Potential of soluble (pro)renin receptor in kidney disease: can it go beyond a biomarker?

Abstract

(Pro)renin receptor (PRR), also termed ATPase H⁺-transporting accessory protein 2 (ATP6AP2), is a type I transmembrane receptor and is capable of binding and activating prorenin and renin. Apart from its association with the renin-angiotensin system, PRR has been implicated in diverse developmental, physiological, and pathophysiological processes. Within the kidney, PRR is predominantly expressed in the distal nephron, particularly the intercalated cells, and activation of renal PRR contributes to renal injury in various rodent models of chronic kidney disease. Moreover, recent evidence demonstrates that PRR is primarily cleaved by site-1 protease to produce 28-kDa soluble PRR (sPRR). sPRR seems to mediate most of the known pathophysiological functions of renal PRR through modulating the activity of the intrarenal renin-angiotensin system and provoking proinflammatory and profibrotic responses. Not only does sPRR activate renin, but it also directly binds and activates the angiotensin II type 1 receptor. This review summarizes recent advances in understanding the roles and mechanisms of sPRR in the context of renal pathophysiology.

INTRODUCTION

The prevalence of chronic kidney disease (CKD) has significantly increased, affecting 37 million Americans (1) and 800 million people worldwide (2), and therefore has risen to a global public health priority. The common causes of CKD are diabetes and high blood pressure. Other less common risk factors include high body mass index, cardiac disease, glomerulonephritis, ureteral obstruction, a family history of CKD, older age, etc. Despite diverse causes of CKD, this disease undergoes a common pathological process characterized by renal fibrosis, eventually leading to end-stage kidney disease requiring renal replacement therapy, namely, dialysis or kidney transplantation. Renal replacement therapy is a life-saving measure but cannot fully restore the quality of life to a normal level. Furthermore, this therapy imposes a major health and economic burden on the affected individuals, families, and society at large. Treating CKD cost over $81.8 billion in 2018, nearly 7% of all United States government Medicare spending. In the same year, renal replacement therapy costs an additional $36.6 billion. Despite intensive investigation, knowledge gaps exist in understanding the molecular pathways that drive CKD progression, particularly the fibrotic process. Furthermore, there is no curative treatment of CKD except renal replacement therapy.

The renin-angiotensin system (RAS) plays a pivotal role in the physiological control of electrolyte and fluid balance, plasma volume, and blood pressure (3). Apart from its well-known physiological functions, angiotensin II (ANG II), the major effector hormone in the RAS, directly induces proinflammatory and prooxidant responses leading to renal damage (4–6). For the past several decades, the anti-RAS regimen, including angiotensin-converting enzyme inhibitors (ACEi) and ANG II type 1 receptor (AT₁R) antagonists (ARBs), represents the cornerstone therapy for the management of CKD (7, 8). However, the efficacy of anti-RAS therapy for the treatment of CKD is suboptimal since it cannot halt CKD progression to end-stage renal disease (9). For example, the residual renal risk in patients with CKD treated with losartan remains high, and nearly half of the patients show progression of the disease over 4–5 years (10). The reason for the incomplete effectiveness of anti-RAS therapy remains unknown, but several factors can be considered. First, the RAS has evolved into a highly complex system consisting of the systemic and intrarenal RAS. Increasing evidence supports an essential role of the intrarenal RAS in the pathogenesis of CKD as well as hypertension (11–14). However, compared with the overwhelming information about the systemic RAS, the intrarenal RAS is relatively less defined. It remains unclear whether ACEi or ABRs can inhibit the two systems with comparable efficacy. Second, suppression of ANG II production or downstream signaling will relieve the feedback inhibition of renin secretion at juxtaglomerular (JG) cells, resulting in increases in plasma renin levels in up to 50% of patients on ACEi/ARBs (15, 16). The hyperreninemic state in these patients is associated with lack of reduced aldosterone concentration, a phenomenon called aldosterone escape (16). Finally, the optimal application of RAS blockade is limited by a common side effect of hyperkaliemia as well as an acute decline of renal function, angioedema, and other respiratory systems (17–19). Due to these toxicities, ∼5–20% of patients discontinue anti-RAS therapy (18).

