High potassium promotes mutual interaction between (pro)renin receptor and the local renin-angiotensin-aldosterone system in rat inner medullary collecting duct cells

Abstract

(Pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD) with unclear functional implication. It is not known whether CD PRR is regulated by high potassium (HK). Here, we aimed to investigate the effect of HK on PRR expression and its role in regulation of aldosterone synthesis and release in the CD. In primary rat inner medullary CD cells, HK augmented PRR expression and soluble PPR (sPRR) release in a time- and dose-dependent manner, which was attenuated by PRR small interfering RNA (siRNA), eplerenone, and losartan. HK upregulated aldosterone release in parallel with an increase of CYP11B2 (cytochrome P-450, family 11, subfamily B, polypeptide 2) protein expression and upregulation of medium renin activity, both of which were attenuated by a PRR antagonist PRO20, PRR siRNA, eplerenone, and losartan. Similarly, prorenin upregulated aldosterone release and CYP11B2 expression, both of which were attenuated by PRR siRNA. Interestingly, a recombinant sPRR (sPRR-His) also stimulated aldosterone release and CYP11B2 expression. Taken together, we conclude that HK enhances a local renin-angiotensin-aldosterone system (RAAS), leading to increased PRR expression, which in turn amplifies the response of the RAAS, ultimately contributing to heightened aldosterone release.

(pro)renin receptor (prr) is a single-transmembrane receptor for renin and prorenin with almost equal binding affinity, and this binding leads to increased catalytic activity (28, 30), contributing to the conversion of angiotensinogen to angiotensin I (ANG I). PRR is cleaved by furin (6) or ADAM19 (a disintegrin and metalloprotease domain 19) (51) to generate intracellularly a truncated soluble form with a NH₂-terminal region (sPRR). sPRR can be secreted into the extracellular space, such as plasma (6) and urine (14), and into a variety of cell types (51), but can also be retained inside the cell through stable and direct or indirect membrane association (40). Both full-length PRR (fPRR) and sPRR bind renin and prorenin (6) to, respectively, increase renin activity and induce nonproteolytic activation of prorenin (14). Although PRR was originally discovered as a potential regulator of renin-angiotensin system, existing literature on this topic has been quite controversial (8a, 28a). The controversy mostly stems from the observations that PRR is associated with vacuolar H⁺-ATPase in Wnt/β-catenin signaling pathway and is involved in embryogenesis, in both mammals and low vertebrates (7, 19, 37). Increasing evidence from our group (22, 23, 45) and others (20, 21) favors the renin-regulatory role of PRR in the kidney and brain. The multiple functional roles of PRR in regulation of development and physiology appear to be conferred by a common pathway mediated by β-catenin (23, 45).

Within the kidney, PRR is highly expressed in the intercalated cells of the collecting ducts (CDs) (1). Renal expression of PRR is altered by changing salt balance, implicating a potential role of PRR in regulation of electrolyte metabolism (16, 25, 36). This notion is also supported by the observation that overexpression of human PRR in transgenic rats elevates aldosterone production (5). Aldosterone is thought to be critical for potassium homeostasis. However, it is unknown whether PRR expression is regulated by potassium. We hypothesized that PRR may regulate aldosterone release through an influence on local renin-angiotensin-aldosterone system (RAAS). Thus the primary goal of this study was to test whether high potassium (HK) influenced PRR expression in primary rat inner medullary CD (IMCD) cells.

MATERIALS AND METHODS

Primary cultures of rat IMCD cells.

