Association Between Serum G Protein–Coupled Estrogen Receptor and the Peripheral Blood Balance of T-Helper/New Effector T-Cells in Patients With Hashimoto's Thyroiditis

Objective:To investigate the mechanism of adipose derived stem cell exosomes（ADSC-exos） regulating Th2/Treg balance in peripheral blood of patients with allergic rhinitis（AR）. Methods:Thirty patients with AR who were treated in Department of Otolaryngology Head and Neck Surgery, the First Affiliated Hospital of Zhengzhou University from March 2022 to October 2022 were selected, and 30 patients with simple deviation of nasal septum who were treated in our department during the same period were selected as the control group. 10 mL peripheral venous blood was collected from all patients. The levels of IL-4 and TGF-β in plasma were analyzed by ELISA. PBMCs were isolated by density gradient centrifugation. Then, protein and RNA were further extracted, and the expression levels of IL-4, TGF-β, GATA3 and Foxp3 genes were detected by qRT-PCR. Western Blotting detected p-PI3K（P85）, p-AKT（Ser473） in PBMCs of AR patients and healthy controls. Protein expression levels of p-mTOR（Ser2448）, p-p70S6K（Thr389）, and the proportion of Th2 and Treg cells were analyzed by flow cytometry. PBMCs of AR patients were stimulated to differentiate and co-cultured with exosomes of adipose stem cells. p-PI3K（P85）, p-AKT（Ser473）, p-mTOR（Ser2448） were detected in exosome treated group and untreated group by Western Blotting. The expression level of p-p70S6K（Thr389） protein, the proportion of Th2 and Treg cells were analyzed by flow cytometry, and the levels of IL-4 and TGF-β in the supernatant of cell culture were detected by ELISA. Results:Compared with the control group, the mTOR pathway in peripheral blood of AR group was significantly activated, the level of IL-4 in plasma was increased, and the level of TGF-β was decreased（P<0.05）. Compared with the control group, the proportion of Th2 cells in peripheral blood was increased, and the proportion of Treg cells was decreased（P<0.01）. Compared with the untreated group, the expression level of mTOR pathway protein decreased, the level of IL-4 decreased, and the level of TGF-β increased. The proportion of Th2 cells decreased, and the proportion of Treg cells increased（P<0.01）. Conclusion:There is an imbalance of Th2 and Treg cells in peripheral blood mononuclear cells of AR patients; the PI3K/AKT/mTOR/p70S6K pathway is activated in peripheral blood mononuclear cells of AR patients Exosomes derived from adipose mesenchymal stem cells may regulate Th2/Treg balance in AR patients through the PI3K/AKT/mTOR/p70S6K pathway.

Predictive value of Th17 and Treg cells at baseline for HBsAg loss in chronic hepatitis B patients with low HBsAg quantification treated with pegylated interferon and nucleos(t)ide analogue

