Mutagenesis of the cleavage site of (pro)renin receptor abrogates aldosterone-salt-induced hypertension and renal injury in mice

Abstract

Soluble (pro)renin receptor (sPRR), the extracellular domain of (pro)renin receptor (PRR), is primarily generated by site-1 protease and furin. It has been reported that sPRR functions as an important regulator of intrarenal renin contributing to angiotensin II (ANG II)-induced hypertension. Relatively, less is known for the function of sPRR in ANG II-independent hypertension such as mineralocorticoid excess. In the present study, we used a novel mouse model with mutagenesis of the cleavage site in PRR (termed as PRR^R279V/L282V or mutant) to examine the phenotype during aldosterone (Aldo)-salt treatment. The hypertensive response of mutant mice to Aldo-salt treatment was blunted in parallel with the attenuated response of plasma volume expansion and renal medullary α-epithelial Na⁺ channel expression. Moreover, Aldo-salt-induced hypertrophy in the heart and kidney as well as proteinuria were improved, accompanied by blunted polydipsia and polyuria. Together, these results represent strong evidence favoring endogenous sPRR as a mediator of Aldo-salt-induced hypertension and renal injury.

NEW & NOTEWORTHY We used a novel mouse model with mutagenesis of the cleavage site of PRR to support soluble PRR as an essential mediator of aldosterone-salt-induced hypertension and also as a potential therapeutic target for patients with mineralocorticoid excess. We firstly report that soluble PRR-dependent pathway medicates the Na⁺-retaining action of aldosterone in the distal nephron, which opens up a new area for a better understanding of the molecular basis of renal handling of Na⁺ balance and blood pressure.

INTRODUCTION

Hypertension, often labeled as “the silent killer,” is a major risk factor for cardiovascular disease, stroke, and renal failure, costing more than $52.4 billion annually (1). Hypertension in nearly half of the patient population is not controlled, and high systolic blood pressure (SBP) is the first leading years of life lost risk factor globally in 2019 (1). The incidence of hypertension is increasing in the United States. The age-adjusted prevalence of hypertension among US adults of ≥20 yr of age was estimated to be 47.3% in 2013–2016 (1). Increased salt intake represents the major environmental factor that contributes to the pathogenesis of essential hypertension. Enhanced sensitivity of blood pressure (BP) to salt intake is present in nearly half of Americans who are afflicted with hypertension (2). Yet, the underlying causes of salt sensitivity in essential hypertension remain elusive. Aldosterone (Aldo) is the principal regulator of transepithelial Na⁺ transport in the kidney, which is central to the maintenance of extracellular fluid volume and blood pressure in mammals (3, 4). Aldo is a major effector hormone in the renin-angiotensin-aldosterone system (RAAS), and its overproduction in primary aldosteronism represents the most common form of secondary hypertension (5, 6). Idiopathic hyperaldosteronism is the most common subtype of primary aldosteronism and accounts for 40–60% of cases (7). Increasing evidence supports the involvement of Aldo in the pathogenesis of essential hypertension. It has been shown that ∼10% of patients with essential hypertension have a high ratio of plasma aldosterone, suggesting inappropriately increased production of Aldo (8, 9). Mineralocorticoid receptor blockade is reported to have antihypertensive and protective effects on cardiovascular and renal injury in animals and humans (10). Finerenone is a newly developed nonsteroidal mineralocorticoid receptor antagonist. Landmark phase III clinical trials, Finerenone in Reducing Kidney Failure and Disease Progression in Diabetic Kidney Disease (FIDELIO-DKD) and Finerenone in Reducing Cardiovascular Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trials, have demonstrated that finerenone has beneficial effects on cardiovascular and renal outcomes in patients with chronic kidney disease and type 2 diabetes (11, 12). Despite the recognized role of Aldo in hypertension and target organ damage, the precise mechanism of action of Aldo is still incompletely understood.

(Pro)renin receptor (PRR) was originally cloned as a specific transmembrane receptor for prorenin and renin implicated in the regulation of the tissue RAAS (13, 14). Besides its association with the RAAS, PRR exerts complex functions in that the cytoplasmic domain is an accessory protein (M8-9), a subunit of V-ATPase (vacuolar-type H⁺-ATPase, also designated ATP6ap2) (15), and it activates multiple signaling transduction pathways including MAPK and β-catenin signaling (16–18). Through multiple mechanisms, PRR participates in a wide variety of developmental and physiological processes involving multiple organs (19, 20). Renal PRR/soluble PRR (sPRR) plays a pivotal role in the maintenance of extracellular fluid volume and BP in mammals largely through fine-tuning urinary Na⁺ excretion that matches Na⁺ intake (21). The majority of studies have shown that deletion of PRR in renal tubules (22) or the collecting duct (CD) (23, 24) impairs urine concentrating capability and attenuates hypertension development during angiotensin II (ANG II) infusion. Renal PRR mediates ANG II-induced hypertension via enhancement of the intrarenal renin-angiotensin system and epithelial Na⁺ channel (α-ENaC) expression (25–27).