Primary cultures enriched in rat IMCD cells were prepared from pathogen-free male Sprague-Dawley rats (150∼200 g) in hypertonic conditions, as previously described (9). The protocols were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University, China. The IMCD cells were cultured in hypertonic medium [Dulbecco's modified Eagle's medium (DMEM)-Ham's F-12, 0.5 μM 8-Br-cAMP, 10 ng/ml epidermal growth factor, 130 mM sodium chloride, 80 mM urea, 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 2 mM l-glutamine, 10,000 U/ml penicillin-streptomycin, 10% fetal bovine serum, 640 mosmol/kgH₂O]. Cells were serum-starved for 12 h and then treated with 7.5, 10, and 15 mM potassium chloride (KCl) for 12 h; or pretreated with a PRR blocker, PRO20 (2 μM; the first 20 amino acid residues of the prorenin prosegment, L¹PTDTASFGRILLKKMPSVR²⁰) (21), or 10 μM eplerenone, or 10 μM losartan for 1 h; or transfected with PRR small interfering RNA (siRNA) (catalog no. SR411813, OriGene, Rockville, MD) and then treated with 10 mM KCl for various time periods; or treated with 10 mM sodium gluconate (NaG), potassium gluconate (KG), KCl, choline chloride (CC), 5 or 10 mM barium chloride (BaCl₂), 100 nM prorenin, and 10 nM sPRR-His for 48 h. sPRR-His was a recombinant sPRR that included sPRR (residues 17–274) and an 8-histidine tag (23). In control groups, d-mannitol was used to clamp hyperosmotic conditions. After the treatment, the cells were harvested for PRR and CYP11B2 (cytochrome P-450, family 11, subfamily B, polypeptide 2) expression analysis and the cell medium for sPRR and aldosterone assay by using a soluble (Pro)renin Receptor Assay kit (catalog no. 27782, Immuno-Biological Laboratories, Gunma, Japan) and aldosterone EIA kit (catalog no. 10004377, Cayman Chemical), according to the manufacturer's instructions, and expressed as picograms per milligram of cellular protein content.

M1 cell line.

The M1 cells were cultured in DMEM/F-12, 10,000 U/ml penicillin-streptomycin, and 10% fetal bovine serum. Cells were serum-starved for 12 h and then treated with 10 mM KG, KCl, CC, or 5 or 10 mM BaCl₂ for 48 h. In the control group, d-mannitol was used to clamp hyperosmotic conditions. After the treatment, the cells were harvested for PRR expression analysis.

Renin activity assay.

Renin activity in medium was determined by the delta value of the ANG I generation using an ELISA kit from the sample incubating at 4°C and 37°C for 1 h, respectively. ANG I generation was assayed by using an ANG I EIA kit (S-1188, Peninsula Laboratories International), according to the manufacturer's instructions. The values were expressed as nanograms per milliliter per hour of generated ANG I.

Immunoblot analysis.

Cells were lysed and subsequently sonicated in RIPA buffer (Biocolors, Shanghai, China) with protease inhibitor cocktail (Roche, Berlin, Germany). Homogenates were centrifuged at 12,000 g for 10 min at 4°C, and protein concentrations were determined with the Pierce BCA Protein Assay Kit (catalog no. NCI3225CH, Thermo Scientific, Rockford, IL), according to the manufacturer's instructions. Thirty micrograms of protein for each sample were denatured in boiling water for 10 min and then separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane (Immobilion-P, Millipore, Bedford, MA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature, followed by incubation with primary antibodies (PRR, 1:1,000 dilution, ab40790, Abcam; CYP11B2, 1:1,000 dilution, MAB6020, Merck Millipore; β-actin, 1:10,000 dilution, A-2066, Sigma-Aldrich), diluted in antibody dilution buffer (1.5 g bovine serum albumin, 0.1 g NaN₃, 50 ml TBST) overnight at 4°C. After washing with TBST, membranes were incubated with secondary antibodies (goat anti-rabbit/mouse horseradish peroxidase-conjugated secondary antibody) (Thermo Scientific) for 1 h at room temperature and visualized with enhanced chemiluminescence (Thermo Scientific). They were then processed for signal detection by using Tanon 5200 Luminescent Imaging Workstation (Tanon, Shanghai, China) and densitometric analysis by using Image-Pro Plus 6.0 software. The expression of protein was calculated in relation to β-actin.