Galactose-deficient IgA1 (Gd-IgA1) is a critical initiating factor in the pathogenesis of IgA nephropathy (IgAN), which plays an important role in the diagnosis and evaluation of this disease. Moreover, the whole pathogenesis process has an intimate association with the immune response of T and B lymphocytes and their inflammatory factors. There is no specific therapy for IgAN at present. Yiqi Yangyin Formula can significantly reduce urinary protein and hematuria in patients with IgAN. Yiqi Yangying Heluo Formula (YYHF) is optimized on the basis of the above prescription, but its specific mechanism remains to be further studied. The effect of YYHF on urinary protein and urinary red blood cell count in patients with IgAN was observed by a self-controlled clinical study before and after treatment. On this basis, flow cytometry was used to detect the proportion of T lymphocyte subsets in peripheral blood of patients with IgAN before and after treatment and healthy controls. Meanwhile, the levels of Gd-IgA1, B cell activation factor (BAFF), and their cytokines (IL-4, IL-6, and IL-17) in peripheral blood were detected by enzyme-linked immunosorbent assay. The changes in mechanism-related indicators of the two groups were observed and subject to correlation analysis. (1) Compared with the levels before treatment, 24-hour urinary protein content decreased by 47.7% and urinary red blood cell number decreased by 67% in patients with IgAN intervened by YYHF after 48 weeks of follow-up. (2) Compared with the healthy control group, patients with IgAN showed a significantly increased proportion of Th1 cells, Th17 cells, Th1/Th2, Th1/Treg, Th2/Treg, and Th17/Treg, obviously reduced proportion of Th2 cells and Treg cells, and evidently elevated levels of Gd-IgA1, BAFF, and their cytokines (IL-4, IL-6, and IL-17) in the peripheral blood. (3) Following 48 weeks of follow-up after intervention treatment with YYHF, the levels of Gd-IgA1, BAFF, IL-6, and IL-17 were significantly lower, but the level of IL-4 was higher in peripheral blood of patients with IgAN than those before treatment and after 24 weeks of treatment; simultaneously, the proportion of Th1 cells, Th17 cells, Th1/Th2, Th1/Treg, Th2/Treg, and Th17/Treg decreased while that of Th2 cells and Treg cells increased after 48 weeks of follow-up compared with that before treatment in peripheral blood of patients with IgAN. (4) The results of correlation analysis revealed that the level of Gd-IgA1 in peripheral blood of patients with IgAN was positively correlated with the level of BAFF, as well as the proportion of Th1 cells, Th17 cells, Th1/Th2, IL-6, and IL-17 levels, and negatively correlated with the proportion of Treg cells. In addition, the level of Gd-IgA1 in peripheral blood was positively correlated with proteinuria, yet without correlation with hematuria. YYHF can reduce the quantitative level of 24 h urinary protein and urinary red blood cell count in patients with IgAN. Patients with IgAN have obvious T cell immune imbalance. YYHF can significantly reduce the level of Gd-IgA1 in patients with IgAN, and its mechanism may be explained by the reduced level of BAFF in peripheral blood and improved immune balance of T cells.

Objective To explore the effect of high mobility group box-1 protein (HMGB1) on the balance of Th17/Treg in patients with immune thrombocytopenia (ITP). Methods A total of 30 patients who were first diagnosed as ITP in the Fifth People's Hospital of Datong from July 2017 to April 2018 were selected as the case group, and another 30 healthy volunteers in the corresponding period were taken as the control group. The proportion of Th17 and Treg cells was detected by using flow cytometry, and the concentration of HMGB1, interleukin (IL)-17 and transforming growth factor β (TGF-β) in plasma was tested by using enzyme-linked immunosorbent assay (ELISA). Isolated peripheral blood mononuclear cells (PBMC) were cultured in vitro. After the treatment with recombinant human HMGB1 (rhHMGB1), real-time polymerase chain reaction (RT-PCR) was applied to detect the mRNA expression changes in Treg cell transcription factor intracellular forkhead helix transcription factor 3 (Foxp3) and Th17 cell transcription factor retinoid related orphan receptor γt (RORγt). The differences of indicators in Treg cell transcription factor peripheral blood between the case group and the control group were compared, and the balance correlation between HMGB1 and Th17/Treg was analyzed. Results Compared with the healthy control group, the proportion of Th17 cells and the expression level of HMGB1 and IL-17 in peripheral blood of ITP patients were increased (all P < 0.01), while the proportion of Treg cells and the level of TGF-β were decreased (all P < 0.01). The proportion of Th17 cells and the expression level of IL-17 and HMGB1 in peripheral blood of ITP patients were positively correlated with the concentration of HMGB1 (all P < 0.01); the proportion of Treg cells and the level of TGF-β were negatively correlated with the expression level of HMGB1 (all P < 0.01). In vitro experiments, the expression of intracellular RORγt mRNA was increased compared with the negative control group (1.50±0.24 vs. 0.93±0.22, t = 9.612, P < 0.01), and the expression of Foxp3 mRNA was decreased compared with the negative control group after the stimulation of PBMC by rhHMGB1 (0.72±0.19 vs. 1.08±0.18, t = 7.387, P < 0.01). Conclusion The high level of HMGB1 in the peripheral blood of ITP patients induces Th17/Treg imbalance and aggravates inflammatory reactions, which may be an important cause of ITP. Key words: Immune thrombocytopenia; High mobility group box 1 protein; Th17/Treg balance; In vitro culture

