Evidence for G-Protein-Coupled Estrogen Receptor as a Pronatriuretic Factor

Abstract

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.

Keywords: GPER; endothelin 1; estrogen; hypertension; kidney; sodium excretion.

Figure 1. Activation of G‐protein–coupled estrogen receptor (GPER) in the renal medulla facilitates urinary Na⁺ excretion in female rats.

Schematic presentation of the surgical procedure and experimental time line employed (A). Urinary Na⁺ excretion (U_{N a}V) (B), urine flow (UV) (C), urinary K⁺ excretion (U_KV) (D), and mean arterial pressure (MAP) (E) in anesthetized intact female Sprague Dawley rats receiving renal medullary interstitial infusions of vehicle (Veh), G1 (GPER agonist, 5 pmol/kg per minute), G1+G15 (GPER antagonist, 5 pmol/kg per minute), or G15 alone, respectively. n=4 to 9 in each group. Statistical comparisons performed by paired Student t test.

Figure 2. Activation of G‐protein–coupled estrogen receptor (GPER) in the renal medulla did not change urinary Na⁺ excretion in male rats.

Effect of renal medullary interstitial infusions of the GPER agonist, G1 (5 pmol/kg per minute) on urinary Na⁺ excretion (U_{N a}V) (A), urine flow (UV) (B), urinary K⁺ excretion (U_KV) (C), and mean arterial pressure (MAP) (D) in anesthetized male Sprague Dawley rats. Relative mRNA expression and protein abundance of GPER in outer medulla (E) and inner medulla (F) from male and female rats (representative Western blots are presented). Gene expression values represent fold change from corresponding female levels. Protein abundance is presented relative to female levels. n=5 to 10 rats in each group. Statistical comparisons performed by paired Student t test (A through D) and unpaired Student t test (E and F).

Figure 3. Pharmacologic and genetic evidence for the interaction between G‐protein–coupled estrogen receptor (GPER) and renal endothelin 1 (ET‐1) system in females.

Effect of renal medullary interstitial infusions of the GPER agonist, G1 (5 pmol/kg per minute), after a bolus injection of the selective endothelin receptor subtype A (ET_A) receptor blocker (ABT‐627) and the selective endothelin receptor subtype B (ET_B) receptor blocker (A‐192621) on urinary Na⁺ excretion (U_{N a}V) (A), urine flow (UV) (B), urinary K⁺ excretion (U_KV) (C), and mean arterial pressure (MAP) (D) in anesthetized intact female Sprague Dawley rats. mRNA expression of renal ET‐1, ET_A, and ET_B receptors (E) in male and female wild‐type (WT) and GPER knockout (KO) mice. n=5 to 12 animals in each group. Statistical comparisons performed by paired Student t test (A through D) and 2‐way ANOVA with Sidak's post hoc test for multiple comparisons (E).

Figure 4. Ovariectomy (OVX) does not impact the natriuretic response to medullary G‐protein–coupled estrogen receptor (GPER) activation.

Urinary Na⁺ excretion (U_{N a}V) and percentage change in U_{N a}V (A), urine flow (UV) and percentage change in UV (B), urinary K⁺ excretion (U_KV) (C), and mean arterial pressure (MAP) (D) in anesthetized OVX Sprague Dawley rats receiving renal medullary interstitial infusions of G1 (GPER agonist, 5 pmol/kg per minute). Percentage change in U_{N a}V and UV in response to intramedullary infusion of G1 in intact females, and OVX is represented relative to baseline values. Relative mRNA expression and protein abundance of GPER in outer medulla (E) and inner medulla (F) from intact female and OVX rats (representative Western blots are presented). Gene expression values represent fold change from corresponding female levels. Protein abundance is presented relative to female levels. n=6 to 12 rats in each group. Statistical comparisons performed by Student t test (A through F).

Figure 5. Systemic activation of G‐protein–coupled estrogen receptor prevents the ovariectomy (OVX)–induced increase in blood pressure.

Experimental time line (A). The 24‐hour mean arterial pressure (MAP) (B), systolic blood pressure (SBP) (C), diastolic blood pressure (DBP) (D), and heart rate (HR) (E) along the experimental time line in conscious female rats subjected to OVX and vehicle (Veh) or G1 (400 μg/kg per day, osmotic minipump). Panels on the right‐hand side show MAP, SBP, DBP, and HR at baseline (before OVX, average of blood pressure at days ‐1 and ‐2) and 2 weeks after OVX (average of blood pressure at days 13 and 14). Data are means±SE of 6 rats in each group. Statistical comparisons performed by repeated measures 2‐way ANOVA with Sidak's post hoc test for multiple comparisons. *P<0.05 vs corresponding baseline values.

Figure 6. Effect of systemic activation of G protein–coupled estrogen receptor on body weight and basal metabolic cage parameters.

Body weight at baseline and 2 weeks after ovariectomy (OVX) (A), food intake (B), water intake (C), urinary Na⁺ excretion (U_{N a}V) (D), urine flow (UV) (E), and urinary K⁺ excretion (U_KV) (F) in OVX Sprague Dawley rats supplemented with vehicle (Veh) or G1 (400 g/kg per day, osmotic minipump) for 2 weeks. Data are mean±SE of 5 to 6 rats in each group. Statistical comparisons performed by repeated measures 2‐way ANOVA with Sidak's post hoc test for multiple comparisons (A) and unpaired Student t test (B through F).