Quantitative RT-PCR.

Cells were homogenized in TRIzol reagent (catalog no. 15596018, Life Technologies). Total RNA concentrations were determined using NANODROP 2000 Spectrophotometer (Thermo Scientific). Total RNA (1 μg) was used as a template for reverse transcriptase by using the Transcriptor First Strand cDNA Synthesis Kit (catalog no. 04379012001, Roche, Berlin, Germany), according to the manufacturer's instructions. Quantitative PCR was performed using the ABI Prism StepOnePlus System (Applied Biosystems, Life Technologies) and the FastStart Universal SYBR Green Master (ROX) (catalog no. 04913914001, Roche, Berlin, Germany), according to the manufacturer's instructions. Primers are as follows: for PRR, 5′-ATCCTTGAGACGAAACAAGA-3′ (sense) and 5′-AGCCAGTCATAATCCACAGT-3′ (antisense); primers for CYP11B2, 5′-TGAGACGTGGTGTGTTCTTGC-3′ (sense) and 5′-GGCCTCCAAGAAGTCCCTTGC-3′ (antisense); primers for GAPDH, 5′-GTCTTCACTACCATGGAGAAGG-3′ (sense) and 5′-TCATGGATGACCTTGGCCAG-3′ (antisense). All reactions were run in duplicate. The data were shown as a relative value normalized by GAPDH.

Statistical analysis.

Data are summarized as means ± SE. Statistical analysis was performed by using one-way analysis of variance with the Bonferroni test for multiple comparisons or by unpaired Student's t-test for two comparisons using IBM SPSS 19 software. P < 0.05 was considered statistically significant.

RESULTS

To examine the direct effect of HK on PRR expression, we established primary cultures of rat IMCD cells in six-well plates. After reaching confluence, the cells were exposed to various concentrations of KCl from 5–15 mM for 12 h. By immunoblotting, fPRR protein was detected as a 42-kDa band, which was enhanced by KCl in a dose-dependent manner with a peak response at 10 mM KCl (Fig. 1, A and B). For time course studies, IMCD cells were exposed to 10 mM KCl for various times ranging from 3–48 h. The increase of fPRR protein expression was noticeable at 12 h and was maximal at 48 h (Fig. 1, C and D). Medium sPRR as assessed by ELISA was elevated after 24 h and more significantly increased at 48 h (Fig. 1E). To examine whether K⁺ or Cl⁻ affected PRR expression, we treated IMCD cells with 10 mM NaG, KG, KCl, CC for 48 h, both fPRR protein (Fig. 2, A and B) and PRR mRNA (Fig. 2C) expression, as well as medium sPRR (Fig. 2D), were increased in cells treated by KG and KCl, but not NaG and CC. High KCl can cause a significant depolarization of cell membrane. To examine whether high KCl-induced upregulation of PRR expression was due to membrane depolarization, we treated IMCD cells with 5 or 10 mM BaCl₂ for 48 h and examined fPRR protein expression. By immunoblotting, fPRR protein abundance unchanged in primary rat IMCD cells exposed to BaCl₂ (Fig. 2, E and F). The same experiments performed using M1 cells yielded similar results (Fig. 3). These results have ruled out involvement of membrane depolarization in regulation of PRR expression.

Fig. 1.

Effect of KCl on PRR expression in primary rat IMCD cells. A and B: dose response of KCl regulation of PRR expression. The cells were exposed to KCl at various concentrations for 12 h, and then PRR protein expression was analyzed by immunoblotting (A) and densitometric analysis (B). The expression was normalized by β-actin. fPRR, full-length PRR. **P < 0.01 and ***P < 0.001 vs. control. #P < 0.05 and ##P < 0.01 vs. 7.5 mM. C and D: time course studies of KCl regulation of PRR expression. The cells were exposed to 10 mM KCl for the indicated time periods, and PRR protein expression was analyzed by immunoblotting (C) and densitometric analysis (D). The expression was normalized by β-actin. **P < 0.01 vs. control. ***P < 0.01 vs. control or 3 h. #P < 0.05 vs. 24 h. E: ELISA measurement of medium sPRR was normalized by total cellular proteins. **P < 0.01 and ***P < 0.001 vs. control. ##P < 0.01 vs. 24 h. Values are means ± SE; N = 6 per group.