BIOLOGY OF (PRO)RENIN RECEPTOR/SOLUBLE (PRO)RENIN RECEPTOR

(Pro)renin receptor (PRR), also known as ATPase H⁺-transporting accessory protein 2 (ATP6AP2), is encoded by the ATP6AP2 gene on the X chromosome (locus p11.4) and was originally cloned as a specific receptor and activator of prorenin and renin (20). As a type I transmembrane receptor, PRR is composed of an intracellular domain (termed M8.9), an extracellular domain, and a transmembrane domain (20). Besides its renin-regulatory property, PRR induces multiple signaling transduction pathways such as MAPK (20), phosphoinositide 3-kinase/AKT, WNT/β-catenin, oxidative stress, and NF-κB (21–27). These diverse properties may partially contribute to the pleiotropic actions of PRR in the regulation of organ development, physiology, and pathophysiology of multiple systems in a complex way dependent or independent of the RAS (28–31).

Within the kidney, PRR expression is found predominantly in the distal nephron, particularly the intercalated cell (IC) of the collecting duct (CD) and to less extent in multiple other renal structures such as mesangial cells, the proximal tubule, etc. (20, 32–34). Conditional gene knockout studies have demonstrated that renal PRR is implicated in physiological control of fluid balance (23, 35–37) as well as the pathogenesis of hypertension induced by ANG II infusion (33, 38, 39), mineralocorticoid excess (40), fructose/salt intake (41), or obesity (42, 43). The prohypertensive role of PRR seems to be mainly dependent on activation of the intrarenal RAS and enhancement of α-epithelial Na⁺ channel (ENaC) expression, particularly during ANG II infusion (33, 44, 45). Besides hypertension, a large body of experimental evidence demonstrates that overactivation of PRR contributes to the pathogenesis of CKD induced by albumin overloading (46), 5/6 nephropathy (47), diabetes (48), and cyclosporine A (49). Targeting PRR likely holds promise to treat CKD and hypertension.

The extracellular domain of PRR is cleaved by proteases to produce 28-kDa soluble PRR (sPRR). Early clinical studies have reported that sPRR is cleaved by proteases to generate 28-kDa sPRR, which is detectable in biological fluids by ELISA. Elevated circulating sPRR has been associated with early pregnancy (50, 51), preeclampsia (52, 53), gestational diabetes mellitus (50, 54), renal dysfunction in patients with heart failure (55), obstructive sleep apnea syndrome (56–58), and CKD due to hypertension and type 2 diabetes (59). In recent years, significant advancements have been made in understanding the identity of the cleavage protease and the function and signaling of sPRR in the kidney. In particular, furin or ADAM19 (60, 61) were originally reported to be the responsible PRR cleavage proteases. However, recent studies by Nakagawa et al. (62) and us (63) using different approaches consistently demonstrated that site-1 protease (S1P) is crucial in releasing sPRR. This initial observation was made in in vitro models and subsequently validated by in vivo studies using pharmacological and genetic approaches (64, 65). However, the role of furin remains inconclusive. Unlike S1P, furin does not appear to determine the release of sPRR but quite likely this protease may modify sPRR following the initial cleavage by S1P (62, 63). Whether furin-mediated modification is necessary for the acquirement of activity of sPRR remains clusive. The present review is focused on the role and mechanism of sPRR in kidney disease.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL FUNCTIONS OF RENAL sPRR

