Renal G Protein-coupled Estrogen Receptor 1 Regulates the Epithelial Sodium Channel Promoting Natriuresis to a Greater Extent in Females

Abstract

Hypertension prevalence is lower in women than men. Enhanced renal sodium (Na⁺) handling in females has been implicated in sex-differences in hypertension. Epithelial Na⁺ channel (ENaC) is a key contributor to Na⁺ homeostasis and is regulated by estrogen. Recent evidence suggests G protein-coupled estrogen receptor 1 (GPER1) evokes a female-specific natriuresis that involves endothelin-1 (ET-1). ET-1 has been shown to downregulate ENaC activity, but whether GPER1 regulates ENaC to modulate natriuresis is unknown. We tested the hypothesis that renal GPER1 functionally interacts with ENaC to promote natriuresis in a sex-specific manner. RNAscope confirmed co-expression of GPER1 and ENaC in rat renal tubules in a sex and region-specific manner. Within the renal medulla, the number of ENaC/GPER1-positive tubules was greater in females than males. Renal medullary inhibition of ENaC or activation of GPER1 evoked comparable natriuresis in female rats. Electrophysiology revealed that pharmacologic GPER1 activation downregulated ENaC activity, whereas genetic deletion of GPER1 from the principal cells of the collecting duct caused ENaC hyperactivity. The hyperactivity of ENaC caused by deletion of GPER1 in the principal cells was greater in female than male mice. RNAscope co-expression of AQP2 and GPER1 confirmed the KO of GPER1 from the PCs in the kidney. Thus, renal GPER1 functionally interacts with ENaC in a sex-specific manner to promote natriuresis.

Graphical Abstract

Introduction

Sodium (Na⁺) homeostasis is critical for blood pressure regulation with disruptions in renal Na⁺ handling leading to hypertension.¹ Hypertension is more prevalent in men than women,² yet the mechanisms underlying female protections are not entirely understood. Previous studies in rodents and humans have shown that females have an augmented capacity for natriuresis in response to Na⁺ loading.^3–5 Evidence implicates estrogen as a driver for sex-differences in hypertension.⁶ Understanding how estrogen signaling impacts key natriuretic pathways is therefore critical.

Epithelial Na⁺ channel (ENaC) is a major contributor to the regulation of Na⁺ homeostasis,⁷ and sex-differences in renal ENaC expression have been reported in rats.^4,8,9 In addition, the ENaC inhibitor benzamil evoked a greater natriuretic response in male Sprague Dawley (SD) rats compared to females.⁹ Estradiol, the most prevalent estrogen, has been shown to have varying effects on ENaC abundance and activity. Renal ENaC protein abundance was decreased in response to estradiol supplementation to ovariectomized SD rats,¹⁰ but not to aldosterone salt-treated ovariectomized Wistar rats.¹¹ In mouse cortical collecting duct (CD) cells, estradiol (0.1 and 100 μM) decreased ENaC abundance and activity,¹⁰ while lower doses of estradiol (25 and 50 nM) elicited an opposite effect.^12,13 In the presence of progesterone, estradiol at a concentration of 25 nM increased ENaC activity, whereas 50 nM estradiol inhibited the stimulated ENaC activity in cultured CD cells.¹² Actions mediated via different estrogen receptors could account for this controversy.

Estradiol signals through several receptors, including the G protein-coupled estrogen receptor 1 (GPER1).⁶ GPER1 activation lowers blood pressure in male and female rat models.^14–16 Importantly, a hypofunctional variant of GPER1 (P16L) has been identified in the human genome and is associated with increased blood pressure in women.¹⁷ Furthermore, activation of GPER1 in the renal medulla promotes natriuresis in female, but not male, SD rats.¹⁵ The female specific increase in natriuresis following the activation of GPER1 in the renal medulla was shown to be via endothelin-1 (ET-1)^15,18 which reduces tubular Na⁺ reabsorption by inhibiting ENaC activity.¹⁹ Whether ENaC plays a role in GPER1-mediated natriuresis is not known. Improving our understanding of the regulation of ENaC by GPER1 would advance sex-specific therapeutic approaches for hypertension treatments. Thus, the current study tested the hypothesis that renal GPER1 functionally interacts with ENaC to promote natriuresis in a sex-specific manner.