Our previous study has shown that the activation of renal PRR contributes to deoxycorticosterone acetate (DOCA)-salt hypertension (28). However, the detailed mechanism of how PRR is involved in DOCA-salt hypertension remains elusive. The extracellular domain of PRR is cleaved by proteases to generate biological active sPRR (29–32). Emerging evidence reveals the biological functions of sPRR in renal handling of Na⁺ and water. In this regard, exogenous administration of histidine-tagged sPRR (sPRR-His) directly upregulated expression of both aquaporin-2 (AQP2) and α-ENaC via activation of β-catenin signaling, contributing to the enhancement of urine concentrating capability (33). Subsequently, the functional role of endogenous sPRR has been suggested by the use of a novel mouse model of mutagenesis of the cleavage site of PRR and characterization of the phenotype during ANG II-induced hypertension (34). What is more, we have shown that sPRR mediates Aldo-induced activation of α-ENaC in cultured CD cells (35). However, there is no in vivo evidence for sPRR mediation of Na⁺-retaining action of Aldo. Therefore, in the present study, we used this novel mouse model with mutagenesis of the cleavage site of PRR to study the function of endogenous sPRR during Aldo-salt-induced hypertension.

METHODS

Animal Care

All animals were cage housed and maintained in a temperature-controlled room with a 12:12-h light-dark cycle, with free access to tap water and standard mice chow. All animal experiments conducted in the present study were approved by the Animal Care and Use Committee of the University of Utah.

Generation of PRR^R279V/L282V Mice

The genotyping of mice with mutagenesis of the cleavage site of PRR (PRR^R279V/L282V mice) has been confirmed by our previous study (34). The brief description is as follows: mutant mice were generated using CRISPR/Cas9-mediated genome engineering through the service from Cygene. PRR^R279V/R282V means that the amino acid arginine at the 279 position and the amino acid leucine at the 282 position of PRR were both mutated to valine. The mouse PRR gene contains nine exons, with the ATG start codon in exon 1 and the TGA stop codon in exon 9. The codons for the furin-recognizing site, arginine-lysine-serine-arginine (RKSR), and site-1 protease-recognizing site, arginine-threonine-isoleucine-leucine (RTIL), are transcribed from exon 8. To mutate these recognizing sites in PRR protein, donor oligos, guidance RNA, and Cas9 mRNA were coinjected into fertilized eggs to generate PRR^{R279V & L282V} knockin C57Bl6 offspring (34).

Mouse Experiments

Male 16- to 20-wk-old PRR^R279V/L282V (mutant) mice and their littermate wild-type (WT) controls were studied. Five days before Aldo-salt treatment, under anesthesia by 2% isoflurane, the radiotelemetric device was implanted via catheterization of the carotid artery. After the baseline BP parameters were recorded, all mice were treated with vehicle or Aldo (A9477, Sigma-Aldrich) at 0.4 mg/kg/day via a subcutaneously implanted minipump (Alzet model 1002, Alza) plus 1% NaCl fluid as drinking fluid for 14 days, and this protocol is referred as Aldo/saline. In a separate study, salt loading was achieved by administering a high-salt diet (HSD) containing 4% NaCl (TD.92034, Envigo) with free access to tap water during Aldo infusion for the same period, referred to as the Aldo/HSD protocol. Mice were not disturbed during the BP recording period. BP was recorded for 4 h per day from 5:00 PM to 9:00 PM. On day 12 of treatment of Aldo/salt treatment, mice were placed in metabolic cage (MMC100, Hatteras Instruments, Cary, NC) for 24-h urine collection. This metabolic cage is featured by the funnel that ensures high urine collection rate. It captures the urine droplets from the mouse, slowly gathering the liquid on the bulb until enough is present to fall into the collection vessel below. At the end of the experiment, under general anesthesia, blood was withdrawn by puncturing vena cava and centrifuged at 4,000 rpm for 10 min to collect plasma for sPRR measurements, and the kidney was harvested, cut into the cortex and inner medulla, and snap frozen.