In 2016, we reported that sPRR exerted a potent antidiuretic action in vitro and in vivo, thus functioning as a key mediator of urine concentrating capability (34). This is the first report on the biological function of sPRR in general and on renal the physiological function of this protein in particular. A clue leading to this discovery came from a routine immunofluorescence experiment using two types of anti-PRR antibodies: the anti-N antibody against the NH₂-terminus, e.g., the extracellular domain, and the anti-C-antibody against the intracellular domain (34). The anti-C antibody exclusively labeled the apical membrane of ICs, as previously reported (32). To our surprise, however, the anti-N antibody exclusively labeled the apical membrane of principal cells. This result raised an intriguing possibility that sPRR may act a paracrine mechanism by which sPRR released from the IC may act on principal cells to regulate tubular transport. Indeed, a recombinant histidine-tagged sPRR, termed sPRR-His, upregulated transcription of aquporine-2 (AQP2) through activation of β-catenin signaling (34). Besides AQP2, sPRR-His stimulated ENaC-mediated Na⁺ transport chronically through enhancement of β-catenin-dependent enhancement of α-ENaC transcription and acutely through Nox4-derived H₂O₂ (66). Moreover, sPRR-His directly induced renin expression via the same β-catenin signaling (67), an effect likely additive to the translational modification of renin. The roles of endogenous sPRR in the regulation of AQP2 and α-ENaC expression and tissue renin have been subsequently validated using inhibitors of S1P (64) and conditional renal tubule-specific deletion of S1P (65) or mutagenesis of the PRR cleavage site (67).

sPRR MEDIATION OF RENAL FIBROSIS

Renal fibrosis is characterized by excessive production of the extracellular matrix in myofibroblasts located in the tubulointerstitium, leading to the destruction of the renal parenchyma and decline of renal function. Antifibrotic remedy holds promise for halting CKD progression. Transforming growth factor (TGF)-β has been recognized as a key player in renal fibrosis in vitro and in vivo (68). In response to TGF-β, tubular epithelial cells transdifferentiate to myofibroblasts, a well-documented phenomenon termed as epithelial-mesenchymal transition (EMT). EMT involves two major events: one is loss of epithelial polarity due to suppressed E-cadherin expression leading to disruption of tubular basement membrane and the second is acquisition of spindle-like morphology due to de novo synthesis of α-smooth muscle actin (α-SMA) and the production of matrix proteins. TGF-β signals via the type 2 TGF-β receptor, which recruits the type 1 TGF-β receptor to activate canonical pathway, namely Smad2/3 as well as noncanonical (non-Smad-based) signaling pathways, including MAPK, TGF-β-activated kinase 1, phosphatidylinositol 3-kinase/AKT, and integrin-linked kinase (69–71).

Accumulating evidence suggests a potential role of PRR in the pathogenesis of renal fibrosis. For example, administration of a decoy PRR inhibitor, handle region peptide, in streptozotocin-induced diabetic rats improves glomerulosclerosis as well as proteinuria without affecting hyperglycemia (72). The antifibrotic action of a second version of the PRR decoy peptide, PRO20, has been more widely observed in various rodent models of CKD induced by 5/6 nephrectomy (47), adriamycin (73), and protein overload (74). A direct profibrotic role of PRR has been demonstrated in cultured human kidney proximal tubular cells (HKC-8) (75). Overexpression of PRR induces expression of fibronectin, plasminogen activator inhibitor 1, and α-SMA through impact on the Wnt1/β-catenin pathway (75). Clinical evidence suggests an association between circulating sPRR and glomerulosclerosis in patients with CKD (76).

Recently, we, for the first time, reported that sPRR exerts a direct profibrotic action in a cultured human renal proximal tubular cell line (HK-2 cells) (70). Administration of sPRR-His in the nanomolar range in HK-2 cells was able to induce expression of fibronectin, α-SMA, and college type I. However, this enhanced fibrogenic response did not lead to the disappearance of epithelial polarity. Therefore, sPRR-His treatment elicited a fibrogenic response but was insufficient to induce full EMT. We further addressed the role of endogenously produced sPRR during TGF-β-induced EMT in cultured HK-2 cells. Following TGF-β treatment, sPRR production was elevated without a change in full-length PRR, indicating enhanced cleavage. Indeed, TGF-β-induced sPRR production was blocked by S1P inhibition with PF-429242, confirming S1P as a major source of sPRR production during TGF-β treatment.

