Lewis K. Dahl Memorial Lecture

Introduction

The classic renin-angiotensin system (RAS) is a well-defined endocrine system consisting of liver production of angiotensinogen which is converted to angiotensin I by renin, followed by a second cleavage to angiotensin II (AngII) by angiotensin converting enzyme (ACE) located on the surface of lung endothelium^{1, 2}. AngII is the major effector hormone of the RAS, playing a key role in in the maintenance of extracellular volume, blood pressure, renal hemodynamics, and tubular sodium transport, as well as in the pathogenesis of cardiovascular and renal diseases^3–5. AngII acts on two types of membrane receptors, AngII type 1 receptor (AT1R) and type 2 receptor (AT2R). The RAS interventions targeting AT1R and ACE are widely used for management of hypertension, renal disease, and cardiovascular disease. During the past several decades the RAS has evolved into a highly complex system as highlighted by the discovery of ACE2/Ang 1-7/Mas pathway and the local RAS found in a number of tissues such as the kidney, the heart, and the brain^{1, 6, 7}. The RAS activity is largely controlled by renin-mediated conversion of angiotensinogen to Ang I, a rate-limiting step in the RAS. Renin is a protease produced by juxtaglomerular (JG) cells of the afferent arteriole as well as collecting duct principal cells⁸, mast cells⁹, adipocytes¹⁰, etc. Prorenin, a precursor of renin, contains a prosegment covering the catalytic domain and undergoes proteolytic cleavage to generate the mature renin. Plasma prorenin levels are elevated in diabetic patients and predict microvascular complications, whereas plasma renin is normal or low¹¹. Animal data suggests that excess prorenin over active renin in diabetes originates from the collecting duct (CD) rather than from the juxtaglomerular apparatus¹². High prorenin state is also seen normal pregnancy^{13, 14}.

In 2002, Nguyen et al. cloned a novel receptor for prorenin and renin, termed (pro)renin receptor (PRR), and assigned a renin-regulatory function to this receptor¹⁵. Renin bound to PRR exhibits a three- to five fold increased renin activity¹⁵. Furthermore, when PRR binds prorenin, the later undergoes conformational changes to unfold the inhibitory prosegment of prorenin, leading to non-proteolytic activation¹⁵. A soluble form of PRR (sPRR) is generated by intracellular cleavage by furin and secreted in plasma¹⁶. Based on the structure, PRR belongs to type I transmembrane receptor family, consisting of a large N-terminal extracellular domain, a single transmembrane protein, and a short cytoplasmic domain^15,17. Amino acid sequence analysis reveals that the intracellular domain of PRR is highly conserved among all species whereas the extracellular domain shares high homology only in mammals. The conservation scores (identity/AA) are 6.8 for the intracellular domain (ICD, M8.9) and 3.8 for the extracellular domain (sPRR) among all species, and 8.7 for the ICD and 8.9 for sPRR in mammals (Fig. 1). This result is compatible with the concept that sPRR may serve an important function in mammals whereas M8.9 may participate in fundamental survival functions across all species.

Fig. 1.

Distinct conservations of different domains of PRR. The protein sequence alignment was generated by the Clustal × 2.0 multiple sequence alignment program using default parameters. The PRR protein sequences were from GenBank accessions numbers NP_005756.2 (human), NP_001126837.1(P. abelii), AAH79339.1(rat), NP_081715.1(mouse), NP_001080880.1(xenopus Laevis), NP_998188.2(Zebrafish), NP_510360.1(C. elegans), and NP_649876.1(drosophila melanogaster). (A) Alignment of amino acid sequence of PRR. The score depicts conservation between different species. The scores on the bottom two rows depict degrees of conservation across all species and the mammals only. TM, transmembrane domain; ICD, intracellular domain. The TM, the ICD, and furin-mediated cleavage site are highlighted. (B) The summary of the conservation scores.

Within the kidney, PRR is expressed in multiple cell types and is most abundantly detected in the intercalated cells of the CD^18–20, a site for fine-tuning urinary excretion of water and electrolytes; the expression of PRR in the CD is stimulated by chronic AngII infusion²¹ or Na⁺ depletion^{22, 23}. During the past decade, understanding the physiological function of PRR has been hindered by the lethal phenotype of complete or conditional deletion of PRR and also the lack of an effective PRR inhibitor with the controversial antagonistic effect of handle region peptide (HRP) on the PRR^24–26. In recent years, the availability of various strains of viable renal PRR knockout (KO) models along with the newly developed PRR decoy inhibitor PRO20 has greatly facilitated the process in unraveling the physiological function of renal PRR. The existing in vivo data suggests that PRO20 acts via inhibition of the RAS during both AngII infusion and deoxycorticosterone acetate (Deoxycorticosterone acetate Deoxycorticosterone acetate DOCA)-salt hypertension^{27, 28} although in vitro data shows the RAS-independent effect of this inhibitor on epithelial Na⁺ channel (ENaC) activity²⁹. The goal of this review is to highlight recent advances in identifying the roles of PRR in the nephron, particularly in the CD, for regulation of Na⁺ and water balance and blood pressure.