Immunoblot Analysis

Renal tissues, including the cortex and inner medulla, were lysed and subsequently sonicated in PBS that contained 1% Triton X-100, 250 μM PMSF, 2 mM EDTA, and 5 mM DTT (pH 7.5). Protein concentrations were determined by the use of Coomassie reagent, and 40-μg protein for each sample was denatured in boiling water for 10 min, separated by SDS-PAGE, and then transferred onto nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline, followed by incubation overnight with primary antibody. After being washed with Tris-buffered saline, blots were incubated with horseradish peroxidase-conjugated secondary antibody and visualized using enhanced chemiluminescence. The blots were quantitated using Imagepro-plus. Primary antibodies were as follows: rabbit anti-α-ENaC antibody (1:1,000 dilution, Cat. No. SPC-403D, Stressmarq Biosciences, Victoria, BC, Canada) and rabbit anti-GAPDH antibody (1:1,000 dilution, Cat. No. 5174S, Cell Signaling Technology, Danvers, MA). These antibodies are highly specific, and the full gels usually show a single band (36).

Quantitative RT-PCR

Total RNA isolation and reverse transcription were performed as previously described (26). Oligonucleotides were designed using Primer3 software (available at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The specific primers were as follows: for α-ENaC, 5′-GCGACAACAATCCCCAAG-3′ (sense) and 5′-TGAAGCGACAGGTGAAGATG-3′ (antisense); for β-ENaC, 5′-AAGCACCTGTAATGCCCAAG-3′ (sense) and 5′-ATAGCCCATCCCCACCAG-3′ (antisense); for γ-ENaC, 5′-CGAAGAAACTGGTGGGATTT-3′ (sense) and 5′-GATGGTGGAAAAGCGTGAAG-3′ (antisense); for fibronectin, 5′- CGAGGTGACAGAGACCACAA-3′ (sense) and 5′-CTGGAGTCAAGCCAGACACA-3′ (antisense); for α-smooth muscle actin (α-SMA), 5′- GAGGCACCACTGAACCCTAA-3′ (sense) and 5′- CATCTCCAGAGTCCAGCACA-3′ (antisense); for transforming growth factor (TGF)-β, 5′- TGCTCCCACTCCCGTGGCTT-3′ (sense) and 5′- TTGGGGGACTGGCGAGCCTT-3′ (antisense); for IL-6, 5′- AAGTGCATCATCGTTGTTCATACA-3′ (sense) and 5′- CAGAATTGCCATCGTACAACTCTTTTCTCA-3′ (antisense); and for GAPDH, 5′- GTCTTCACTACCATGGAGAAGG-3′ (sense) and 5′- TCATGGATGACCTTGGCCAG-3′ (antisense). The efficiency of primer pair amplification was determined as previously described (37, 38); all primer pairs resulted in a high amount of specific products (34, 38–40).

Enzyme Immunoassay

sPRR in biological fluids was determined using the following commercially available enzyme immunoassay kits according to the manufacturer’s instructions: kits for sPRR (Cat. No. JP27782, IBL, Toronto, ON, Canada). Urine albumin was determined using a DAC Vantage Analyzer (Siemens).

Plasma Volume Determination

Under general anesthesia with isoflurane (2 mL/min), FITC-dextran 500000 conjugate (FITC-d; 2 mg/100 g, Cat. No. 46947-100MG-F, MilliporeSigma) was injected into the jugular vein. Seven minutes later, blood was withdrawn from the vena cava. Plasma was separated by centrifugation of the blood at 2,700 g for 10 min in the dark. Fluorescence levels were measured at an excitation wavelength of 485 nm and emission wavelength of 520 nm (Synergy Neo2 Hybrid Multi-Mode Reader, BioTek Instruments), and the FITC-d concentration per milliliter of plasma was calculated based on a standard curve generated by serial dilution of the 2 mg/mL FITC-d solution. The standard curve was linear and highly reproducible. The plasma volume data are shown as relative values normalized by body weight (41).

Statistics

GraphPad Prism software (v. 8.4) was used for data analysis. Data are summarized as means ± SE. All data points represent animals that were included in the statistical analyses. Sample sizes were determined on the basis of similar previous studies or pilot experiments. For the BP experiment, statistical significance was determined using two-way ANOVA with repeated measurements. The other animal experiments were performed using regular two-way ANOVA with the Bonferroni test for multiple comparisons or using an unpaired two-tailed Student’s t test for two comparisons. Values of P < 0.05 were considered statistically significant (33, 36).