The functional role of sPRR in mediating TGF-β-induced fibrosis was tested using PF-429242 together with supplement of sPRR-His. PF-429242 effectively attenuated TGF-β-induced fibrosis, which was reversed by exogenous sPPR-His, confirming a causal role of sPRR in mediating TGF-β-induced fibrosis. However, TGF-β-induced conversion of epithelial polarity to spindle-like morphology was unaffected by PF-429242. Together, the two sets of experiments to test the roles of exogenous and endogenous sPRR consistently demonstrated a profibrotic action of sPRR in cultured renal epithelial cells. It is further evident that sPRR specifically mediates TGF-β-induced fibrosis but not transdifferentiation of the cells.

The signaling pathway underlying the profibrotic action of sPRR has been examined in detail (70). We have previously shown that sPRR directly binds and activates AT₁Rs to activate NF-κB, leading to a proinflammatory response in cultured human endothelial cells as a potential mechanism of endothelial dysfunction and obesity-related hypertension (43). sPRR acts via the same AT₁Rs to induce fibrosis in cultured HK-2 cells given the effectiveness of losartan in attenuating the profibrotic action of sPRR (70). However, sPRR regulates energy metabolism through an AT₁R-independent mechanism. In a mouse model of high-fat diet-induced obesity, sPRR-His treatment significantly improved multiple components of metallic syndrome including hyperglycemia, insulin resistance, hepatic steatosis, albuminuria, and obesity. The beneficial effects of sPRR on metabolic syndrome are largely contributable to increased energy expenditure. Likely, AT₁R mediates the detrimental proinflammatory and profibrotic effects of sPRR in the cardiorenal system, whereas a distinct, as-yet-unknown receptor may contribute to the metabolic action of this soluble protein. As a membrane receptor, AT₁R is expected to play an important role in transducing the signal from sPRR at the plasma membrane. The AKT/β-catenin/Snail pathway has further been shown to be downstream of AT₁R in eliciting a profibrotic response to sPRR. In this regard, PF-429242 completely blocked TGF-β-induced activation of AKT and β-catenin signaling accompanied with suppressed expression of Snail, a key transcription factor in fibrosis (77, 78).

The mechanism of how TGF-β regulates the PRR cleavage process remains elusive. Since S1P is a key protease for the generation of sPRR during TGF-β treatment as well as several other physiological and pathological conditions (46, 49, 79), it seems conceivable that expression and/or activity of S1P might be under the control of TGF-β. Another possibility is that the cleavage site of PRR may be modified by TGF-β rendering enhanced efficiency in the generation of sPRR. This subject warrants further investigation in the future.

sPRR MEDIATION OF RENAL INFLAMMATION

Inflammation is a common feature of kidney disease at all stages irrespective of the etiology. Inflammation can be induced in response to the initial insult as protective and reparative responses. However, when reparative processes fail, the inflammatory response causes disruption of the kidney structure and induces fibrosis, eventually leading to functional decline (80–83). The inflammatory response is mediated by different types of immune infiltrating cells, including monocytes/macrophages, neutrophils, dendritic cells, mast cells, and natural killer cells (CD8⁺ and CD4⁺ lymphocytes) (80), resulting in the production of proinflammatory cytokines such as TNF-α, IL-6, IL-1β, etc. Indeed, anti-inflammatory therapies such as glucocorticoids (84, 85) and mineralocorticoid receptor antagonists (86) are effective in the management of kidney disease.