PRR regulation of Na⁺ homeostasis

A series of recent studies from our group and others have identified the prorenin/PRR pathway as a potential regulator of ENaC in the CD. Since prorenin/renin and PRR are co-expressed in the CD, we hypothesized that their interactions in an autocrine or paracrine fashion may affect ENaC activity. To address this possibility, we performed electrophysiological studies using mpkCCD cell line grown on Transwells. Using this system, we made an initial observation that prorenin treatment in the nanomolar range induced a rapid and transient increase in ENaC-mediated Na⁺ transport (Fig. 2) through Nox4-derived H₂O₂²⁹. Interestingly, renin treatment was largely ineffective. In agreement of this observation, multiple studies suggest that prorenin has higher affinity to PRR than renin^{30, 31}. The dependence of The Na⁺ stimulatory effect of prorenin on PRR was validated by using a PRR decoy inhibitor PRO20²⁹. This effect was independent of canonical RAS since AT1 receptor inhibition with losartan was ineffective (Fig. 2). These results suggest that prorenin via PRR induces Nox4-derived ROS resulting in a direct activation of ENaC. In agreement with this finding, Ramkumar et al. observed a similar stimulatory effect of prorenin on ENaC activity in isolated split-open CD³². Moreover, the prorenin effect was similarly unaffected by AT1 blockade³². Besides the ENaC regulation at activity level, PRR also upregulates α-ENaC mRNA expression in mIMCD3 cells exposed to low salt media or AngII through serine/threonine-protein kinase-1 and phosphorylated Nedd4-2 {also known as Nedd4L (neural precursor cell-expressed developmentally downregulated gene 4-like)}³³. Together, the in vitro results suggest that PRR can modulate ENaC expression and activity through distinct mechanisms.

Fig. 2.

Effect of prorenin/PRR on ENaC activity in cultured CD cells. Confluent mpkCCD cells grown on Transwells were pretreated for 30 min with PRO20 (1.5 μM) or losartan (1.0 μM) and then treated with prorenin (10 nM). I_eq was monitored for a 15-min period by using an epithelial volt-ohmmeter (EVOM). In a separate experiment, the time point of peak stimulation of Na⁺ transport at 1 min was chosen for measuring amiloride-sensitive I_eq, an index of ENaC activity. (A) Timecourse of I_eq changes in Control, Prorenin, and Prorenin + PRO20 groups. (B) Corresponding amiloride-sensitive I_eq for experiments in (A). (C) Timecourse of I_eq changes in Control, Prorenin, and Prorenin + Losartan groups. (D) Corresponding amiloride-sensitive I_eq for experiments in (C). Los, losartan. * p < 0.05 vs. basal in the same group; # p < 0.05 vs. prorenin. N = 6–12 per group. Data are means ± SE. Adapted from Fig. 1 of a previous study²⁹.

Besides the above-mentioned in vitro data, strong in vivo data from 3 independent studies all support an important role of PRR in ENaC regulation under basal and/or pathophysiological conditions^{29, 32, 33}. To our surprise, PRR regulation of ENaC appears to occur only in the renal medulla but not the cortex. In this regard, we found that the renal inner medullary α-ENaC under basal condition or following AngII infusion was reduced in CD PRR KO mice³⁴. In contrast, renal cortical α-ENaC expression was unaffected by AngII or CD PRR KO. Moreover, the other ENaC subunits (β & γ) remain unchanged. In agreement with our results, Quadri et al. showed that in vivo delivery of shRNA against PRR to the kidney of Sprague-Dawley rats selectively reduced α-ENaC expression in the renal medulla but not in the cortex despite the gene knockdown in the whole kidney³³. These results suggest that PRR may primarily target renal medullary (or inner medullary) ENaC to regulate Na⁺ balance and BP.

