate, with normal renal function (8). An alternate approach to elucidate the role of the PRR in mediating pathology was attempted with the development of a specific PRR inhibitor. A peptide corresponding to amino acids 10 to 19 of the prorenin prosegment (handle region peptide or HRP) was developed by Ichihara et al. and hypothesized to bind to PRR, and thus block (pro)renin binding to PRR (9). This peptide has been shown to be protective in several disease models including diabetic nephropathy and retinopathy (10,11). However, several other groups were unable to reproduce these results (12,13), and it has recently been shown that HRP administration is damaging to retinal neurons and glial cells (11) and even counteracts beneficial treatment of direct renin inhibition with aliskiren in a rat model of hypertension (14). More concerning is that a radioimmunoassay developed to measure intact HRP levels was unable to detect significant circulating amounts, suggesting that HRP is rapidly metabolized and may modulate its effects at the site of infusion, and not by inhibiting PRR at the organ of interest (11). Thus, the efficacy and means by which HRP acts as a PRR inhibitor remain unclear and require further investigation.

Due to the controversy described above surrounding the PRR transgenic animal studies and those with HRP, questions have begun to arise as to what is the true biological function of PRR. Unfortunately, attempts to generate conventional complete PRR knock-out mice failed due to the inability of the embryonic stem cells to generate chimeras when injected into blastocysts (15). This is in contrast to knock-out mice of other components of the RAS, which are not embryonic lethal (16). PRR is expressed in all cell types including neurons, cardiomyocytes, pancreatic β-cells, podocytes, and T-cells (1), suggesting it has a common and basic cellular role. In humans, the only described mutation in prr/atp6ap2 has been found to result in intellectual disability and epilepsy, suggesting an important role for PRR in neuronal development (17). Together, these results suggest that PRR may have an important function in cell homeostasis, differentiation, and development.

In line with this thinking, a paradigm shift in the field over the last 12 months proposes a new role for PRR, separate from the RAS, in contributing to cellular homeostasis and signal transduction pathways. Initially, PRR was thought to have no homology to other proteins (1); however, it is now apparent that the nucleotide sequence of PRR is identical to that of ATP6ap2, a gene identified as an accessory protein of the H⁺ vacuolar-ATPase (V-ATPase). The V-ATPase is a multi-subunit complex organized into two domains: an ATP hydrolysis domain acting like a molecular engine (V₁), and a proton-pumping pore domain (V₀) (18). The V-ATPase is responsible for establishing and maintaining intracellular pH gradients along the secretory and endocytic pathways in all cell types. Acidification of vacuoles along these pathways is essential for many cellular functions including the processing of hormones such as insulin (19), receptor endocytosis and recycling (20), and membrane fusion events (18).

An important mechanistic connection between PRR, V-ATPase activity, and cellular development/homeostasis was published in Science in 2010. In this study, PRR was found to interact with components of the V-ATPase and the canonical Wnt receptor-signalling complex (21) (Figure 1, right panel). Wnts are a family of secreted glycoproteins, and canonical Wnt signalling has been shown to be essential for many aspects of cell development and growth, with mutations in Wnt molecules resulting in carcinogenesis (22,23) and also heart (24) and renal diseases (25,26). In the canonical pathway, Wnt signalling is initiated upon complex formation between soluble Wnt proteins, Frizzled and the plasma membrane receptor, LRP5/6, resulting in the downstream accumulation of β-catenin and gene transcription. Cruciat et al. showed that PRR ablation disrupts the formation of proton gradient by the V-ATPase, subsequently preventing the phosphorylation of LRP5/6 and the initiation of Wnt signal transduction. This ablation also resulted in developmental abnormalities in Xenopus embryos (21).

In additional to the canonical pathway, it has also been shown that PRR is important for non-canonical Wnt signalling (also known as the planar cell polarity (PCP) pathway) (27,28). The non- canonical Wnt pathway shares several components with the canonical pathway, namely Frizzled and Dishevelled (29). After receptor activation, the PCP pathway diverges from the non-canonical pathway and is considered to be β-catenin-independent (30). The end result of PCP activation is the planar polarization of epithelial cells, important for processes such as vertebrate gastrulation (31). Both studies by Buechling et al. and Hermle et al. show that PRR is essential for the PCP pathway, with deletion of PRR resulting in disturbances in PCP gene and protein expression, and malformation of wings as a result of follicle polarization defects. Additionally, these studies show that PRR interacts physically with the receptor Frizzled (27,28), and Hermle et al. provide some evidence that the effect of PRR deletion is mediated via a loss of V-ATPase function (28). Taken together, these studies suggest that PRR has a broader cellular function than simply β-catenin signalling.

The first conditional PRR knock-out, which utilized the cre/loxP technology to circumvent the failure in generating complete PRR knock-out mice, deleted the PRR specifically in cardiomyocytes. This resulted in animal lethality after 3 weeks of birth, due to heart failure as a result of impaired ventricular function. Closer analysis of the cardiomyocytes revealed the formation and accumulation of autophagic vacuoles, which the authors conclude is a result of the disturbance of intracellular pH due to decreased expression of V₀ subunits (32). These results were mimicked by treatment with bafilomycin, a selective chemical inhibitor of V-ATPase. The authors therefore conclude that ablation of PRR disrupts cardiomyocyte function due to a loss of V-ATPase function.