Fig. 2.

Effect of K⁺ vs. Cl⁻ on PRR expression in primary rat IMCD cells. The cells were exposed to 10 mM sodium gluconate (NaG), potassium gluconate (KG), KCl, and choline chloride (CC) for 48 h, and PRR protein expression was analyzed by immunoblotting (A) and densitometric analysis (B). The expression was normalized by β-actin. N = 5 per group. C: IMCD cells were exposed to 10 mM NaG, KG, KCl, or CC for 24 h. Quantitative RT-PCR analysis of CYP11B2 mRNA expression was normalized by GAPDH. N = 5 per group. D: IMCD cells were exposed to 10 mM NaG, KG, KCl, or CC for 48 h. ELISA measurement of medium sPRR was normalized by total cellular proteins. N = 5 per group. IMCD cells were exposed to 5 or 10 mM BaCl₂ for 48 h, and PRR protein expression was analyzed by immunoblotting (E) and densitometric analysis (F). The expression was normalized by β-actin. N = 4 per group. Values are means ± SE. *P < 0.05 and ***P < 0.001 vs. control.

Fig. 3.

Effect of K⁺ and Ba²⁺ on PRR expression in M1 cells. The cells were exposed to 10 mM sodium gluconate (NaG), potassium gluconate (KG), KCl, choline chloride (CC), 5 mM BaCl₂, and 10 mM BaCl₂ for 48 h, and PRR protein expression was analyzed by immunoblotting (A) and densitometric analysis (B). The expression was normalized by β-actin. Values are means ± SE; N = 9–12 per group. ***P < 0.001 vs. control.

To investigate the function of PRR, we developed a siRNA against PRR. siRNA-mediated knockdown of PRR in rat IMCD cells effectively suppressed HK-induced PRR protein expression (Fig. 4, A and B) and medium sPRR concentration (Fig. 4C). With this approach, together with a PRR decoy inhibitor PRO20, we examined the role of PRR in the local renin response to HK. HK induced medium renin activity, which was consistently suppressed by PRR siRNA and PRO20 (Fig. 4D).

Fig. 4.

Validation of siRNA-mediated PRR knockdown and its effect on renin activity. The rat IMCD cells were transfected with PRR siRNA and then treated with 10 mM K⁺ for 48 h. PRR protein expression was analyzed by immunoblotting (A) and densitometric analysis (B). C: ELISA analysis of medium sPRR. D: measurement of medium renin activity in IMCD cells. In this experiment, besides PRR siRNA, PRO20 was used to inhibit PRR. Values are means ± SE; N = 5 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control. ###P < 0.001 vs. HK.

Aldosterone is one of the chief hormones of the RAAS and the primary hormone regulating K⁺ homeostasis. We examined medium aldosterone concentration by using ELISA. The exposure to KG and KCl, but not NaG or CC, increased medium aldosterone (Fig. 5A). CYP11B2 is the key enzyme for aldosterone synthesis, catalyzing the final step of aldosterone synthesis wherein deoxycorticosterone is converted to aldosterone (34). We hypothesized that CYP11B2 may represent the source of local aldosterone generation. Therefore, we examined the effect of HK on CYP11B2 expression in cultured rat IMCD cells. Both KG and KCl treatment, but not NaG and CC treatment, increased CYP11B2 protein (Fig. 5, B and C) and mRNA (Fig. 5D) expression.

Fig. 5.