In vivo anti-inflammatory actions of PRR antagonism have been demonstrated in several rodent models of hypertension and kidney disease. In ANG II-infused uninephrectomized rats, intramedullary administration of PRO20 completely blocked the rise of renal TNF-α expression associated with improved hypertension, albuminuria, and renal fibrosis (33). Subsequently, a similar anti-inflammatory action of PRO20 was demonstrated in multiple rodent models of CKD induced by 5/6 nephrectomy (47), protein overloading (74), and cyclosporine A (49). Inhibition of the renal inflammatory response at least in part accounts for the beneficial effects of PRO20 in ANG II-induced hypertension and renal injury.

A direct proinflammatory action of sPRR was first demonstrated in albumin-loaded HK-2 cells (46). Production of IL-6 and IL-8 in cultured HK-2 cells was induced by albumin overload, which was significantly suppressed by PF-429242 and rescued by the addition of sPRR-His (46). This result suggests a direct proinflammatory action of sPRR during albumin overload. A more in-depth analysis of the proinflammatory action of sPRR was conducted in cultured human umbilical vein endothelial cells (43). Exposure of human umbilical vein endothelial cells to sPRR-His induced IκBα degradation concurrent with NF-κB p65 activation, resulting in increased expression of IL-6, IL-8, VCAM-1, and ICAM-1. These responses were secondary to sPRR-His binding to AT₁Rs and subsequent elevations in Nox4-derived H₂O₂ production (43). The proinflammatory action of sPRR accounts for the endothelial dysfunction that underlies obesity-induced hypertension. It is conceivable that the sPRR/AT₁R/Nox4/NF-κB pathway elucidated in the vasculature may be similarly operative in the kidney during CKD progression.

sPRR ACTIVATION OF THE INTRARENAL RAS

During the past several decades, there has been a paradigm shift in understanding the role of the intrarenal RAS compared with the systemic RAS (11, 12, 87–89). A large body of experimental evidence has confirmed the existence and importance of the intrarenal RAS under various physiological and pathophysiological conditions. The intrarenal RAS has been increasingly appreciated as an important driver of CKD progression in rodent models of 5/6 nephropathy, adriamycin nephropathy, unilateral ureteral obstruction, and polycystic kidney disease (90–93). The upregulation of multiple RAS components in these models is usually detected in the absence of increased plasma renin activity or plasma ANG II (92). The activity of the intrarenal RAS in kidney disease is regulated positively by PRR (47, 49, 74, 75) and β-catenin signaling (94) but negatively by Klotho (90) and vitamin D (13).

As a key component of the intrarenal RAS, renin is expressed in principal cells of the CD, providing a source of urinary renin during hypertension induced by ANG II infusion (95) or two kidney-one clip-induced renovascular hypertension (96). Interestingly, PRR is predominantly expressed in ICs in the same CD. The expression of renin and PRR in neighboring cell types of the CD provides anatomic evidence to support their intrinsic relationship during activation of the intrarenal RAS. Functional evidence favoring this notion has been obtained using PRO20. Multiple studies from our group have shown that PRO20 treatment was able to suppress indexes of the RAS in animal models of nephropathy induced by albumin overload (46, 74), 5/6 nephrectomy (47), and cyclosporin A (49). In particular, in albumin-overloaded rats, PRO20 specifically reduced urinary renin without affecting circulating renin, evidence of intrarenal renin as the target of PRO20 (74). It is interesting to note that through unknown mechanisms, PRO20 treatment reduced sPRR production in vitro and in vivo (49, 74), an unpredicted action based on the known decoy property of the peptide. Irrespective of the mechanism, these results may be taken as indirect evidence to support a pathogenic role of sPRR in CKD progression. This issue warrants further functional study using more specific methods to manipulate the production of sPRR such as conditional deletion of S1P or mutagenesis of the cleavage site of PRR.