Objective To investigate the distribution of T helper cell 1 (Th1), Th2 and Th17 in peripheral blood of patients with carotid atherosclerotic ischemic stroke. Methods There were a total of 180 patients with carotid atherosclerotic ischemic stroke and 60 normal controls enrolled in this study. According to the degree of carotid artery stenosis, 180 patients were divided into 3 subgroups: mild stenosis group (N = 60), moderate stenosis group (N = 60) and severe stenosis group (N = 60). Flow cytometry (FCM) was used to test the proportion of Th1, Th2 and Th17 cells in peripheral blood. Results Compared with control group, the proportion of Th1 [(5.76 ± 1.81)% vs (3.54 ± 0.29)%, P = 0.000] and Th17 [(0.36 ± 0.13)% vs (0.18 ± 0.03)% , P = 0.000] cells in ischemic stroke group was significantly increased. There were statistical differences in the proportion of Th1 cells [(4.56 ± 0.55)%, (4.88 ± 0.42)% and (7.83 ± 1.69)%; P = 0.000] and Th17 cells [(0.23 ± 0.04)%, (0.34 ± 0.02)% and (0.50 ± 0.09)%; P = 0.000] in peripheral blood of patients with different degrees of carotid stenosis. The proportion of Th1 cells ( P = 0.001, 0.001) and Th17 cells ( P = 0.000, 0.001) in severe stenosis group were higher than those in mild and moderate stenosis groups, and the proportion of Th17 cells was higher in moderate stenosis group than that in mild stenosis group ( P = 0.000). Conclusions Th1 and Th17 cells participate in the pathological process of carotid atherosclerosis. As the degree of carotid atherosclerosis in patients with ischemic stroke is increased, the proportion of peripheral blood Th17 cells is increased, suggesting that cellular immune mechanism is involved in the occurrence and development of atherosclerosis and provide theoretical basis for the immune treatment of carotid atherosclerotic ischemic stroke. DOI: 10.3969/j.issn.1672-6731.2016.09.008

Background: Endocrine therapy is an important therapeutic choice for patients with estrogen receptor (ER)a-positive breast carcinoma and functions by blocking proliferative estrogen signalling through the classical nuclear ERa. G protein-coupled estrogen receptor 1 (GPER1) is a novel membrane estrogen receptor responsible for unique estrogen responses in vitro and in vivo and that is activated by tamoxifen. The aim of this study was to determine the correlation of GPER1 with clinicopathological variables and distant disease-free survival (DDFS) in breast cancer patients treated with and without adjuvant tamoxifen, and whether the prognostic impact is dependent on ERa-status. Material and Methods: GPER1 was investigated by immunohistochemistry in tissue microarrays of breast tumors from 208 premenopausal node-negative patients (median age 47 years; range 30–57), mainly (87%) not subjected to any adjuvant systemic treatment, and from 273 stage II patients (median age 63 years; range 26–81), all treated with adjuvant tamoxifen for 2 years. Because almost 90% of the samples had a high percentage (&amp;gt;50%) GPER1-stained cells, we decided to evaluate only the staining intensity (negative, very weak, weak, moderate, and strong) for this variable. Pearson's χ2 test for trend was used for analyzing association between GPER1 and categorical clinicopathological variables, a test for trend based on ranks for association with age and tumor size, and a log-rank test for trend for evaluating the impact of GPER1 on DDFS after 5 years of follow-up. Results: GPER1 positively correlated with ERa (P=0.0005 and P=0.01, respectively) and progesterone receptor expression (P=0.004 and P=0.01, respectively) in both the premenopausal and tamoxifentreated groups, but not with HER2 expression (P=0.45 and P=0.42, respectively). In the premenopausal group, GPER1 negatively correlated with tumor size (P=0.02) and positively with age (P=0.003), whereas in the tamoxifen group GPER1 did not correlate with either tumor size or age. During 5 years of follow up, 64 patients were diagnosed with distant recurrences in the tamoxifen group and 34 patients in the premenopausal group. In univariate analysis, GPER1 positively correlated with DDFS in the tamoxifen group (P=0.04), but non-significantly in the premenopausal group (P=0.08). When stratifying for ERa-status, GPER1 was a prognostic factor in the ERa-positive subgroup (P=0.02 in tamoxifen group and P=0.08 in premenopausal group), but not in the ERa-negative subgroup (P=0.57 and P=0.95, respectively). Conclusion: We propose that GPER1 is a prognostic marker for increased DDFS in ERa-positive breast cancer. While our results suggest that GPER1 is also a tamoxifen-predictive factor, this needs to be further studied, ideally in a randomized trial comparing clinical outcome for patients treated with and without adjuvant tamoxifen. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-02.