Effect of K⁺ vs. Cl⁻ on aldosterone release and CYP11B2 expression in primary rat IMCD cells. The cells were exposed to 10 mM NaG, KG, KCl, or CC for 48 h. A: ELISA analysis of medium aldosterone concentration. The value was normalized by total cellular proteins. Immunoblotting (B) and densitometric analysis (C) of CYP11B2 protein expression are shown. The expression was normalized by β-actin. D: IMCD cells were exposed to 10 mM NaG, KG, KCl, or CC for 24 h. Quantitative RT-PCR analysis of CYP11B2 mRNA expression was normalized by GAPDH. Values are means ± SE; N = 5–8 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

Following exposure to HK, medium aldosterone gradually increased over time with a maximal induction at 48 h (Fig. 6A). In the presence of PRO20, the HK-induced aldosterone release at 48 h was significantly suppressed (Fig. 6A). HK treatment time-dependently induced increases in CYP11B2 protein (Fig. 6, B and C), coinciding with the time course of aldosterone release (Fig. 6A). In a separate experiment, we examined the effect of PRR siRNA on HK-induced aldosterone release and CYP11B2 expression in cultured rat IMCD cells. The induction of both aldosterone release and CYP11B2 expression was effectively suppressed by PRR siRNA (Fig. 6, D and E), an observation analogous to that obtained with PRO20.

Fig. 6.

Effect of PRO20 and PRR siRNA on HK-induced aldosterone secretion and CYP11B2 expression in primary rat IMCD cells. A–C: the cells were exposed to 10 mM K⁺ for the indicated time periods. The effect of PRO20 was examined at 48 h. A: ELISA analysis of medium aldosterone concentration. Immunoblotting (B) and densitometric analysis (C) of CYP11B2 protein expression are shown. The expression was normalized by β-actin. N = 6 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control. #P < 0.05 vs. 12 h. ^&&P < 0.01 and ^&&&P < 0.001 vs. 48 h. D–F: the cells were transfected with PRR siRNA and then treated with 10 mM K⁺ for 48 h. D: ELISA analysis of medium aldosterone concentration. Immunoblotting (E) and densitometric analysis (F) of CYP11B2 protein expression are shown. The expression was normalized by β-actin. Values are means ± SE; N = 5 per group. **P < 0.01 and ***P < 0.001 vs. control. #P < 0.05 and ###P < 0.001 vs. HK.

To examine the direct effect of sPRR and prorenin on aldosterone release and CYP11B2 expression, IMCD cells were exposed to 100 nM prorenin or 10 nM sPRR-His for 48 h, followed by ELISA analysis of medium aldosterone and immunoblotting analysis of CYP11B2 protein. Both sPRR and prorenin increased aldosterone release (Fig. 7A) and CYP11B2 protein expression (Fig. 7, B and C). PRR siRNA suppressed prorenin-induced upregulation of aldosterone release (Fig. 7A) and CYP11B2 expression (Fig. 7, B and C).

Fig. 7.

Effect of sPRR-His and prorenin on aldosterone release and CYP11B2 expression in primary rat IMCD cells. The cells were treated with 10 nM sPRR or 100 nM prorenin for 48 h. A: ELISA analysis of medium aldosterone concentration. Immunoblotting (B) and densitometric analysis (C) of CYP11B2 protein expression are shown. The expression was normalized by β-actin. Values are means ± SE; N = 5 per group. ***P < 0.001 vs. control. ###P < 0.001 vs. prorenin.

The above result suggested PRR as a regulator of the local RAAS in the IMCD cells during HK treatment. A possibility may exist that the local RAAS may regulate PRR expression in this setting. HK may promote the mutual interaction between PRR and the local RAAS. Therefore, we examined the effect of inhibition of mineralocorticoid receptor (MR) and ANG II type 1 receptor on HK-induced PRR expression. HK induced parallel increases in fPRR protein expression and sPRR release, as assessed by immunoblotting and ELISA in the IMCD cells, respectively, both of which were attenuated by eplerenone and losartan (Fig. 8, A–C). Interestingly, the HK-induced increase of medium renin activity was also suppressed in parallel (Fig. 8D).