RESULTS

BP Response to Aldo/Saline

Pharmacological inhibition of PRR with PRO20 attenuated DOCA-salt-induced hypertension in rats (28). However, the functional role of endogenous sPRR during mineralocorticoid excess remains elusive. Therefore, we examined the BP phenotype of PRR^R279V/L282V (mutant) mice during Aldo/saline treatment. Radiotelemetry was used to monitor daily changes in mean arterial pressure (MAP), SBP, diastolic BP (DBP), and heart rate (HR) during the first 12 days. Aldo infusion plus 1% NaCl as drinking fluid gradually elevated MAP, SBP, and DBP and decreased HR in WT mice [day 12: change in (Δ)MAP, 22.4 ± 4.1 mmHg; ΔSBP, 24.5 ± 3.8 mmHg; ΔDBP, 22.4 ± 5.0 mmHg; and ΔHR, −92.4 ± 10.4 beats/min). In contrast, the changes in BP in the mutant-Aldo/saline group were much less (day 12: ΔMAP, 11.7 ± 2.5 mmHg; ΔSBP, 11.9 ± 3.8 mmHg; ΔDBP, 10.8 ± 2.6 mmHg; and ΔHR, −68.2 ± 9.1 beats/min) (Fig. 1). These results suggest that mutant mice exhibited a blunted hypertensive response to Aldo/saline treatment.

Figure 1.

Role of endogenous soluble (pro)renin receptor (sPRR) in aldosterone (Aldo)/saline-induced hypertension. Male wild-type (WT) and male PRR^R279V/L282V (mutant) mice were instrumented with radiotelemetric devices and treated with vehicle or Aldo/saline. The Aldo/saline protocol consisted of subcutaneous implantation of an Aldo minipump in combination with 1% NaCl as drinking fluid. Mean arterial blood pressure (MAP; A), systolic blood pressure (SBP; B), diastolic blood pressure (DBP; C), and heart rate (HR; D) over the first 12 days of Aldo/saline treatment. Statistical significance was determined using two-way ANOVA with repeated measurements, and the P values are indicated. Data are means ± SE. WT-Aldo/saline: n = 6 mice; mutant-Aldo/saline: n = 5 mice.

Role of Endogenous sPRR in Aldo/Saline-Induced Cardiac Hypertrophy and Renal Hypertrophy

Cardiac hypertrophy and renal hypertrophy are well-known consequences of Aldo/saline treatment (42, 43). The ratio of heart to body weight was used as an index of cardiac hypertrophy. Indeed, the heart-to-body weight ratio was increased by Aldo/saline treatment, which was attenuated in mutant mice (Fig. 2A). Moreover, we examined cardiac fibrosis by determining the mRNA expression of fibrosis markers in the heart. As provided in Supplemental Fig. S1, cardiac fibronectin, α-SMA, and TGF-β mRNA expressions were increased in Aldo/saline-WT mice, and these increases were attenuated in Aldo/saline-mutant mice. A similar pattern of changes was observed for the kidney-to-body weight ratio (Fig. 2B), an index of renal hypertrophy. Thus, these mutant mice showed improved cardiac hypertrophy and renal hypertrophy. By ELISA, plasma sPRR was reduced by 57% in mutant mice under basal conditions as previously reported (34). Furthermore, plasma sPRR was increased after Aldo/saline treatment in WT mice, which was almost completely blocked in mutant mice (Fig. 2C). These data support endogenous sPRR as a mediator of Aldo action. As shown in Supplemental Fig. S2, A and B, mutant mice had reduced basal levels of renal renin and plasma ANG II compared with WT mice. Levels of renal renin and plasma ANG II were decreased in WT mice after Aldo/saline treatment, whereas mutant mice showed no response to the treatment.

Figure 2.

Role of endogenous soluble (pro)renin receptor (sPRR) in aldosterone (Aldo)/saline-induced cardiac hypertrophy and renal hypertrophy. A: heart weight/body weight. B: kidney weight/body weight. C: ELISA analysis of plasma sPRR concentrations. Statistical significance was determined using two-way ANOVA with the Bonferroni test for multiple comparisons, and the P values are indicated. Data are means ± SE. Wild-type (WT)-Aldo/saline: n = 6 mice; PRR^R279V/L282V (mutant)-Aldo/saline: n = 5 mice; WT/control (CTR): n = 6 mice; mutant/CTR: n = 6 mice.

Role of Endogenous sPRR in Aldo/Saline-Induced Plasma Volume Expansion

Plasma volume expansion is a hallmark of Na⁺-retaining action of Aldo. With the use of FITC-d, we performed direct measurement of plasma volume. Expansion of plasma volume was observed in WT mice following Aldo/saline treatment, which was attenuated in mutant mice (Fig. 3A). As Aldo/saline has no noticeable effect on erythropoiesis, the change in hematocrit can be used as an indirect marker of plasma volume expansion. Consistent with the plasma volume data, hematocrit was decreased in WT mice following Aldo/saline treatment, which was reversed in mutant mice (Fig. 3B). These data indicate that the activation of endogenous sPRR contributes to Aldo/saline-induced plasma volume expansion.

