Background The novel estrogen receptor, G‐protein–coupled estrogen receptor ( GPER ), is responsible for rapid estrogen signaling. GPER activation elicits cardiovascular and nephroprotective effects against salt‐induced complications, yet there is no direct evidence for GPER control of renal Na + handling. We hypothesized that GPER activation in the renal medulla facilitates Na + excretion. Methods and Results Herein, we show that infusion of the GPER agonist, G1, to the renal medulla increased Na + excretion in female Sprague Dawley rats, but not male rats. We found that GPER mRNA expression and protein abundance were markedly higher in outer medullary tissues from females relative to males. Blockade of GPER in the renal medulla attenuated Na + excretion in females. Given that medullary endothelin 1 is a well‐established natriuretic factor that is regulated by sex and sex steroids, we hypothesized that GPER activation promotes natriuresis via an endothelin 1–dependent pathway. To test this mechanism, we determined the effect of medullary infusion of G1 after blockade of endothelin receptors. Dual endothelin receptor subtype A and endothelin receptor subtype B antagonism attenuated G1‐induced natriuresis in females. Unlike males, female mice with genetic deletion of GPER had reduced endothelin 1, endothelin receptor subtype A, and endothelin receptor subtype B mRNA expression compared with wild‐type controls. More important, we found that systemic GPER activation ameliorates the increase in mean arterial pressure induced by ovariectomy. Conclusions Our data uncover a novel role for renal medullary GPER in promoting Na + excretion via an endothelin 1–dependent pathway in female rats, but not in males. These results highlight GPER as a potential therapeutic target for salt‐sensitive hypertension in postmenopausal women.

Background Premenopausal women are less likely to develop hypertension and salt‐related complications than are men, yet the impact of sex on mechanisms regulating Na + homeostasis during dietary salt challenges is poorly defined. Here, we determined whether female rats have a more efficient capacity to acclimate to increased dietary salt intake challenge. Methods and Results Age‐matched male and female Sprague Dawley rats maintained on a normal‐salt (NS) diet (0.49% NaCl) were challenged with a 5‐day high‐salt diet (4.0% NaCl). We assessed serum, urinary, skin, and muscle electrolytes; total body water; and kidney Na + transporters during the NS and high‐salt diet phases. During the 5‐day high‐salt challenge, natriuresis increased more rapidly in females, whereas serum Na + and body water concentration increased only in males. To determine if females are primed to handle changes in dietary salt, we asked the question whether the renal endothelin‐1 natriuretic system is more active in female rats, compared with males. During the NS diet, female rats had a higher urinary endothelin‐1 excretion rate than males. Moreover, Ingenuity Pathway Analysis of RNA sequencing data identified the enrichment of endothelin signaling pathway transcripts in the inner medulla of kidneys from NS‐fed female rats compared with male counterparts. Notably, in human subjects who consumed an Na + ‐controlled diet (3314–3668 mg/day) for 3 days, women had a higher urinary endothelin‐1 excretion rate than men, consistent with our findings in NS‐fed rats. Conclusions These results suggest that female sex confers a greater ability to maintain Na + homeostasis during acclimation to dietary Na + challenges and indicate that the intrarenal endothelin‐1 natriuretic pathway is enhanced in women.

Recent evidence indicates a crucial role for G protein-coupled estrogen receptor 1 (GPER1) in the maintenance of cardiovascular and kidney health in females. The current study tested whether GPER1 activation ameliorates hypertension and kidney damage in female Dahl salt-sensitive (SS) rats fed a high-salt (HS) diet. Adult female rats were implanted with telemetry transmitters for monitoring blood pressure and osmotic minipumps releasing G1 (selective GPER1 agonist, 400 μg/kg/day, intraperitoneal) or vehicle. Two weeks after pump implantation, rats were shifted from a normal salt diet (NS, 0.4% NaCl) to a matched HS diet (4.0% NaCl) for 2 weeks. 24-hour urine samples were collected during both diet periods and urinary markers of kidney injury were assessed. Histological assessment of kidney injury was conducted after the 2-week HS diet period. Compared with values during the NS diet, 24-hour mean arterial pressure markedly increased in response to HS, reaching similar values in vehicle-treated and G1-treated rats. HS also significantly increased urinary excretion of protein, albumin, nephrin (podocyte damage marker) and KIM-1 (proximal tubule injury marker) in vehicle-treated rats. Importantly, G1 treatment prevented the HS-induced proteinuria, albuminuria and increase in KIM-1 excretion but not nephrinuria. Histological analysis revealed that HS-induced glomerular damage did not differ between groups. However, G1 treatment preserved proximal tubule brush border integrity in HS-fed rats. Collectively, our data suggest that GPER1 activation protects against HS-induced proteinuria and albuminuria in female Dahl SS rats by preserving proximal tubule brush border integrity in a blood pressure-independent manner.