Fig. 8.

Effect of eplerenone and losartan on HK-induced PRR expression and renin activity in primary rat IMCD cells. The cells were pretreated with 10 μM eplerenone or 10 μM losartan for 1 h and then treated with 10 mM K⁺ for 48 h. Immunoblotting (A) and densitometric analysis (B) of PRR protein expression are shown. C: ELISA analysis of medium sPRR. D: measurement of medium renin activity. Values are means ± SE; N = 5 per group. **P < 0.01 and ***P < 0.001 vs. control. ##P < 0.01 and ###P < 0.001 vs. HK.

DISCUSSION

In the present study, we investigated the effect of HK on PRR expression and sPRR release and further explored the role of PRR in in regulation of local RAAS in primary cultured rat IMCD cells. In these cells, HK increased PRR protein expression in a dose- and time-dependent manner, in parallel with the enhancement of medium renin activity and aldosterone release and CYP11B2 expression, indicative of activation of local RAAS. HK-induced activation of local RAAS was blocked by PRR inhibition. In addition, both sPRR-His and prorenin increased aldosterone release and CYP11B2 expression. Interestingly, HK-induced increase in PRR expression and sPRR release were attenuated by inhibition of MR and AT1 receptor. Therefore, these results suggest that HK induces PRR and the local RAAS, the two of which are dependent on each other.

PRR is expressed in a number of renal structures, including the CD, the distal convoluted tubule, renal vasculature, etc. (1). Renal medullary PRR is shown to regulate local renin response and thus blood pressure during ANG II infusion (44). Emerging evidence suggests that activation of PRR stimulates epithelial Na⁺ channel (ENaC) activity in cultured mpkCCD cells (22) and increases ENaC expression in rats (32) and deletion of nephron PRR causes urine concentrating defect (35). However, whether renal PRR is involved in potassium homeostasis is not known. We examined the regulatory mechanism of PRR in cultured CD cells in response to HK treatment. The present study demonstrated a direct stimulatory effect of K⁺ but not Cl⁻ on PRR expression and aldosterone synthesis in cultured IMCD cells, which is independent of membrane depolarization. This observation has extended our laboratory's recent report on the in vivo effect of high K⁺ intake on renal PRR expression (48).

It is usually thought that aldosterone plays a pivotal role in determining K⁺ secretion from the CD besides the regulation of ENaC. The basic concept is that an increase in dietary K⁺ intake leads to an elevation of plasma K⁺, which stimulates adrenal generation of aldosterone. However, several dilemmas still exist. For example, it is unclear how circulating aldosterone coordinates the regulation of both Na⁺ and K⁺ excretion by the kidney. Second, under some conditions, renal K⁺ excretion can be matched to K⁺ intake in the absence of measurable change in plasma K⁺ or aldosterone (31, 33, 42). Lastly, Adisonian patients can maintain K⁺ homeostasis, despite the absence of MR (8, 41). These results challenge the essential role of circulating aldosterone in regulation of K⁺ homeostasis. In contrast, severe K⁺ retention is demonstrated in mice with systemic or nephron-specific deletion of MR (3, 43). What is more, our laboratory's previous study (48) showed administration of PRO20 and spironolactone in K⁺-loaded adrenalectomy rats' elevated plasma K⁺ level and decreased urinary K⁺ excretion and blunted HK-induced upregulation of renal outer medullary K⁺ channel, calcium-activated potassium channel subunit α1, α-Na⁺-K⁺-ATPase, and ENaC subunit-β (β-ENaC). These findings seem to suggest a possibility that PRR-dependent extra-adrenal generation of aldosterone may be involved in K⁺ homeostasis.