Methods

Animal Studies

All animals used were housed in temperature‐controlled rooms (22–24°C) with a 12:12‐hour light‐dark cycle, with access to food and water ad libitum. All animal protocols were in accordance with the Guide for the Care and Use of Laboratory Animals. All rat protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. All mouse protocols were approved by the Vanderbilt University Medical Center or the University of Texas Health Science Center (San Antonio and Houston) Institutional Animal Care and Use Committees

Intramedullary infusion:

Female SD rats (Envigo Indianapolis, IN) 17–21 weeks of age were anesthetized using inactin hydrate (100 mg/kg, IP, T133; Sigma‐Aldrich Co) between 8 am and 11 am, and prepared for intermedullary infusion studies as previously described.¹⁵ Briefly, catheters were inserted into the femoral artery to monitor blood pressure, the femoral vein to maintain euvolemia, the left ureter to collect urine, and the medulla of the left kidney to infuse drugs directly into the renal medullary interstitium. After the surgery, animals were allowed to equilibrate for 80 minutes before a 20‐minute baseline urine collection. This was followed by two 20-minute urine collection periods during which the ENaC inhibitor amiloride (Aml, 17.4 μmol/kg per minute,²⁰ A7410; Sigma‐Aldrich Co), the GPER1 agonist G1 (5 pmol/kg per minute,^15,21 41004001, purity ≥98%; Sandia Biotec Inc), or a solution of both reagents, was infused into the renal medulla. Urine from the baseline and the second drug infusion collection period was analyzed. During baseline urine collection, vehicle was infused (saline for Aml or 0.02% dimethyl sulfoxide, D8418; Sigma‐Aldrich Co in saline for G1) into the renal medulla. At the end of each experiment, the infused kidney was dissected to confirm proper catheter positioning. Throughout the study, mice received i.v. supplementation with 6% BSA saline via the femoral vein, and the femoral artery blood pressure was monitored using a pressure transducer and a PowerLab data-acquisition system (AD Instruments).

Principal cell GPER1 knockout mouse generation:

A Gper1^{tm1a(KOMP)Wtsi} construct was obtained from the University of California, Davis Knockout Mouse Project (KOMP). To obtain the floxed-mouse ES cell clone EPD0334_2_H06 was injected into morulae or blastocysts. The L1L2_Bact_P cassette was inserted at position 139410951 of Chromosome 5 upstream of exon 3 (Build GRCm39). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site. This first loxP site is followed by neomycin resistance gene under the control of the human beta-actin promoter, SV40 polyA, a second FRT site and a second loxP site. A third loxP site is inserted downstream of exon 3 at position 139412865. Exon 3 is thus flanked by loxP sites. A “conditional ready” (floxed) allele was created by flp recombinase expression in mice carrying this allele to remove the lacZ sequence and neo selection cassette, leaving loxP sites flanking exon 3. Resulting chimeras were mated to C57Bl/6N mice and heterozygous tm1a (Knockout First) animals were generated. Heterozygous mice were bred to a ubiquitous Flp recombinase mouse line for recombination of the FRT sites to generate tm1c (FLPed Knockout First) allele mice. The resulting litter was a mix of wild type and heterozygous for the CSD FLPed KO First allele. Male and female heterozygous mice were used to generate a homozygous Gper1-floxed mouse.

Aqp2Cre mice^22,23 were then crossed with the Gper1 floxed mouse to generate principal cell (PC)-specific Gper1-knockout (KO) mice (PC-Gper1 KO mice). Aqp2Cre mice were bred from the female lineage to generate Cre-positive floxed experimental mice and Cre-negative floxed wildtype (WT) mice.

Patch clamping:

Cortical CDs were isolated from male and female PC-Gper1 WT and KO mice 12–16 weeks of age as previously described.^19,24,25 Briefly, mice were euthanized, the kidneys were removed immediately then thinly sliced (<1 mm) and placed into ice-cold physiological saline solution containing (in mM): 150 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, 5 glucose and 10 HEPES (pH 7.35). CDs were then manually isolated with forceps and adhered to poly-L-lysine coated cover-glasses. A cover-glass containing a CD was placed in a perfusion chamber mounted on an inverted Nikon Eclipse Ti-S microscope and perfused with bath solution at room temperature. CDs were further split-opened with two sharpened micropipettes, controlled with different micromanipulators to gain access to the apical membrane. The CDs were used within 2 hours of isolation. CDs were chosen at random and single channel patch clamp was performed as previously described.^19,24,25 In brief, the Gap-free single channel current data from gigaOhm seals were acquired under voltage clamp conditions (−Vp =− 60 mV) in cell-attached patches on the apical membrane of the principal (polygonal-shape) cells and further analyzed with Axopatch 200B (Molecular Devices) patch clamp amplifier interfaced via a Digidata 1440 (Molecular Devices) to a PC running the pClamp 10.7 suite of software (Molecular Devices). Currents were low-pass filtered at 1 kHz with an eight-pole Bessel filter (Warner Instruments). Events were inspected visually prior to acceptance. Channel activity in individual patches, defined as NP_o, was calculated using the following equation: NP_o = (t₁ + 2t₂ + … + nt_n), where N and P_o are the number of ENaC in a patch and the mean open probability of these channels, respectively, and t_n is the fractional open time spent at each of the observed current levels. Total ENaC activity (fNP_o) was estimated by normalizing the NP_o to the frequency of observed patches with at least 1 active channel (f = number of patches with active channels/total number of patches). ENaC activity was measured in principal cells isolated from an average of 3 different mice, 6 different CDs with 5–8 different patch clamp recordings from each CD for each experimental group giving the overall number of 26–38 total recordings per group. In a subset of CDs isolated from male and female WT mice, the samples were pretreated with G1 (1 μM, 41004001, purity ≥98%; Sandia Biotec Inc) or vehicle for 40 minutes before starting the experiments.

Kidney collection for RNAscope:

Kidneys from male and female SD rats (Envigo Indianapolis, IN) and PC-Gper1 WT and KO mice at 16–20 weeks of age were perfused by phosphate-buffered saline (PBS, P4417; Sigma‐Aldrich Co) containing 10% heparin, followed by 4% paraformaldehyde (PFA, J19943-K2; Thermo Fisher Scientific). After fixation, tissues were put in 4% PFA for 24 hours at 4°C. Subsequently, the samples were transferred to 30% sucrose (84097; Sigma‐Aldrich Co) for 24 hours at 4°C and then embedded in Tissue Plus optimal cutting temperature compound (O.C.T., NC1029572; Fisher Scientific).

Metabolic cage experiments:

Mice were individually placed into metabolic cages (Techniplast) and allowed to acclimate with access to food and water ad libitum for 2 days. Urine was then collected for a 24-hour period as previously described.²⁴ Collection surfaces in the cages in contact with urine were coated with Sigmacote (Sigma-Aldrich), and the urine was collected under mineral oil (M8410; Sigma‐Aldrich Co) to eliminate evaporation.

Assays

RNAscope:

RNAscope Multiplex Fluorescent Reagent Kit v2 (323100, Advanced Cell Diagnostics) was used following the manufacturer’s protocol to determine the expression patterns of GPER1 and ENaCα in SD rats and GPER1 and aquaporin 2 (AQP2) in PC-Gper1 WT and KO mice. Cryosections from OTC-frozen kidneys were permeabilized, subjected to antigen retrieval, then hybridized with the relevant probes (Advanced Cell Diagnostics); rat GPER1 probe (487781), rat αENaC probe (422601), mouse GPER1 probe (475251) and mouse AQP2 probe (452411). The appropriate amplifier was then hybridized and signal development performed for each probe. Then, rat slides were incubated with PECAM1 and DAPI for structural landmarks of the vasculature and nuclei respectively and mouse slides were incubated with DAPI. Rat sections were imaged using a Keyence BZ-X800 microscope at 20x magnification. Mouse sections were imaged using an Aperio Versa 200 automated slide scanner at 40x magnification and zoomed images were captured using an Olympus IX81 inverted microscope. For quantitative image analysis, 4–6 evenly distributed areas were selected across the kidney cortex or medulla. For rat sections, the number of ENaC-positive only, GPER1-positive only, and ENaC/GPER1-positive tubules were then counted in each area. For mouse sections, the number of AQP2/GPER1-positive tubules were counted in each area.

Urinary electrolyte measurement:

Urinary Na⁺ and potassium (K⁺) concentrations were determined with an atomic absorption spectrometer (iCE 3000 series paired with a CETAC ASX‐520 AutoSampler; Thermo Fisher Scientific).