This study was also mirrored by recent work in our lab where we examined the conditional knock-out of PRR in podocytes (33). Podocyte-specific PRR knock-out mice developed a severe renal phenotype with nephrotic syndrome and acute kidney failure leading to death of the mice at the age of 2–3 weeks. We identified a decrease in vacuolar acidification in podocytes, most likely by suspended V-ATPase activity, which finally led to a complete block in protein degradation and processing by inhibiting the autophago-lysosomal system. This block in protein degradation resulted in the accumulation of toxic proteins and enhanced ER stress, ultimately leading to disintegration and massive podocyte cell death. Our results were confirmed in an identical study by Oshima et al., who also observed a severe renal phenotype as a result of podocyte dysfunction related to a loss of V-ATPase function (34).

Based on the observations that PRR is essential for V-ATPase function and subsequently Wnt signalling (21,27,28), it can be predicted that PRR is essential for Wnt-related developmental mechanisms, such as controlling the genetic programmes of embryonic development, organogenesis, and tissue homeostasis (35,36). This provides an explanation as to why complete PRR knock-out animals are unviable. However, comparisons of PRR conditional knock-out models to those for canonical Wnt molecules have several discrepancies. In contrast to our PRR podocyte study (33), β-catenin podocyte-specific knock-out mice had no difference to wild-type littermates in body weight and kidney/body weight ratio, and urine albumin was detected up to 12 months after birth (25). Additionally, β-catenin knock-out animals were protected against a disruption in podocyte integrity after experimental injury of glomerulosclerosis, indicating that pathologic activation of the Wnt pathway occurs in podocyte disorders. Also, mice with cardiomyocyte-specific ablation of β-catenin only show a mild cardiac hypertrophy which is not accompanied by a cardiac malfunction or any other cardiac deterioration up to the age of 6 months (37). This study also utilized a cardiac specific αMHC promoter-driven cre, like that used by Kinouchi et al. (32), excluding the possibility that any developmental related discrepancies are responsible for the phenotype differences between β-catenin and PRR cardiomyocyte knock-outs.

The comparison of these canonical Wnt-related phenotypes to PRR phenotypes suggests that knock-out of PRR results in a much more severe biological effect. This is most likely due to the involvement of PRR in V-ATPase function. The V-ATPase is an indispensable component of almost all eukaryotic cells, with its primary function being the regulation of pH along the endocytic and secretory pathways (38) (although this can also be modulated by other factors such as proton diffusion, ClC chloride channels, and sodium-potassium pumps (39,40)).

Intracellular pH along the endocytic pathway is very tightly regulated, ranging from neutral pH in the endoplasmic reticulum, weakly acidic pH (6.8–5.9) in early endosomes, and highly acidic pH (6.0–4.9) in late endosomes/lysosomes (41). In general, endocytosis functions to internalize extracellular macromolecules and fluid, and also plasma membrane proteins, receptors, and their bound ligands. It is well established that the acidification of organelles/vesicles is obligatory for plasma membrane receptor trafficking along endocytic pathways. The internalization and trafficking of plasma membrane receptors is believed to be essential for the activation and regulation of several different signal transduction pathways (42,43). In the case of Wnt signalling, a recent study has shown that the internalization of the Wnt-Fz-LRP6 receptor complex results in the formation of a ‘signalosome’ which is essential for Wnt signal activation (44). Importantly, in this study inhibition of Wnt receptor complex internalization did not affect LRP6 phosphorylation, suggesting that both events are required for successful Wnt signal activation. This is supported by the study of Cruciat et al., who also showed that silencing of PRR and a V₀ subunit of the V-ATPase prevented signalosome formation (21). Another recent study has proposed that the V-ATPase acts as a bifunctional protein to establish and also to sense vesicular pH. Here, a subunit of the V₀ domain was found to interact with cytosolic adaptor molecules of the endocytic transport machinery at a certain pH, and this interaction was subsequently shown to be important for the regulation of membrane receptor internalization and transport from early to late endosomes (45). The molecular mechanism by which PRR is important for V-ATPase function remains unknown.

In addition to Wnt, other signalling pathways important for cell development and homeostasis, such as Notch, require V-ATPase activity (46). One example is signalling by PRR itself: in cultured collecting duct cells, PRR knock-down by siRNA was mimicked by pharmacological V-ATPase inhibition and attenuated the increase in mitogen-activated protein kinase (MAPK) ERK1/2 phosphorylation induced by either renin or prorenin stimulation (47), suggesting that (pro)renin signalling through the PRR is also dependent on vesicular acidification. We can thus speculate that other pathways that require plasma membrane receptor internalization for signal transduction, e.g. Notch (46), LIF/STAT3 (48), pre-T-cell receptor signalling (49), and pathways such as Sonic-Hedgehog (50,51), where ligand internalization is required, may similarly be reliant on V-ATPase activity and thus PRR. These above-mentioned signalling pathways are essential for the development and proliferation of all cell types, thus providing an explanation as to why complete PRR KO embryos are unviable, and why such different cell types are severely affected by PRR loss (32,33).

The biological function of PRR becomes even more complex with the recent discovery by Nguyen et al. of a soluble form of PRR (52). Soluble PRR (sPRR) is generated by cleavage of the full-length protein by furin, resulting in the production of a 28 kDa soluble protein. Soluble PRR has been detected in the conditioned media of various cell types (52,53) and in plasma (52). A recent study showed that levels of sPRR are increased in the urine and renal medulla of rats infused with Ang II (54), providing a prima facie case that sPRR has an important function. The biological role of sPRR is also unknown, but it has been speculated to have a role in vivo by binding to specific receptors and initiating signal transduction pathways (15). Thus, it may be likely from the conditional PRR studies summarized above that part of the phenotype observed is due to the loss of sPRR. Future studies to distinguish between those cellular functions attributable to full-length PRR and those belonging to sPRR will be essential for understanding the role of PRR in physiology and pathology.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.