As compared with the overwhelming information about Na⁺ transport process in the early part of the CD^35–38, little attention has been paid to the terminal part of the CD, e.g. the inner medullary collecting duct (IMCD). Part of the reason is that the IMCD in rabbits or rats is highly branched or difficult to dissect due to tightly adherent tissues and therefore is not easily accessible to microperfusion as for the cortical CD (CCD). The literature concerning net Na⁺ transport in the IMCD is controversial. It has been difficult to demonstrate net Na⁺ reabsorption in isolated perfused IMCD by measurement of Na⁺ concentrations in the perfusate³⁹. However, other studies reported robust Na⁺ reabsorption in this nephron segment^{40, 41}. Furthermore, the apical membrane conductance in the IMCD due to passive movement of Na⁺ is detected and attributed to amiloride-sensitive conductive pathway⁴². In addition, Na⁺ reabsorption in the IMCD has been demonstrated by using micropuncture techniques. The IMCD is accessible to micropuncture through a retrograde placement of thin pipettes from the tip of the IMCD. Early micropuncture studies using this technique have demonstrated active net Na⁺ reabsorption in the IMCD^{43, 44}. More importantly, Na⁺ reabsorption in the IMCD fluctuates in response to altered plasma volume so that it is increased during hypovolemia but suppressed during volume expansion^44–47 despite a conflicting report⁴⁸. Overall, these results suggest a potential role of Na⁺ transport in the IMCD in the control of Na⁺ excretion during alteration of plasma volume. Unfortunately, this area has almost remained uninvestigated during the past decade. It is anticipated that the demonstration of PRR as a regulator of renal medullary ENaC will provide an impetus for re-evaluation of importance of Na⁺ transport in the IMCD.

As discussed above, PRR regulation of renal medullary α-ENaC contributes to pathogenesis of AngII-induced hypertension³⁴. In addition, dysregulation of renal medullary ENaC has been implicated in the development of salt-sensitive hypertension in Dahl salt-sensitive (S) rats⁴⁹. Based on the conductive permeability to Na⁺ and the rate of Na⁺ transport, the IMCD cells from Dahl S rats transport twice as much Na⁺ as the cells from Dahl salt-resistant (R) rats^{50, 51}. Consistent with this finding, the mRNA of β- and γ-ENaC and the protein abundance of all 3 ENaC subunits are increased in the renal medulla of Dah S rats under basal condition and following high salt intake as compared with Dahl R rats⁴⁹. Additionally, aberrant activation of intrarenal RAS has been suggested to play a role in the Dahl S model which is traditionally considered as a low renin hypertension model. Despite the similar suppression of plasma angiotensinogen (AGT) in both strains, high salt enhances renal and urinary levels of AGT in Dahl S but not Dahl R rats⁵². Future studies need to address the precise location of the intrarenal RAS activation and possible involvement of PRR in salt-sensitive hypertension in the Dahl S model.

PRR regulation of water homeostasis

Most mammals can produce highly concentrated urine when water consumption is reduced so that a constant plasma osmolality can be maintained. Such urine concentrating capability resides in the renal medulla and is chiefly determined by arginine vasopressin (AVP)^{53, 54}. The antidiuretic action of AVP is mediated by enhancement of water permeability of the CD as well as the generation of osmotic gradient due to increased NaCl reabsorption in the thick acceding limb (TAL)^{54, 55}. In the CD, AVP via arginine vasopressin receptor 2 (V₂R) activates cAMP/PKA pathway that induces aquaporin-2 (AQP2) trafficking to the apical membrane and expression leading to increased water permeability⁵³. We discovered that AVP-stimulated AQP2 expression depends on prorenin activation of PRR⁵⁶. In primary rat IMCD cells on Transwells, AVP induced a marked increase in AQP2 protein expression. To address the involvement of PRR, we employed three independent approaches to inhibit PRR: a pharmacological inhibitor, a siRNA, and a neutralizing PRR antibody. Inhibition of PRR with either one of these approaches was highly effective to suppress AQP2 upregulation by AVP. Furthermore, activation of PRR with prorenin but not renin induced a marked increase in AQP2 expression (Fig. 3). AVP also stimulated release of prorenin, and sPRR in primary rat IMCD cells⁵⁶. In M-1 CD cell line, a similar enhancement of renin mRNA, prorenin and renin is observed following desmopressin (ddAVP) treatment⁵⁷. Together, the in vitro data demonstrates a potential role of PRR in mediating AVP-induced AQP2 expression in the CD cells. However, whether PRR regulates AVP-induced AQP2 trafficking remains uninvestigated.

Fig. 3.