Critical role of Th17 cells in development of autoimmune hemolytic anemia

Obstructive sleep apnea (OSA) can be considered a chronic inflammatory disease that impacts all bodily systems, including the immune system. This study aims to assess the Th17/Treg pattern in patients with OSA and the effect of continuous positive airway pressure (CPAP) treatment. OSA patients and healthy controls were recruited. OSA patients recommended for CPAP treatment were followed up for three months. Flow cytometry was employed to determine the proportion of Th17 and Treg cells. Real-time quantitative polymerase chain reaction (PCR) and western blotting were utilized to detect the mRNA and protein levels of receptor-related orphan receptor γt (RORγt) and forkhead/winged helix transcription factor (Foxp3), respectively, in peripheral blood mononuclear cells (PBMCs). Enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum levels of interleukin-17 (IL-17), IL-6, transforming growth factor-β1 (TGF-β1), and hypoxia-induced factor-1α (HIF-1α). A total of 56 OSA patients and 40 healthy controls were recruited. The proportion of Th17 cells, Th17/Treg ratio, mRNA and protein levels of RORγt, and serum IL-17, IL-6, and HIF-1α levels were higher in OSA patients. Conversely, the proportion of Treg cells, mRNA and protein levels of Foxp3, and serum TGF-β1 levels were decreased in OSA patients. The proportion of Th17 and Treg cells in OSA can be predicted by the apnea hypopnea index (AHI), IL-6, TGF-β1 and, HIF-1α. 30 moderate-to-severe OSA patients were adherent to three-month CPAP treatment, with improved Th17/Treg imbalance, IL-17, IL-6, TGF-β1, and HIF-1α levels compared to pre-treatment values. There was a Th17/Treg imbalance in OSA patients. The prediction of Th17 and Treg cell proportions in OSA can be facilitated by AHI, as well as serum IL-6, TGF-β1, and HIF-1α levels. Furthermore, CPAP treatment can potentially improve the Th17/Treg imbalance and reduce proinflammatory cytokines in OSA patients.