Hypertension is more prevalent in men compared to premenopausal women. Maintenance of Na+ homeostasis is an essential component of blood pressure regulation, and defects in the renal capacity to appropriately excrete Na+ are fundamental mechanisms involved in the initiation of salt‐sensitive hypertension. We recently reported that female rats elicit a more rapid natriuretic response to a high salt (HS) diet compared to males. The overall goal of the current study was to determine the natriuretic signaling pathway(s) involved in the enhanced ability of females to handle HS challenges. Male and female Sprague Dawley rats (16–18 weeks old) were placed into metabolic cages on normal salt (NS, 0.49% NaCl) followed by HS (4% NaCl) diet for 5 days. 24‐hour urine samples were assessed for endothelin‐1 (ET‐1), aldosterone (ALD) and norepinephrine (NE). On NS, urinary ET‐1 was significantly higher in females, compared to males; no sex‐differences were detected in urinary ALD or NE. In males, urinary ET‐1 was increased (10.2±0.8 vs. 5.6±1.0 pg/kg/day, n=8 each, p=0.002) and urinary ALD was decreased on day 1 HS relative to NS (6.1±0.7 vs. 15.2±2.6 μg/kg/day, n=5 each, p=0.02); no significant changes were observed in ET‐1 or ALD in females on day 1 HS. Urinary NE remained unchanged during the HS challenge in both sexes. Ingenuity Pathway Analysis (IPA) of RNA sequencing data from inner medullae from males and females on NS identified an enrichment (p=3.11E‐04) of transcripts in the ET‐1 signaling pathway in females, with the abundance of 35/190 (18.4%) of pathway transcripts being at least 1.5‐fold different in abundance between sexes. Further, IPA predicted that ET‐1 signaling is activated in female inner medulla, relative to males. Previous studies showed that interleukin‐1β (IL‐1β) stimulates renal ET‐1 production. Thus we tested whether renal IL‐1β is upregulated in response to HS in a sex‐dependent manner. Renal IL‐1β was assessed by ELISA and IHC. Renal cortical IL‐1β was significantly increased on day 1 HS, relative to NS, only in males (8.1±0.3 vs. 6.4±0.3 pg/mg protein, n=9 each, p=0.03). Our data suggest that activation of IL‐1β/ET‐1 and inhibition of ALD signaling pathways contribute to acclimation to HS challenges in males. We further suggest that the already activated renal ET‐1 signaling in females under NS may contribute to the enhanced ability to acclimate to HS challenges observed in females.Support or Funding InformationFunded by AHA 18CDA34110010 and 15POST25090329 to EYG, and P01 HL136267 to JSP and DMP. We appreciate Drs. Celso and Elise Gomez‐Sanchez's help with ALD assay.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The novel estrogen receptor, G protein‐coupled estrogen receptor (GPER), is a membrane‐bound receptor linked to acute signaling pathways. GPER activation elicits cardiovascular and nephroprotective effects against salt‐induced complications. However, its specific role in renal sodium handling is not yet defined. We hypothesized that activation of GPER in the renal medulla facilitates sodium excretion. Isosmotic saline was infused into the renal medullary interstitium (500 μl/h) of anesthetized male and female Sprague‐Dawley rats for an 80 min equilibration period and 20 min baseline urine collection period. Then, G1 (GPER agonist, 50 pmol/kg/min) or vehicle was infused into the renal medulla for an additional 20 min period. Compared with vehicle, G1 significantly increased urinary sodium excretion in females (from 0.6 ± 0.1 to 1.0 ± 0.1 μmol/min n=9, p<0.05). G1 did not change blood pressure or urinary potassium excretion in comparison with vehicle control (106 ± 3 vs. 105 ± 4 mmHg, 0.5 ± 0.1 vs. 0.4 ± 0.1 μmol/min, n=9). Medullary blood flow remained unchanged during these experiments. In males, G1 did not change urinary sodium excretion (from 0.6 ± 0.1 to 0.7 ± 0.1 μmol/min, n=9). No sex‐differences in medullary GPER mRNA expression was observed, suggesting that sex‐differences in G1 effects on sodium excretion could be related to GPER downstream signaling cascade. Given that endothelin‐1 (ET‐1) is a well‐established natriuretic factor that is regulated by sex and sex steroids, we hypothesized that GPER activation promotes natriuresis via an ET‐1‐dependent pathway. To test this idea, we studied the effect of medullary infusion of G1 after dual blockade of ETA and ETB receptors achieved by i.v. bolus injection of ABT‐627 (5 mg/kg) and A‐192621 (10 mg/kg), respectively. We found that the natriuretic response to medullary GPER activation in females was inhibited by dual blockade of ET‐1 receptors (from 0.5 ± 0.2 to 0.5 ± 0.2 μmol/min n=5). These data uncover a novel role for renal medullary GPER in promoting sodium excretion via an ET‐1‐dependent pathway in females, but not in males.Support or Funding InformationFunded by AHA 15POST25090329 to EYG and P01 HL136267 to DMP and JSPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.