The direct role of prorenin/PRR in regulation of AQP2 expression in the CD cells. Primary rat IMCD cells were pretreated with PRO20 or anti-PRR-N antibody or pre-transfected with PRR siRNA, and then treated for 24 h with 10 nM prorenin. AQP2 protein expression was determined by immunoblotting and normalized by β-actin. (A) Effect of prorenin alone or in combination with PRO20 on AQP2 expression (n = 6 per group). (B) Effect of prorenin alone or in combination with anti-PRR-N antibody on AQP2 expression (n = 6 per group). (C) Effect of prorenin alone or in combination with PRR knockdown on AQP2 expression (n = 6 per group). Adapted from Fig. 4 of a previous study⁵⁶.

In agreement with the in vitro data as discussed above, compelling in vivo data is also available to support antidiuretic action of renal PRR. First, renal fPRR protein abundance is consistently elevated in rats following 48-h or 72-h water deprivation (WD)^{56, 58}. Interestingly, this maneuver increases urinary sPRR excretion⁵⁶ but decreases plasma sPRR⁵⁵. This result may suggest differences in the origin of urinary and plasma sPRR. Second, pharmacological inhibition of intrarenal PRR results in impaired urine concentrating capability⁵⁶. In this regard, PRO20 is administered via an infusion catheter chronically implanted in the kidney of nephrectomized rats followed by 48-h WD. Intrarenal PRO20 infusion partially attenuates the reduction of urine volume and the increase of urine osmolality and the expression of AQP2 in the WD rats. Lastly, conditional deletion of PRR⁵⁶ in the CD and the whole nephron^{59, 60} causes a common polyuria phenotype. CD PRR KO mice generated by using AQP2-Cre are not associated with renal developmental abnormality seen in the null model produced by using Hoxb7-Cre⁶¹. The whole nephron PRR deletion is generated by using an inducible way wherein PRR ablation occurs in adulthood so that the developmental stage is bypassed. All of these KO models have increased urine volume and decreased urine osmolality under basal condition and exhibit impaired ability to concentrate urine following WD. Evidence is available to suggest impairment of AVP signaling in these models. For example, renal AQP2 expression is consistently downregulated in these models and AVP sensitivity is also blunted in the nephron PRR KO mice. Besides AQP2, renal expression of Na/K/2Cl cotransporter (NKCC2), another target of AVP in the TAL⁵⁵, is also suppressed in the null mice. These results suggest that PRR is indispensable for AVP signaling in both the CD and the TAL. However, it is important to note that interpretation of the phenotype of the nephron PRR KO model is complicated by renal structural abnormalities due to accumulation of autophagosome particularly in the TAL. This result raises a possibility that the downregulation of transporter proteins such as NKCC2 in this model may be secondary to an autophagic defect in the renal epithelial cells.

EP₄ regulation of PRR

Further evidence suggests that prostaglandin EP₄ receptors are required for enhancement of PRR and hence AQP2 following AVP treatment. PGE₂ is one of the major cyclooxygenase-derived metabolites of arachidonic acid produced in the kidney, particularly the IMCD^{62, 63}. PGE₂ acts on four distinct G protein-coupled receptors, designated EP_1–4, to induce a wide spectrum of biological functions⁶⁴. PGE₂ exerts a prominent, yet complex, role in the regulation of water homeostasis through distinct EP receptors. In particular, PGE₂ and sulprostone, an EP_1/3 agonist, blunt AVP-induced AQP2 trafficking and urine concentration^65–68 whereas EP₂ and EP₄ agonists induces AQP2 trafficking to the apical membrane and alleviate the symptoms of nephrogenic diabetes insipidus (NDI) in rodents⁶⁹.A nonbiased gene expression analysis of G protein coupled receptor (GPCR) in mouse IMCD cells identified the prostaglandin EP₄ subtype as the highly expressed GPCR in addition to the V₂R⁶⁹. In microdissected nephron segments, EP₄ mRNA was abundantly detected in the cortical collecting duct⁷⁰ where PGE₂ stimulated cAMP production⁷¹. Following 24-hour water deprivation, EP₄ protein expression is remarkably upregulated contrasting to unchanged expression of the other 3 EP subtypes⁷², indicating a unique role of EP₄ receptors in regulation of water homeostasis. This notion has been confirmed by generation of nephron- (Ksp-EP₄-/-) and CD (AQP2-EP₄-/-) -specific deletion of EP₄, both of which impair urine concentrating capability⁷². An earlier study from our group showed that EP₄ acting downstream of COX-2 mediates upregulation of renal medullary PRR and the intrarenal RAS during AngII-induced hypertension^{73, 74}. Therefore, we proposed a hypothesis that PGE₂/EP₄ pathway may mediate the antidiuretic action of AVP via targeting PRR and the intrarenal RAS. We found that in primary rat IMCD cells, activation of EP₄ receptors was able to stimulate AQP2 expression and conversely inhibition of this receptor subtype effectively attenuates AVP-induced AQP2 expression⁵⁶. These results suggest the EP₄ receptor as a mediator of AVP-induced AQP2 expression. Since EP₄ agonist stimulated PRR expression⁷⁴ and EP₄-induced AQP2 expression was blunted by inhibition of PRR, the EP₄ receptor should act downstream of AVP and upstream of PRR in regulation of AQP2 expression.