Figure 3.

Role of endogenous soluble (pro)renin receptor (sPRR) in aldosterone (Aldo)/saline-induced plasma volume expansion. A: direct measurement of plasma volume. Plasma volume was normalized by body weight. B: blood samples were harvested and subjected to analysis of hematocrit. Statistical significance was determined using two-way ANOVA with the Bonferroni test for multiple comparisons, and the P values are indicated. Data are means ± SE. Wild-type (WT)-Aldo/saline: n = 6 mice; PRR^R279V/L282V (mutant)-Aldo/saline: n = 5 mice; WT/control (CTR): n = 6 mice; mutant/CTR: n = 6 mice.

To further explore the mechanism of endogenous sPRR in regulating plasma volume expansion during Aldo/saline treatment, we analyzed physiological data from the metabolic cage experiment. The WT-Aldo/saline group exhibited significant increases in Na⁺ intake (Fig. 4A) and urinary Na⁺ excretion (Fig. 4B). In contrast, the dysregulation of Na⁺ metabolism as reflected by these two parameters was attenuated in mutant-Aldo/saline mice (Fig. 4, A and B). There was no difference in food intake. Na⁺ intake (WT/control: 0.22 ± 0.01 mmol/24 h, mutant/control: 0.22 ± 0.01 mmol/24 h, WT-Aldo/saline: 3.17 ± 0.12 mmol/24 h, and mutant-Aldo/saline: 2.29 ± 0.07 mmol/24 h) and Na⁺ excretion (WT/control: 0.16 ± 0.01 mmol/24 h, mutant/control: 0.15 ± 0.01 mmol/24 h, WT-Aldo/saline: 3 ± 0.12 mmol/24 h, and mutant-Aldo/saline: 2.17 ± 0.07 mmol/24 h). Similarly, Aldo/saline-induced increases in water intake and urine volume were less in mutant mice compared with WT controls (Fig. 4, C and D). These results support a significant role of endogenous sPRR in mediating Aldo/saline-induced dysregulation of Na⁺ and water metabolism.

Figure 4.

Summary of physiological data from metabolic cage experiments. A: Na⁺ intake. B: urinary Na⁺ excretion. C: water intake. D: urine volume. Statistical significance was determined using two-way ANOVA with the Bonferroni test for multiple comparisons, and the P values are indicated. Data are means ± SE. Wild-type (WT)-aldosterone (Aldo)/saline: n = 6 mice; PRR^R279V/L282V (mutant)-Aldo/saline: n = 5 mice; WT/control (CTR): n = 6 mice; mutant/CTR: n = 6 mice.

Analysis of Renal ENaC Expression

We have previously shown that sPRR induces ENaC activation and functions as a mediator of the antinatriuretic action of Aldo (35). Therefore, we performed quantitative RT-PCR and immunoblot analysis of the expression of various subunits of ENaC in both the renal cortex and inner medulla. Renal cortical and inner medullary mRNA expression of α-, β-, and γ-subunits of ENaC were all increased in WT mice after Aldo/saline treatment, but only the increase of α-ENaC in the inner medulla was blunted in mutant mice (Fig. 5, A and B). These changes in renal α-ENaC were confirmed at the protein level by immunoblot analysis (Fig. 5C). These results support the concept that endogenous sPRR selectively affects renal inner medullary α-ENaC to regulate ENaC activity during Aldo/saline treatment.

Figure 5.

Assessment of renal regional expression of epithelial Na⁺ channel (ENaC) subunits. mRNA expression of the three subunits of ENaC in the renal cortex (A) and inner medulla (B) of vehicle or aldosterone (Aldo)/saline-treated male wild-type (WT) and PRR^R279V/L282V (mutant) mice was determined by quantitative RT-PCR and normalized by GAPDH. Statistical significance was determined using two-way ANOVA with the Bonferroni test for multiple comparisons, and the P values are indicated. C: protein expression of α-ENaC in the two regions was verified by immunoblot analysis. The protein abundances were analyzed by densitometry and normalized by GAPDH. The values are shown underneath the blots. Statistical significance was determined using two-way ANOVA with the Bonferroni test for multiple comparisons; **P < 0.01 vs. the WT group and ##P < 0.01 vs. the WT-Aldo/saline group. Data are means ± SE. WT-Aldo/saline: n = 6 mice; PRR^R279V/L282V (mutant)-Aldo/saline: n = 5 mice; WT/control (CTR): n = 6 mice; mutant/CTR: n = 6 mice.