Aldosterone is thought to primarily derive from the adrenal cortex and regulated mainly by ANG II, corticotropin, and potassium (2, 26, 50). Besides the adrenal glands, local biosynthesis of aldosterone in the kidney has been reported (39, 49), although the physiological relevance of this finding still remains elusive. In line with the previous in vivo study that high K⁺ intake increased urinary and intrarenal aldosterone levels (48), herein, we showed that, in cultured IMCD cells, HK increased aldosterone synthesis and release, and this increase was completely blocked by siRNA-mediated PRR knockdown and PRO20. Along this line, activation of PRR by prorenin or sPRR-His induced local aldosterone production. These results suggest the local aldosterone generation depended on activation PRR.

CYP11B2 is a rate-limiting enzyme responsible for aldosterone synthesis mainly in the adrenal gland (17) and has been found in blood vessels (40a), heart (12, 18), brain (13, 24), and the kidney (39, 49). Renal expression of CYP11B2 is elevated by low-salt, high-K⁺ ANG II treatment and diabetes (39, 48, 49). In the present study, we found that HK induced CYP11B2 expression in cultured IMCD cells, which was consistently blocked by PRO20 and PRR siRNA, in line with the previous in vivo study (48). The changes in CYP11B2 are consistent with those of aldosterone production. These findings support CYP11B2 as a potential enzymatic source of PRR-dependent local generation of aldosterone in IMCD cells.

The intrarenal renin-angiotensin system has been increasingly appreciated for its role in the pathogenesis of hypertension and kidney disease (38, 44). In this local system, prorenin and renin are produced from the CD and function as endogenous ligands of PRR. In the present study, not only did we demonstrate a role of PRR in mediating HK-induced activation of local RAAS in cultured IMCD cells, but we also showed an effect of the local RAAS on PRR. We found that the increases in PRR expression and sPRR release in response to HK were blunted by inhibition of MR or ANG II type 1 receptor. Likewise, these results suggest a mutually stimulatory relationship between PRR and the local RAAS during HK condition. Such a positive feedback regulation may be necessary for heightened aldosterone production in the kidney for promoting renal potassium excretion during HK intake.

A sizable amount of literature has documented that serum sPRR levels are elevated in patients with heart failure (10), kidney disease (15, 46), hypertension (27), and preeclampsia (47). In particular, serum sPRR is associated positively with serum creatinine, blood urea nitrogen, and urine protein, and inversely with estimated glomerular filtration rate in patients with chronic kidney disease due to hypertension and type 2 diabetes (15). A relatively small number of studies showed a negative correlation of serum sPRR with serum renin level in these conditions (29). Recently, our laboratory described a novel function of sPRR in regulation of aquoporin-2 expression in cultured IMCD cells and urine concentrating capability in rats (23). In the present study, we demonstrated that sPRR-His stimulated aldosterone synthesis in cultured IMCD cells, suggesting that sPRR may, in part, mediate the K⁺-regulatory role of PRR through releasing aldosterone. We suspect that activation of PRR/sPRR may represent a common pathway leading to enhancement of the intrarenal RAAS that plays an integrative role in coordinating the fine-tuning of urinary excretion of electrolytes and water.

In summary, the present study provides in vitro evidence that PRR functions as a regulator of the local RAAS to control aldosterone synthesis during HK treatment. Interestingly, the activation of local RAAS in turn mediates upregulation of PRR. These results reveal a mutually stimulatory relationship between PRR and the local RAAS, which likely plays a role in regulation of K⁺ homeostasis.

GRANTS

This work was supported by National Natural Science Foundation of China Grants 91439205 and 31330037, National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-094956 and DK-104072, and VA Merit Review. T. Yang is a Research Career Scientist in the Department of Veterans Affairs.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

C.X., H.F., L.Z., and A.L. performed experiments; C.X., H.F., L.Z., and A.L. analyzed data; C.X. interpreted results of experiments; C.X. prepared figures; C.X. drafted manuscript; T.Y. conception and design of research; T.Y. edited and revised manuscript; T.Y. approved final version of manuscript.