Urinary creatinine measurement:

Urinary creatinine was measured by the UAB-UCSD O’Brien Center for Acute Kidney Injury Research using underivitized, stable isotope dilution liquid chromatography mass spectrometry (LC-MS/MS) as previously described.²⁶ LC-MS/MS was performed on an Agilent Infinity 1260 LC, Infinity 1290 autosampler with a 6460 Triple Quad mass spectrometer using a TSK-Gel Amide-80 column from Tosoh Bioscience. Separation was achieved with an isocratic flow of 10 mM ammonium acetate (A637 Fisher Scientific) in 65% acetonitrile (A996 Fischer Scientific).

ET-1 measurement:

Urinary levels of ET-1 were measured using a commercially available QuantiGlo ELISA kit following the manufacturer’s protocol (QET00B, R&D Systems). The minimum detectable dose of the assay is 0.064 pg/ml. The intra-assay precision coefficient of variability (CV) value is 3.1%, while the inter-assay precision CV value is 6.7%. Plates were read on a Synergy H4 hybrid multi-mode microplate reader (BioTek)

Statistical analysis

Values are presented as mean ± SEM in all figures and tables. Statistical tests used for each data set are specified in the figure/table legend. Data were evaluated via a repeated measures one-way ANOVA or a two-way ANOVA. P values for each data set are displayed in the associated figure or table. A post hoc Holm Sidak’s test was performed after the ANOVA analysis. P≤0.05 was considered significant. Significant values from the post hoc analysis are displayed on the graph.

Results

ENaC and GPER1 co-expression

RNAscope fluorescent assays were used to detect the expression sites and tubular expression of ENaC and GPER1 within the renal cortex and medulla of male and female SD rats to determine co-expression patterns within the kidney (Figure 1A). The number of tubules positive for only ENaC was greater in the medulla than the cortex of both sexes. Post-hoc analysis did not reveal significant sex-differences in tubules positive for ENaC only in the cortex however female SD rats had more ENaC-positive only tubules in the medulla than males (Figure 1B). In contrast, the number of GPER1-positive only tubules was comparable in the cortex and medulla in males. However, there was significantly more GPER1-positive cortical tubules in females compared to the medulla. There was no sex-difference in GPER1-postitive tubules in the medulla, however, in the cortex there were more GPER1-positive only tubules vs males (Figure 1C).

Figure 1:

ENaC and GPER1 are coexpressed in the renal cortex and medulla. (A) Representative RNAscope images of the renal cortex and medulla of male and female SD rats. DAPI in blue, ENaC in white, and GPER1 in pink displayed in individual channels and the merged image. PECAM1 in green was used as a structural marker and is displayed only in the merged image. Images taken at 20x magnification. Scale bars represent 200 μm. Quantification of tubules positive for (B) GPER1, (C) ENaC, or (D) ENaC and GPER1 in the cortex and medulla of male and female SD rats. N=5–6/group. Two-way ANOVA with post hoc Holm Sidak’s test. ANOVA results are displayed in the table under each graph. P values ≤0.05 from post hoc testing are displayed on each graph. Abbreviations: 4′,6-diamidino-2-phenylindole (DAPI), epithelial sodium channel (ENaC), G protein-coupled estrogen receptor 1 (GPER1), platelet endothelial cell adhesion molecule (PECAM1), Sprague Dawley (SD).

Tubules co-expressing ENaC and GPER1 were detected within the cortex and medulla of male and female SD rats, with more detection within the medulla than the cortex in both sexes (Figure 1D). The number of ENaC/GPER1-positive tubules within the cortex did not differ by sex; however, the number of ENaC/GPER1-positive tubules within the medulla was greater in females than males (Figure 1D).

GPER1 activation and/or ENaC inhibition induce a comparable natriuretic response

Intramedullary infusion studies were performed to assess Na⁺ excretion following pharmacological activation of GPER1 or inhibition of ENaC. Given that renal medullary GPER1 activation induces natriuresis in female, but not male, SD rats,¹⁵ the current intramedullary infusion studies were performed in female SD rats only. Infusion of the ENaC inhibitor Aml, the GPER1 agonist G1, or Aml+G1 into the renal medulla of female SD rats comparably increased natriuresis (Figure 2A). None of the infusions changed the urinary K⁺ levels or mean arterial pressure (Figure 2B, D). Infusion of G1, but not Aml or Aml+G1, increased urine volume (Figure 2C).