The biological function of sPRR

PRR contains a protease cleavage site at the N-terminal domain near the transmembrane domain¹⁵. sPRR was originally shown to be generated by intracellular cleavage by furin and detected in the conditioned medium of cultured cells¹⁶. This conclusion was based on the observations that LoVo colon carcinoma cells deficient in furin didn’t produce sPRR despite the expression of fPRR and that overexpression of α1-anttrypsin Portland variant, an inhibitor of furin completely suppressed sPRR production in Chinese hamster ovary cells¹⁶. However, the role of furin as the cleaving enzyme was not confirmed by another study⁷⁵. In the later study, ADAM19 but not furin is shown to be responsible for the cleavage process⁷⁵. Moreover, in cultured vascular smooth muscle cells (VSMC), PRR is mainly localized to the endoplasmic reticulum (ER) and the Golgi, and shed by ADAM19 in the Golgi, leading to the secretion of sPRR to the extracellular space where it enhances renin activity of prorenin⁷⁵. Thus the relative importance of furin versus ADAM19 in this cleavage event remains elusive.

A large number of clinical studies suggest importance of circulating sPRR. sPRR can be measured by ELISA⁷⁶ which offers a simple tool for assessment of sPRR in the body fluid. By ELISA, serum levels of sPRR are increased during pregnancy^{77, 78} and in patients with heart failure⁷⁹, gestational diabetes mellitus⁸⁰, obstructive sleep apnea syndrome⁸¹, primary epithelial ovarian cancer⁸². In 374 patients with chronic kidney disease (CKD) due to hypertension and type 2 diabetes, serum sPRR is positively associated with serum creatinine, blood urea nitrogen, and proteinuria, and inversely with estimated glomerular filtration rate (eGFR)⁸³. It is puzzling however that in this study serum sPRR levels were lower in patients with primary hypertension or diabetes than in those without these conditions⁸³. In 25 patients who underwent uninephrectomy, plasma sPRR further correlated with renal tubulointerstitial fibrosis⁸⁴. Despite the lack of correlation of plasma sPRR with circulating renin, prorenin, and aldosterone in healthy subjects⁸⁵, sPRR correlates positively with urinary AGT in patients with essential hypertension⁸⁶. Urinary AGT is quite widely considered as an index of intrarenal RAS⁸⁷ despite some evidence for its hepatic origin⁸⁸. Overall, most of the previous studies favor plasma sPRR as a biomarker of certain diseases likely due to its association with the intrarenal RAS.

A clue suggesting a biological function of renal PRR came from our immunostaining studies comparing antibodies against the different PRR domains. A commercial antibody against the C-terminus of PRR (anti-PRR-C antibody)^{18, 20} reproducibly labeled intercalated cells. However, to our surprise, an antibody against the N-terminus of PRR (sPRR) only stained the apical membrane of principal cells. The anatomical evidence suggested an intriguing possibility that sPRR might be secreted from intercalated cells and secreted to the luminal side and then bind an as yet unknown protein on the apical membrane of principal cells, revealing a paracrine loop linking the two cell types in the CD; this paracrine mechanism may play a role in regulation of Na⁺ and water transport in the principal cells of the CD. To test this hypothesis, we generated a histidine-tagged recombinant sPRR, termed as sPRR-His, and examined its effect on AQP2 expression in primary IMCD cells. sPRR-His at the nanomolar range remarkably induced AQP2 mRNA and protein expression (Fig. 4)⁸⁹. The parallel increase in AQP2 promoter activity (Fig. 4) suggested that the transcriptional mechanism may account for the upregulation of AQP2 expression by sPRR-His⁸⁹. We employed SPRING to predict proteins that interact with sPRR. This led to the discovery of frizzled-8 (FZD8) that interacted with sPRR to activate Wnt/β-catenin pathway and thus enhancement of AQP2 gene transcription⁸⁹. However, this mechanism doesn’t appear to contribute to AQP2 redistribution following an acute AVP treatment⁸⁹. Since AVP stimulated sPRR release, we speculate that the sPRR/β-catenin/AQP2 pathway may primarily mediate the upregulation of AQP2 transcription during chronic AVP treatment. While previous studies extensively focus on the detailed signaling mechanism of AQP2 trafficking between intracellular vesicles and apical plasma membrane which occurs within minutes of AVP treatment^{53, 54, 90, 91}, little is known about the mechanism of transcriptional regulation of AQP2 gene under chronic condition. Our results uncovered a novel role of sPRR in transcriptional regulation of AQP2 expression.