Aldo also plays a key role in the regulation of renal K⁺ excretion (3, 4). Therefore, we examined plasma K⁺ concentration and urinary K⁺ excretion. After the 2-wk Aldo/saline treatment, plasma K⁺ concentration was decreased associated with increased urinary K⁺ excretion, which was attenuated in mutant mice (Supplemental Fig. S2, C and D).

Assessment of Renal Injury Markers

Besides hypertension, hyperaldosteronism is also associated with renal injury (42). Albuminuria was most evident in the Aldo/saline-WT group and was attenuated in the Aldo/saline-mutant group (Fig. 6A). We further examined renal injury by determining mRNA expression of fibrosis markers and inflammation markers in the kidney. As shown in Fig. 6, B–F, fibronectin, α-SMA, IL-6, monocyte chemoattractant protein-1, and TGF-β mRNA expressions were increased in Aldo/saline-WT mice, and these increases of mRNA expressions were prevented in Aldo/saline-mutant mice. Moreover, we performed Masson’s trichrome staining to evaluate renal fibrosis. Aldo/saline-treated WT mice showed increased accumulation of the extracellular matrix, which was attenuated in mutant mice (Supplemental Fig. S3A). A semiquantitative tubulointerstitial fibrosis index of kidney sections (Supplemental Fig. S3B) confirmed this finding. These results indicate that the loss of endogenous sPRR can relieve Aldo/saline-induced renal injury.

Figure 6.

Assessment of renal injury. A: the urinary albumin-to-creatinine ratio was detected in urine. The renal cortex was subjected to quantitative RT-PCR analysis of mRNA expression of fibronectin (B), α-smooth muscle actin (α-SMA; C), interleukin (IL)-6 (D), monocyte chemoattractant protein (MCP)-1 (E), and transforming growth factor (TGF)-β (F) normalized by GAPDH. Statistical significance was determined using two-way ANOVA with the Bonferroni test for multiple comparisons, and the P values are indicated. Values are means ± SE. Wild-type (WT)-aldosterone (Aldo)/saline: n = 6 mice; PRR^R279V/L282V (mutant)-Aldo/saline: n = 5 mice; WT/control (CTR): n = 6 mice; mutant/CTR: n = 6 mice.

Assessment of Phenotype in Aldo/HSD-Treated WT Mice and Mutant Mice

WT and mutant mice were treated with Aldo/HSD for 2 wk. Although Na⁺ intake became comparable between the two genotypes (Supplemental Fig. S4A), the hypertensive response of mutant mice to Aldo/HSD treatment was still blunted (Fig. 7) in parallel with attenuated hypertrophy in the heart and kidney as well as proteinuria (Supplemental Fig. S4, B–D). Hematocrit was decreased in WT mice following Aldo/HSD treatment, which was reversed in mutant mice (Supplemental Fig. S4E). As provided in Supplemental Fig. S4F, with the 4% NaCl diet, Na⁺ intake remained unchanged in the two genotypes but suppression of renal medullary α-ENaC mRNA expression was consistently observed. This result suggests that the alteration of renal medullary α-ENaC not be secondary to altered Na⁺ intake.

Figure 7.

Roles of endogenous soluble (pro)renin receptor (sPRR) in aldosterone (Aldo)/high-salt diet (HSD)-induced hypertension. Male wild-type (WT) and male PRR^R279V/L282V (mutant) mice were instrumented with radiotelemetric devices and treated with Aldo/HSD. The Aldo/HSD protocol consisted of subcutaneous implantation of an Aldo minipump in combination with 4% NaCl diet as food. Mean arterial blood pressure (MAP; A), systolic blood pressure (SBP; B), diastolic blood pressure (DBP; C), and heart rate (HR; D) over the first 12 days of Aldo/HSD treatment. Statistical significance was determined using two-way ANOVA with repeated measurements, and the P values are indicated. Values are means ± SE. WT-Aldo/HSD: n = 6 mice; mutant-Aldo/HSD: n = 6 mice.

DISCUSSION

Activation of renal PRR has been implicated in the pathogenesis of hypertension induced by mineralocorticoid access (28). However, less is known about the involvement of sPRR under this condition. The availability of a novel mouse model with the mutagenesis of the cleavage site of PRR offers a unique opportunity to define the biological function of endogenously produced sPRR. Accordingly, the present study examined the role of sPRR during mineralocorticoid excess by analyzing the phenotype of mutant mice during Aldo-salt treatment. Radiotelemetry demonstrated that the mutant mice exhibited a blunted hypertensive response to Aldo-salt treatment accompanied by attenuation of cardiac and renal hypertrophy, plasma volume expansion, and indexes of renal injury. Furthermore, we provided evidence for renal medullary α-ENaC as a major molecular target of sPRR during Aldo-salt hypertension. Together, these results indicate endogenous sPRR as a mediator of Aldo-salt-induced hypertension and renal injury.