Figure 2:

ENaC inhibition and/or GPER1 activation induce natriuresis to a similar extent in SD rats. (A) Timeline for the renal medullary infusion studies. (B) U_NaV, (C) U_KV, (D) urine volume, and (E) MAP in female SD rats after renal medullary drug infusion. Data presented are from baseline and the second drug infusion timepoint. N=6–9/group. Repeated measures one-way ANOVA with post hoc Holm Sidak’s test. ANOVA results are displayed in the table below each graph. P values ≤0.05 from post hoc testing are displayed on each graph. Abbreviations: Epithelial sodium channel (ENaC), G protein-coupled estrogen receptor 1 (GPER1), amiloride (Aml), GPER1 agonist (G1), urinary sodium excretion (U_NaV), urinary potassium excretion (U_KV), mean arterial blood pressure (MAP), Sprague Dawley (SD).

GPER1 downregulates ENaC activity in the CD

Given the potential functional interaction of GPER1 and ENaC, patch clamp studies were performed to evaluate ENaC activity following pharmacological or genetic manipulation of GPER1. ENaC activity did not differ by sex in vehicle-treated (Figure 3A–B) nor naïve (Figure 3E–F) CD tubules isolated from WT mice. G1 treatment decreased ENaC activity in CD tubules from male and female WT mice to a similar extent (Figure 3A–B). ENaC activity was increased in CD tubules from male and female PC-Gper1 KO mice, and this increase was more substantial in females (Figure 3E–F). Similar changes were seen when evaluating ENaC number of active channels and open probability represented in Figure 3C–D and G–H.

Figure 3:

GPER1 negatively regulates ENaC activity in the CD of mice to a greater extent in females. Patches were formed on the apical membrane of PCs in split-open CDs isolated from male and female mice. (A) Representative current traces from cell-attached patches recorded from PCs of WT male and female mice treated with vehicle or G1. Quantification of the total ENaC (B) activity fNP_o, (C) N and (D) P_o from cell-attached patches recorded from PCs of WT male and female mice treated with vehicle or G1. N=6–8/group. (E) Representative current traces from cell-attached patches recorded from PCs of male and female WT or PC-GPER1 KO mice. Quantification of the total ENaC (F) activity fNP_o, (G) N and H) P_o from cell-attached patches recorded from PCs of male and female WT or PC-GPER1 KO mice. N=26–38/group. Clamping voltage =− 60 mV. Two-way ANOVA with post hoc Holm Sidak’s test. ANOVA results are displayed in the table below each graph. P values ≤0.05 from post hoc testing are displayed on each graph. Post hoc evaluation was performed within each factor. Abbreviations: G protein-coupled estrogen receptor 1 (GPER1), epithelial sodium channel (ENaC), collecting duct (CD), GPER1 agonist (G1), total channel activity (fNP_o), number of channels in a patch (N), open probability (P_o), wildtype (WT), principal cell (PC), knockout (KO).

PC-GPER1 deletion was confirmed with RNAscope

RNAscope fluorescent assays were used to determine the KO of GPER1 from the PC (Figure 4A). The number of tubules positive for AQP2 and GPER1 in the cortex and medulla of male (Figure 4B) and female (Figure 4C) PC-Gper1 KO mice was significantly reduced compared to corresponding WT mice.

Figure 4:

GPER1 deletion from the PC eliminate sex differences in urinary ET-1 excretion in mice. (A) Representative RNAscope images of the renal cortex and medulla of male and female PC-Gper1 WT and KO mice. DAPI in blue, AQP2 in green, and GPER1 in red. Images taken at 60x magnification. Scale bars represent 20 μm. Quantification of tubules positive for AQP2 and GPER1 in the cortex and medulla of (B) male and (C) female PC-Gper1 WT and KO mice N=3/group. (D) Urinary ET-1 excretion in male and female PC-Gper1 WT and KO mice N=5–7/group. Two-way ANOVA with post hoc Holm Sidak’s test. ANOVA results are displayed in the table below the graph. P values ≤0.05 from post hoc testing are displayed on the graph. Abbreviations: 4′,6-diamidino-2-phenylindole (DAPI), aquaporin 2 (AQP2), G protein-coupled estrogen receptor 1 (GPER1), principal cell (PC), wildtype (WT), knockout (KO), endothelin 1 (ET-1).