Fig. 4.

Characterization of function of sPRR. A recombinant PRR, termed sPRR-His, was expressed in a mammalian expressing system as a fusion protein that contained sPRR, a histidine tag in the C terminus, and a secretion signal peptide in the N terminus. (A) sPRR-His was purified from the medium as a single 29.6 kDa band in 12% SDS polyacrylamide gel electrophoresis (PAGE). E1-4 denotes sequential fractions from the elution. (B&C) Primary rat IMCD cells grown in Transwells were exposed to 10 nM sPRR-His for 24 h, and AQP2 expression was analyzed by qRT-PCR and immunoblotting (n=5 per group). (D) In a separate experiment, the cultured CD cells were transfected with an AQP2-luciferase construct and allowed to grow to confluence, and then treated with vehicle or 10 nM sPRR-His for 24 h and the luciferase activity was assayed (n=5 per group). (E) SPRING is a template-based algorithm for protein-protein structure prediction. We employed SPRING to identify proteins that interact with sPRR. This analysis revealed 10 hits: CTSG (cathepsin G), ACE2 (angiotensin 1 converting enzyme 2), CMA1 (chymase 1), MME (membrane metallo-endopeptidase), ACE (angiotensin 1 converting enzyme 1), CTSZ (cathepsin Z), ENPEP (glutamyl aminopeptidase), FZD8 (frizzled family receptor 8), WNT3A (wingless-type MMTV intergration 3A), and LRP6 (low density lipoprotein receptor-related protein 6). Adapted from Fig. 2 of a previous study⁸⁹.

The Wnt/β-catenin pathway is a multifunctional network critically involved in embryogenesis⁹². Dephosphorylated β-catenin translocates to the nucleus where it recruits several transcription factors, such as lymphoid enhancer–binding factor 1/T-cell-specific transcription factor⁹³. In low vertebrates including zebrafish and Xenopus, PRR functions as a key component of the membrane receptor complex in Wnt signaling to control embryogenesis⁹⁴. In addition, the Wnt/β-catenin pathway is also involved in physiological and pathophysiological processes. Aberrant activation of this pathway contributes to pathogenesis of CKD^95–97. Our recent work demonstrates that the coupling of PRR with β-catenin signaling is operative in the setting of physiological regulation of water transport in the distal nephron⁸⁹. In support of this observation, Jung et al. reported that tankyrase-mediated β-catenin pathway mediated AVP-induced AQP2 expression in mpkCCDc14 cells⁹⁸.

The mechanism of how AVP regulates sPRR production, particularly the involvement of V₁R versus V₂R and intercalated cells versus principal cells largely remains elusive. It has been shown that within the CD, V₁R is mainly expressed in the intercalated cells regulating urine acidification⁹⁹. Given colocalization of V₁R and PRR to the intercalated cells, it seems possible that V₁R may mediate AVP-induced sPRR production. Another possibility also exists thatV₂R in principal cells may affect intercalated cell sPRR production through an intermediate mediator such as prostaglandins. Indeed, we found involvement of the prostaglandin EP₄ receptor in mediating AVP-induced activation of PRR⁵⁶.