During the past few years, significant progress has been made in understanding the mechanism of sPRR regulation of renin. In vitro evidence demonstrates that binding of sPRR or PRR to prorenin or renin increases catalytic activity likely via changes in the conformation of the binding partner, a process known as the nonproteolytic activation of prorenin (20). The in vivo renin-regulatory role of sPRR has been examined during ANG II-induced hypertension. In this regard, ANG II infusion elevated renal medullary and urinary renin activity, evidence of activation of local renin. ANG II-induced intrarenal renin activation was blunted by inhibition of S1P, an effect partially reversed by supplement of sPRR-His (64). Similar results were obtained using mice with mutagenesis of the cleavage site of PRR in terms of changes in renal medullary and urinary renin activity (67). Furthermore, albumin overload induced upregulation of renin expression in cultured renal epithelial cells, which was attenuated by S1P inhibition (63).

SUMMARY AND PERSPECTIVES

In recent years, the discovery of the biological function of sPRR represents breakthrough progress in the PRR research field. Within the kidney, sPRR provokes multiple mechanisms involving stimulation of expression of α-ENaC, AQP2, and renin in the CD to expand plasma volume and elevate blood pressure. Apart from the physiological regulation of renal function, emerging evidence has suggested a potential pathophysiological role of sPRR in experimental models of CKD. Strong in vitro data demonstrate that sPRR specifically mediates the TGF-β-induced profibrotic response without affecting morphology. Furthermore, sPRR directly interacts and activates AT₁R to drive the proinflammatory response. Finally, sPRR enhances renin expression via activation of β-catenin signaling to enhance the intrarenal RAS. Together, the profibrotic, proinflammatory, and renin-regulatory properties of sPRR may make this soluble protein an attractive mediator of CKD progression (Fig. 1). However, several issues remain to be addressed in the future. The mechanism of how the cleavage process is regulated during CKD largely remains unknown. Is the activity or expression of renal S1P altered during CKD? Moreover, in vivo studies with specific manipulation of sPRR production using mice with mutagenesis of the PRR cleavage site or renal cell-specific deletion of S1P will provide definitive evidence for the function of renal sPRR. Finally, given the IC as a major cell type of renal PRR expression, it seems reasonable to speculate that IC-derived sPRR may participate in the disease process. ICs are specificized epithelial cells in the CD and are traditionally associated with the regulation of acid-base homeostasis (97). Interestingly, increasing evidence has uncovered unexpected roles of ICs in the innate immune system during renal injury (98–101), hypertension (102–104), and urinary tract infection (105–107). In particular, the proinflammatory function of ICs has been studied in detail and depends on P2Y₁₄-mediated purinergic signaling (98). It seems to be an intriguing possibility that ICs may serve as a predominant source of sPRR that triggers proinflammatory, profibrotic, and local renin responses during renal injury. A better understanding of the signaling mechanisms in ICs may offer novel diagnostic and therapeutic targets for kidney diseases.

Figure 1.

Schematic illustration of the proposed role of soluble (pro)renin receptor (sPRR) in the progression of chronic kidney disease. In response to various types of insults, sPRR production is elevated, contributing to progression of chronic kidney disease via multiple mechanisms. sPRR directly activates the angiotensin II type receptor to induce inflammation and possibly fibrosis. Moreover, sPRR acts via β-catenin signaling to stimulate local renin expression to drive the intrarenal renin-angiotensin system (RAS) that amplifies the proinflammatory and profibrotic actions of the soluble protein. AT₁R, ANG II type 1 receptor; CKD, chronic kidney disease; Cyto, cytoplasmic domain; ECD, extracellular domain; PRR, (pro)renin receptor; S1P, site-1 protease; TM, transmembrane domain.

GRANTS

This work was supported by National Institutes of Health Grants HL139689, DK104072, HL135851, and HL160020 and by a Veterans Affairs Merit Review from the Department of Veterans Affairs. T.Y. is a Senior Research Career Scientist in the Department of Veterans Affairs.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

T.Y. prepared figures; drafted manuscript; edited and revised manuscript; approved final version of manuscript.