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; G protein–coupled estrogen receptor 1 (GPER1), previously named GPR30, is a membrane receptor reported to mediate nongenomic estrogen responses. We investigated if GPER1 expression correlates with any clinicopathologic variables and distant disease-free survival (DDFS) in patients with breast cancer, if any prognostic impact of the receptor is dependent on estrogen receptor-α (ER-α) status, and if the receptor impacts apoptotic signaling in ER-positive breast cancer cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; GPER1 expression was analyzed by immunohistochemistry in breast tumors from 273 pre- and postmenopausal stage II patients, all treated with adjuvant tamoxifen for 2 years (cohort I) and from 208 premenopausal lymph node-negative patients, of which 87% were not subjected to any adjuvant systemic treatment (cohort II). GPER1-dependent proapoptotic signaling was analyzed in MCF7 cells with and without GPER1 knockdown, T47D cells, HEK293 cells (HEK), and HEK stably expressing GPER1 (HEK-R).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; GPER1 positively correlates with ER and progesterone receptor expression. Multivariate analysis showed that GPER1 is an independent prognostic marker of increased 10-year DDFS in the ER-positive subgroup. HEK-R has higher basal proapoptotic signaling compared with HEK including increased cytochrome C release, caspase-3 cleavage, PARP cleavage, and decreased cell viability. Treating HEK-R with the proteasome inhibitor epoxomicin, to decrease GPER1 degradation, further increases receptor-dependent proapoptotic signaling. Also, GPER1 knockdown decreases basal and agonist-stimulated proapoptotic receptor signaling in MCF7 cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; GPER1 is a prognostic indicator for increased DDFS in ER-positive breast cancer, which may be associated with constitutive GPER1-dependent proapoptotic signaling in ER-positive breast cancer cells. &lt;i&gt;Clin Cancer Res; 19(7); 1681–92. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; G protein–coupled estrogen receptor 1 (GPER1), previously named GPR30, is a membrane receptor reported to mediate nongenomic estrogen responses. We investigated if GPER1 expression correlates with any clinicopathologic variables and distant disease-free survival (DDFS) in patients with breast cancer, if any prognostic impact of the receptor is dependent on estrogen receptor-α (ER-α) status, and if the receptor impacts apoptotic signaling in ER-positive breast cancer cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; GPER1 expression was analyzed by immunohistochemistry in breast tumors from 273 pre- and postmenopausal stage II patients, all treated with adjuvant tamoxifen for 2 years (cohort I) and from 208 premenopausal lymph node-negative patients, of which 87% were not subjected to any adjuvant systemic treatment (cohort II). GPER1-dependent proapoptotic signaling was analyzed in MCF7 cells with and without GPER1 knockdown, T47D cells, HEK293 cells (HEK), and HEK stably expressing GPER1 (HEK-R).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; GPER1 positively correlates with ER and progesterone receptor expression. Multivariate analysis showed that GPER1 is an independent prognostic marker of increased 10-year DDFS in the ER-positive subgroup. HEK-R has higher basal proapoptotic signaling compared with HEK including increased cytochrome C release, caspase-3 cleavage, PARP cleavage, and decreased cell viability. Treating HEK-R with the proteasome inhibitor epoxomicin, to decrease GPER1 degradation, further increases receptor-dependent proapoptotic signaling. Also, GPER1 knockdown decreases basal and agonist-stimulated proapoptotic receptor signaling in MCF7 cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; GPER1 is a prognostic indicator for increased DDFS in ER-positive breast cancer, which may be associated with constitutive GPER1-dependent proapoptotic signaling in ER-positive breast cancer cells. &lt;i&gt;Clin Cancer Res; 19(7); 1681–92. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

G protein-coupled estrogen receptor 1 (GPER1), previously named GPR30, is a membrane receptor reported to mediate nongenomic estrogen responses. We investigated if GPER1 expression correlates with any clinicopathologic variables and distant disease-free survival (DDFS) in patients with breast cancer, if any prognostic impact of the receptor is dependent on estrogen receptor-α (ER-α) status, and if the receptor impacts apoptotic signaling in ER-positive breast cancer cells. GPER1 expression was analyzed by immunohistochemistry in breast tumors from 273 pre- and postmenopausal stage II patients, all treated with adjuvant tamoxifen for 2 years (cohort I) and from 208 premenopausal lymph node-negative patients, of which 87% were not subjected to any adjuvant systemic treatment (cohort II). GPER1-dependent proapoptotic signaling was analyzed in MCF7 cells with and without GPER1 knockdown, T47D cells, HEK293 cells (HEK), and HEK stably expressing GPER1 (HEK-R). GPER1 positively correlates with ER and progesterone receptor expression. Multivariate analysis showed that GPER1 is an independent prognostic marker of increased 10-year DDFS in the ER-positive subgroup. HEK-R has higher basal proapoptotic signaling compared with HEK including increased cytochrome C release, caspase-3 cleavage, PARP cleavage, and decreased cell viability. Treating HEK-R with the proteasome inhibitor epoxomicin, to decrease GPER1 degradation, further increases receptor-dependent proapoptotic signaling. Also, GPER1 knockdown decreases basal and agonist-stimulated proapoptotic receptor signaling in MCF7 cells. GPER1 is a prognostic indicator for increased DDFS in ER-positive breast cancer, which may be associated with constitutive GPER1-dependent proapoptotic signaling in ER-positive breast cancer cells.