The in vitro capability of sPRR-His to regulate AQP2 expression as discussed above predicts an antidiuretic action of sPRR. This prediction has been confirmed by in vivo studies using two types of DI modes induced by inhibition of V₂R and liver X receptor (LXR)⁸⁹. Administration of sPRR-His via an intravenous route effectively attenuated polydipsia and polyuria, and partially restored urine osmolality in both models⁸⁹. These results demonstrate therapeutic potential of sPRR-His for treatment of diabetes insipidus, particularly NDI. Central diabetes insipidus (CDI) is caused by deficiency of AVP and thus can be treated with supplementing the missing hormone^{100, 101}. NDI results from the insensitivity of the kidney to AVP usually due to genetic abnormalities with 90% of families having a mutation in V₂R^{102, 103} or with AQP2 mutations accounting for rest of the other families¹⁰⁴. Patients with NDI usually develop very severe polyuria and polydipsia. It is particularly astonishing that children with congenital NDI can produce 20 liters of urine daily and must drink a comparable amount of water to avoid dehydration¹⁰¹. Infants with this disease may fail to thrive. So far, a specific therapy for NDI is lacking. The existing therapy for NDI consisting of a thiazide diuretic, a low salt diet, and indomethacin is only minimally effective¹⁰¹. Our results suggest that patients with NDI may benefit from administration of sPRR-His. This calls for future clinical evaluation of the antidiuretic potential of sPRR-His in NDI patients. It is also needed to screen NDI patients for mutations of PRR that resides on X chromosome. Indeed, the congenial NDI is mostly an X-linked disease.

PRR regulation of proton transport

Prior to the cloning of the full-length PRR, a truncated fragment, termed M8.9, was identified from adrenal secretory vesicle membrane as part of the vacuolar H⁺-ATPase (V-ATPase) and thus named ATPap2 (ATPase 6 accessory protein 2)¹⁰⁵. V-ATPase is a multimeric protein complex consisting of a Vo domain responsible for proton translocation, and a V1 domain for ATP hydrolysis to provide energy for proton transport. Therefore, V-ATPase acts as an ATP-dependent proton pump involved in many cellular functions. V-ATPase is expressed in all cell types and are mainly expressed in the intracellular compartments where it pumps proton into the vesicle to maintain the acidic range needed for cellular degradation, autophagy, endocytosis, protein tracking, Wnt/β-catenin signaling, etc.¹⁰⁶. Defective lysosomal acidification due to impairment of V-ATPase contributes to multiple neurodegenerative diseases such as Alzheimer disease, Parkinson disease, and frontotemporal lobar degeneration¹⁰⁶. In some cell types such as the intercalated cells of the CD, V-ATPase is expressed on the apical membrane responsible for pumping proton to the urine. Mutations of renal specific V-ATPase subunits cause distal renal tubular acidosis¹⁰⁷.

In vitro evidence demonstrates that PRR is requited for integrity of V-ATPase in cultured Madin-Darby canine kidney (MDCK) cells¹⁰⁸. In this study, prorenin at low nanomolar concentrations increased V-ATPase activity, which was blunted by the V-ATPase inhibitor bafilomycin or small siRNA against PRR¹⁰⁸. However, the acute stimulation with prorenin in freshly isolated CD cells fails to affect H⁺-ATPase activity¹⁰⁹. Given these conflicting results, it remains unclear as to whether PRR regulation of H⁺-ATPase is ligand-dependent. The association of PRR with V-ATPase appears to be essential for cell survival. Impairment of V-ATPase has been proposed as an important mechanism for lethal heart failure due to suppressed autophagic function in mice with cardiomyocyte-specific deletion of PRR¹¹⁰. This mechanism may also explain severe podocyte injury and chronic renal failure induced by podocyte-specific deletion of PRR¹¹¹.

The abundant expression of PRR on the apical membrane of the intercalated cells of the CD¹⁸ implies its functional association of V-ATPase for regulation of proton secretion to the urine. This issue was examined recently by using a mouse model of inducible deletion of PRR in the nephron⁶⁰. Under basal condition, blood pH, P_CO2, and [HCO₃⁻] were not different between KO and control animals. However, when challenged with acid load, the KO mice developed severe acidosis due to impaired ability to decrease urine pH, contrasting to well-maintained acid-base balance in the acid-loaded control animals⁶⁰. Moreover, the transport activity of V-ATPase in the individual intercalated cells of isolated perfused cortical CDs was also measured by determining the recovery in sodium-free solutions of intracellular pH after an acute acid load. The majority of the intercalated cells of the KO mice exhibited blunted V-ATPase activity as compared wild-type controls⁶⁰. Overall, these results represent quite convincing evidence supporting a role of PRR in regulation of V-ATPase-dependent proton secretion from the intercalated cells of the CD.