The present study took advantage of the recently generated novel mouse model of mutagenesis of the cleavage site of PRR generated by CRISPR/Cas9 strategy. sPRR production in mutant mice exhibited an over 90% reduction in the kidney and 50% in circulation, making the model suitable for investigating the functional role of renal sPRR (34). In the previous study, we speculated that the cleavage efficiency may vary depending on the type of tissues expressing a specific type of cleavage protease. Furthermore, the site-1 protease may function more effectively in the kidney than in other tissues based on the assumption that furin may play a minimal role in the cleavage process (34). We performed a comprehensive analysis of the phenotype during Aldo-salt treatment. The Aldo-salt-induced hypertensive response was directly evaluated using radiotelemetry and indirectly reflected by the extent of cardiac and renal hypertrophy. The mutant mice consistently exhibited blunted hypertensive and cardiac and renal hypertrophic responses to Aldo-salt treatment compared with WT controls. Aldo-salt-induced hypertension features plasma volume expansion. Therefore, we performed two independent techniques with direct measurement of plasma volume using FITC-d and indirect assessment of plasma volume by the determination of hematocrit. The consistent data generated by the different methods confirmed attenuated plasma volume expansion in Aldo-salt-treated mutant mice. Finally, metabolic cage experiments revealed predicted changes in Na⁺ and water balance in these animals. Together, the compelling data from measurements of BP, plasma volume, and Na⁺ balance demonstrate a nonredundant role of sPRR in Na⁺ reabsorption and hypertension induced by Aldo-salt treatment.

The alteration of Na⁺ intake in mutant mice following Aldo/saline treatment suggests the involvement of a central mechanism in mediating Na⁺-retaining and prohypertensive actions of sPRR. This finding is consistent with a previous report on the important role of central PRR in the regulation of Na⁺ and water intake during DOCA/saline treatment (44). Specifically, neuron-specific deletion of PRR attenuated Na⁺ and water intake under this condition (44). Along this line, activation of PRR in the central nervous system increases sympathetic outflow in anesthetized rats (45). To address this issue, we administered salt via 4% NaCl diet rather than 1% saline. In this way, Na⁺ intake became comparable between the two genotypes, and we were able to observe the same phenotype observed previously with the Aldo/saline protocol. This result favors an important role of a renal versus central mechanism in mediating the prohypertensive action of sPRR, at least in the current experimental models.

ANG II and Aldo are the two important effector hormones within the RAAS, and they induce a hypertensive response through overlapping but distinctive mechanisms. ANG II-induced hypertension depends on the activation of intrarenal renin, whereas the action of Aldo is renin-independent. In addition, the renal cyclooxygenase (COX)-2/EP4 pathway mediates the activation of intrarenal renin as well as PRR during ANG II-induced hypertension (26, 46). In contrast, renal PRR expression induced by DOCA-salt was independent of COX-2 (28). Therefore, distinctive mechanisms underlie the activation of renal PRR during ANG II infusion and mineralocorticoid excess. Although the COX-2/EP4 pathway is known to mediate ANG II-induced activation of PRR and intrarenal renin, the mechanism of how Aldo-salt induces renal PRR expression remains elusive.

Despite differences in the mechanisms upstream of renal PRR during ANG II infusion and mineralocorticoid excess as discussed earlier, sPRR is similarly involved. A majority of previous studies have demonstrated the involvement of renal sPRR in the pathogenesis of ANG II-induced hypertension (34, 36, 47). These studies further showed that sPRR acts via activation of intrarenal renin and renal medullary α-ENaC. Indeed, CD renin contributes to ANG II-induced hypertension (48). Renin expression in the CD and urinary excretion of renin are elevated by ANG II infusion. Transgenic overexpression of renin in the CD increases BP, and CD-specific deletion of renin abrogates the hypertensive response to ANG II. On the other hand, activation of renal medullary ENaC similarly contributes to ANG II-induced hypertension (24). However, the relative importance of intrarenal renin versus renal medullary ENaC in mediating the prohypertensive action of sPRR remains elusive. Another possibility is that sPRR may indirectly stimulate renal medullary α-ENaC expression or activity via activation of intrarenal renin. Indeed, ANG II exerts a direct stimulatory effect on ENaC activity in the CD (49). In the present study, we, for the first time, report that sPRR targets renal medullary α-ENaC in a renin-independent manner to mediate Aldo-salt-induced hypertension. Consistent with this finding, sPRR directly induced α-ENaC transcription via β-catenin signaling in cultured CD cells in which an intact RAS is unlikely to exist (35). Together, these results represent strong evidence for the notion that sPRR can act in both a renin-dependent and -independent manner to induce a hypertensive response to diverse hypertensive stimuli. Therefore, targeting sPRR generation may hold promise for the development of a novel antihypertensive therapy in patients with hypertension with different etiologies.