PC-GPER1 deletion eliminates sex-differences in urinary ET-1 excretion

Given that it has been previously shown that GPER1-natriuretic effects are mediated via ET-1,^15,18 which in turn regulates ENaC,¹⁹ we assessed the effect of genetic deletion of GPER1 in PC on urinary ET-1 excretion in mice. Importantly, urinary excretion of ET-1 is a reflection of intrarenal production of this peptide.²⁷ Female WT mice excreted more ET-1 than male WT mice (Figure 4D). Genetic deletion of GPER1 in the PC attenuated urinary ET-1 excretion in female mice and had no impact on urinary ET-1 excretion in male mice, eliminating the sex-difference in ET-1 excretion (Figure 4D).

Discussion

The renal abundance of ENaC, a key regulator of Na⁺ homeostasis and blood pressure, differs by sex,^4,8,9 and in response to estradiol treatment^12,13 which suggest estradiol as a regulator of Na⁺ handling. Previous work pointed to estrogen signaling through GPER1 in the renal medulla as a female-specific natriuretic mechanism¹⁵ but did not fully characterize the pathway. This study reveals that GPER1 downregulates ENaC activity, to a greater extent in females, which promotes Na⁺ excretion in a sex-specific manner.

We show that ENaC is co-expressed with GPER1 in the cortical and medullary tubules of male and female SD rat kidneys indicative of their potential functional interaction. Notably, this co-expression was more apparent in females than males particularly in the medullary region. It is important to note that the images being analyzed are a single section through the tissue, representing one section through each tubule. While we are interpreting ENaC or GPER1-only positive tubules as having a singular expression, it is possible that GPER1 and ENaC are co-expressed in a different section of these tubules. The implied potential for functional interaction between GPER1 and ENaC to a greater degree in female compared to male SD rats could explain the greater natriuretic capacity of GPER1 in females. Consistent with our previous study,^15,28 renal medullary infusion of G1 in female SD rats increased Na⁺ excretion, and this response was comparable to that prompted by medullary infusion of Aml. Simultaneous medullary ENaC inhibition and GPER1 activation was not additive, suggesting Aml and G1 may be inducing natriuresis through the same pathway. Though only GPER1 activation evoked diuresis, it is important to note that these are short term infusion studies and compensatory reservations of urine volume could have led to no changes in urine flow with the infusion of Aml. Our previous studies showed that GPER1 promotes natriuresis via ET-1,^15,18 which is recognized as an ENaC inhibitor.¹⁹ Taken together, this suggests GPER1 activation evokes natriuresis by inhibiting ENaC activity.

At the cellular level, G1-mediated activation of GPER1 in CDs from male and female WT mice suppressed ENaC activity, whereas genetic deletion of GPER1 in the PCs induced ENaC hyperactivity, which was more robust in female mice. G1 treatment evoked a near total suppression of ENaC activity with no sex-difference, however it is possible that lower doses of G1 may uncover sex-specific differences in the acute inhibitory effect on ENaC. Of note, genetic deletion GPER1 in the PCs increased the number of ENaC per patch suggesting a prolonged translational effect. Future studies are needed to investigate the regulatory mechanisms involved in the inhibitory effect of GPER1 on ENaC in acute vs chronic settings.

Consistent with our previous findings in SD rats,^18,29 urinary ET-1 excretion was greater in female than male WT mice. Interestingly, GPER1-KO in PCs attenuated urinary ET-1 excretion in female mice, eliminating the sex-difference. This corroborates our finding in global GPER1 KO mice¹⁸ and indicates that PC GPER1 is responsible for the greater renal ET-1 production in females. We also demonstrated a functional interaction between ET-1 receptors (ET_A and ET_B) in mediating the natriuretic response to medullary GPER1 activation in the female rat kidney.²¹ Importantly, RNAscope confirmed the KO of GPER1 from the PCs in both male and female mice. Overall, these data strongly suggest that PC GPER1 regulates ENaC activity and natriuresis through ET-1-mediated mechanisms. A role for β1-Pix/14–3-3/Nedd4–2 pathway in mediating the inhibitory effect of ET-1 on ENaC has been established.³⁰ Future studies are needed to explore whether this pathway is involved in the GPER1-mediated regulation of ENaC. In addition, GPER1 has been linked to the regulation of purinergic receptor P2Y receptors in lung epithelial cells.³¹ We have previously identified a sex-specific link between P2Y₂ receptors and ET-1 in mediating natriuresis,^32,33 and it is established that P2Y₂ regulates the activity of ENaC.³⁴ However, the contribution of P2Y₂ receptors in mediating the inhibitory effect of GPER1 on ENaC remains to be tested. Future studies are required to identify the signaling pathways involved in renal GPER1-ENaC interaction. Of note, we assessed ENaC activity in cortical CDs not medullary CDs, however a previous assessment in SD rats found similar Aml sensitive Na⁺ currents in the cortical CD compared to the outer medullary CD with a reduced current from the inner medullary CD.³⁵ Future studies on varying populations of CD’s can be performed to confirm these results as necessary.