Perspectives

Our data suggests an intriguing possibility that sPRR may be derived from the intercalated cells and secreted to the lumen and acts on the principal cells, revealing a paracrine pathway mediating the communication between the two CD cell types (Fig. 5). However, to establish this concept, many questions need to be answered with solid experimental data. For example, the concept is heavaly relied on immunobting with antibodies reocngzining the extrcaelular and intracellular domains. The generation of intercalated cell-specific deletion of PRR will be necessary in answering the source of sPRR. However, this attempt was unsuccessful with the use of V-ATPase B1-Cre¹¹² since the null mice died around perinatal period (data not shown). The AQP2-Cre non-selectively targets both intercalated and principal cells^{56, 113}. Therefore, novel approaches are needed to define the contribution of each of these cell types to sPRR production along the CD. We have shown dependence of PRR regulation by the EP₄ receptor during antidiuresis. How and where does AVP stimulate PGE₂ synthesis and activate the EP₄ receptor? Specifically, will this occur in intercalated cells or principal cells via V₁R or V₂R? Additionally, autophagosome accumulation has been postulated to contribute to downregulate NKCC2 as well as AQP2 expression in the nephron PRR KO mice⁶⁰. Future studies are needed to clarify the causality between the autophagic defect and downregulation of the transporter proteins.

Fig. 5.

Schematic illustration of the mechanism of action of sPRR. sPRR is generated from the intercalated cells and secreted to the lumen. Then, sPRR binds FZD8 in a receptor complex on apical membrane of principal cells, resulting in activation of β-catenin which promotes cAMP accumulation, ultimately leading to increased AQP2 transcription and enhanced urine concentration. Adapted from Fig. 8 with modifications of a previous study⁸⁹.

Recent studies have demonstrated that PRR selectively targets α-ENaC in the renal medulla. This is unexpected in light of the traditional view of CCD as a major site for active sodium transport along the CD. It is however unclear as to what signaling mechanism mediates PRR regulation of α-ENaC expression and whether this phenomenon has a broader implication in other types of hypertension in addition to the AngII infusion model.

Na⁺ and water reabsorption in the CD is traditionally thought to be independently controlled by Aldo and AVP. The capability of PRR to simultaneously target both ENaC and AQP2 suggests a common regulatory pathway mediated by PRR that coordinates renal handling of Na⁺ and water. While PRR is shown to mediate the antidiuretic action of AVP, it remains elusive whether PRR expression in the CD is also regulated by Aldo. If so, what mechanisms coordinate the actions of AVP and aldosterone in modulating the expression/activity of PRR?

The discovery of prorenin as a possible physiological ligand of PRR in regulation of Na⁺ and water transport sheds new lights on the long-debated relationship between PRR and the RAS. Strong in vitro data demonstrates that prorenin in the nanomolar range stimulates ENaC activity and AQP2 expression in the CD cells whereas renin is largely effective. Furthermore, these effects of prorenin are independent of the canonical activity of the RAS. However, the in vivo data to support prorenin as a true physiological ligand of PRR is insufficient. In particular, the conditional deletion of renin and PRR in the CD or the whole nephron doesn’t produce the same phenotype. This observation may indicate an additional as yet unknown physiological ligand of PRR may exist in regulation of water homeostasis. Another possibility is that prorenin from extrarenal source may act in an endocrine fashion to activate renal PRR.

PRR is associated with V-ATPase with a truncated fragment of PRR, M8.9, as an important component of the H⁺-ATPase complex. PRR has been shown to be an important determinant of V-ATPase activity in the intercalated cells, promoting urine acidification following an acid load. In addition, nephron PRR deletion causes renal structural abnormalities characterized by prominent autophagosome accumulation in the TAL. In the latter case, it is likely that V-ATPase activity of PRR in the TAL may contribute to cell survival through regulation of organelle acidification and autophagy.

The diversity of PRR’s biological functions may be ascribed to the protease-mediated cleavage that generates the two distinct subunits, sPRR and M8.9. It is likely that following the cleavage, sPRR serves a paracrine function to regulate Na⁺ and water transport in the principal cells whereas M8.9 retains in the intercalated cells to control proton secretion (Fig. 6). I anticipate that a further investigation of the functions of PRR’s different domains will provide novel insights into the complex role of PRR in the kidney.

Fig. 6.

The proposed functions of PRR’s different domains generated by protease-mediated cleavage. After the cleavage, sPRR serves a paracrine function to control water and Na⁺ reabsorption in the principal cells via regulation of AQP2 and ENaC. M8.9 likely retains in the intercalated cells to control proton secretion to the urine. In addition, V-ATPase activity of M8.9 may also regulate vesicle acidification needed for some basic cellular functions such as autophagy, signal transduction, etc.