A series of our studies consistently demonstrated that pharmacological antagonism of renal medullary PRR (25), CD-specific deletion of PRR (24), and mutagenesis of the cleavage site of PRR (34) selectively blunted renal medullary expression of α-ENaC but not the β- or γ-subunits. In agreement with our findings, Quadri et al. (50) showed that adenovirus-mediated delivery of short hairpin RNA against PRR to the rat kidney selectively reduced renal medullary expression of α-ENaC but not the β- or γ-subunits. In cultured A6 renal epithelial cells, Aldo increased the abundance of all three ENaC subunit mRNAs but had a selective effect to increase the rate of synthesis of α-subunit protein as measured in pulse-chase experiments, suggesting regulation of translation (51). Furthermore, salt restriction or Aldo infusion in rats selectively induces renal protein expression of α-ENaC (52). Indeed, among the three subunits, the synthesis of α-ENaC is a limiting factor in the assembly of the ENaC complex (51). In agreement with these findings, the present study showed that both renal cortical and renal medullary expression of α-ENaC was elevated by Aldo-salt treatment in WT mice, but only the renal medullary expression of α-ENaC was blunted in mutant mice. It seems reasonable to speculate that sPRR-mediated activation of renal medullary α-ENaC may in part contribute to Aldo-salt-induced hypertension, and sPRR medicates the Na⁺-retaining action of Aldo in the distal nephron.

Apart from the physiological role of PRR/sPRR in the regulation of Na⁺ reabsorption and BP, PRR/sPRR has been implicated in pathophysiology of renal disease. It has been shown that PRR can promote kidney injury and fibrosis in vivo by amplifying Wnt/β-catenin signaling (53). Another study from our laboratory has reported that sPRR promotes the fibrotic response in vitro by activating Akt/β-catenin/Snail signaling (54). It is likely that sPRR also mediates Aldo-salt-induced renal injury via the β-catenin signaling pathway. Another possibility is that renal injury may be secondary to hypertension in this model.

In summary, the present study examined the regulation and function of endogenous sPRR in a mouse model of Aldo-salt-induced hypertension that is renin independent. Mice with mutagenesis of the cleavage site of PRR were largely resistant to Aldo-salt-induced hypertension, plasma volume expansion, and renal injury. The underlying mechanism involves sPRR mediation of selective activation of renal medullary α-ENaC during Aldo-salt treatment. The present study has opened a new area concerning a novel role of sPRR in activating a specific ENaC subunit within a specific kidney region during mineralocorticoid excess.

Perspectives and Significance

The prevalence of hypertension is increasing, with nearly one in two adults in the United States suffering from this disease and BP being uncontrolled for most of the cases (55). It is of high significance to understand the disease mechanism as well as the therapeutic target. Hyperaldosteronism due to adenoma in the adrenal gland is a common form of secondary hypertension. Furthermore, dysregulation of Aldo production or signaling directly/indirectly contributes to the pathogenesis of essential hypertension as well as its cardiac and renal complications. Despite intensive investigation, the mechanism of action of Aldo remains incompletely understood. The present study provides key in vivo evidence supporting sPRR as an essential mediator of Aldo-salt-induced hypertension and also as a potential therapeutic target to treat human disease due to overactivation of Aldo.

DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request.

SUPPLEMENTAL DATA

Supplemental Figs. S1–S4: https://doi.org/10.6084/m9.figshare.20639463.v4.

GRANTS

This work was supported by National Institutes of Health Grants HL139689 (to T.Y.), DK104072 (to T.Y.), HL135851 (to T.Y.), and HL160020 (to T.Y.), Merit Review IK6BX005223 and BLR&D Research Career Scientist Award IK6BX005223-04 from the Department of Veterans Affairs (to T.Y.).

DISCLOSURES

None of the authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

T.Y. and Z.F. conceived and designed research; Z.F., H.Z., K.K., J.L., and Y.C. performed experiments; Z.F., H.Z., and J.L. analyzed data; Z.F. interpreted results of experiments; Z.F. and H.Z. prepared figures; Z.F. drafted manuscript; Z.F. and T.Y. edited and revised manuscript; Z.F. and T.Y. approved final version of manuscript.