This study contradicts two previous studies which documented a stimulatory effect of estradiol on ENaC in cultured CD cells,^12,13 but is in line with results from a study by Zhang et. al. which found that estradiol dose-dependently suppresses ENaC activity in cultured CD cells.¹⁰ Similarly, Chang et. al. found that increasing estradiol concentration almost completely inhibited the stimulated ENaC activity in the presence of progesterone in cultured CD cells.¹² These studies used commercially obtained cultured cells, while ours used ex-vivo whole CDs. Further, the supraphysiologic concentrations of estradiol used in the previous studies likely activated estrogen receptor α (ERα) and ERβ as well as GPER1. Physiological ranges of estrogens in females range from 0.37 to 0.92 nanomole per liter,³⁶ whereas these studies utilized 25 nanomole per liter to 100 micromole per liter concentrations. Our studies with G1, which is specific to GPER1,³⁷ and PC-GPER1 KO mice indicated the specific contribution of GPER1. In fact, our findings highlight the possibility that the role of GPER1 in renal Na⁺ handling and hypertension is distinct from that of other estrogen receptors. Interestingly, however, Greenlee et.al. showed in alveolar cells, estradiol increases ENaC activity via GPER1,³⁸ suggesting the effects of GPER1 on ENaC activity could be tissue-specific.

Evidence suggests a potential contribution for GPER1 hypofunction in postmenopausal hypertension.¹⁷ It is established that the prevalence of hypertension increases in females after menopause.³⁹ Of note, the renal expression of GPER1 increases with age in female mice.⁴⁰ Given the decline in estrogen levels with menopause,⁴¹ GPER1-mediated regulation of ENaC may be a contributor to the enhanced risk for hypertension evident in postmenopausal women. To improve our understanding of the clinical implications of this natriuretic pathway to postmenopausal hypertension, additional work is required to investigate the impact of aging and menopause on the inhibitory effect of GPER1 on ENaC activity.

Overall, we have provided initial evidence that GPER1 regulates renal ENaC activity in a sex-specific manner. Limitations of our study include the lack of full characterization of the PC-Gper1 KO mouse model. RNAscope image analysis did not include an assessment of the total tubule number. While this study reveals that GPER1 negatively regulates ENaC, the potential molecular mechanisms mediating this interaction remains to be determined.

Conclusion

Overall, this study revealed that (1) ENaC and GPER1 colocalize in the renal tubule (2) GPER1 downregulates ENaC activity in the CD, and (3) GPER1-mediated natriuresis occurs at least in part through ENaC regulation. Patch clamping revealed that GPER1 negatively regulates ENaC activity in PCs to a higher degree in females than males. This GPER1-mediated regulation of ENaC, especially in females, may result from the increased renal production or release of ET-1. Further characterization of the GPER1/ENaC natriuretic axis could shed light on sex-differences in the prevalence of hypertension and reveal novel avenues for sex-specific antihypertensive therapeutic development.

New and Noteworthy.

This study identified GPER1 as a sex-specific upstream regulator of ENaC. We found that GPER1 and ENaC were co-expressed in the rat renal tubules in a sex and a region-specific manner. Activation of GPER1 inhibited ENaC activity in isolated mouse collecting ducts, while deletion of GPER1 from the principal cells caused ENaC hyperactivity to a greater extent in female mice. Our data suggest GPER1 functionally interacts with ENaC in a sex-specific manner to promote natriuresis.

Data Sharing Statement

All data and information are available in the main text. Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request.