The G protein–coupled estrogen receptor 1 (GPER1) mediates rapid estrogenic signaling. We recently reported that activation of GPER1 in the renal medulla evokes endothelin-1–dependent natriuresis in female, but not male, rats. However, the involvement of the ET receptors, ETA and ETB, underlying GPER1 natriuretic action remain unclear. In this study, we used genetic and pharmacologic methods to identify the contributions of ETA and ETB in mediating this female-specific natriuretic effect of renal medullary GPER1. Infusion of the GPER1-selective agonist G1 (5 pmol/kg per minute) into the renal medulla for 40 minutes increased Na+ excretion and urine flow in anesthetized female ETB-deficient (ETB def) rats and littermate controls but did not affect blood pressure or urinary K+ excretion in either group. Pretreatment with the selective ETA inhibitor ABT-627 (5 mg/kg, intravenous) abolished G1-induced natriuresis in ETB def rats. To further isolate the effects of inhibiting either receptor alone, we conducted the same experiments in anesthetized female Sprague-Dawley (SD) rats pretreated or not with ABT-627 and/or the selective ETB inhibitor A-192621 (10 mg/kg, intravenous). Neither antagonism of ETA nor antagonism of ETB receptor alone affected the G1-induced increase in Na+ excretion and urine flow in SD rats. However, simultaneous antagonism of both receptors completely abolished these effects. These data suggest that ETA and ETB receptors can mediate the natriuretic and diuretic response to renal medullary GPER1 activation in female rats.SIGNIFICANCE STATEMENTActivation of G protein–coupled estrogen receptor 1 (GPER1) in the renal medulla of female rats evokes natriuresis via endothelin receptors A and/or B, suggesting that GPER1 and endothelin signaling pathways help efficient sodium excretion in females. Thus, GPER1 activation could be potentially useful to mitigate salt sensitivity in females.

<b>Abstract ID 14042</b> <b>Poster Board 508</b> Hypertension, which is a leading cardiovascular risk factor impacting 40% of American women, is more frequent in aged compared to younger women. Postmenopausal women undergo a decline in the circulating level of estrogen, along with the natural aging process. It has been shown that G protein-coupled estrogen receptor 1 (GPER1) contributes to the cardiovascular protective actions of estrogen in different experimental models. Pregnancy augments GPER1-induced vasodilation in rats. However, the potential interaction between GPER1 and pregnancy in impacting the cardiovascular health later in life is not clear. We hypothesize that GPER1 signaling and pregnancy protects against hypertension in aged female mice. In our study, 16-20 months-old GPER1 knock-out (KO) and wild-type (WT) female mice with and without a history of former pregnancies were implanted with radio-telemeters for blood pressure recording. Animals were placed into metabolic cages for collection of 24h urine samples. Then, animals were euthanized and plasma was collected. To examine whether GPER1 and/or former pregnancies affects circadian blood pressure rhythm in aged female mice, blood pressure was analyzed by cosinor analysis. No significant differences in body weight were observed between groups. GPER1 deletion in virgin mice did not impact food intake or urinary Na⁺excretion. Assessment of plasma sex hormone levels revealed that genetic deletion of GPER1 did not alter plasma estradiol or progesterone levels in virgin mice. However, previous pregnancies increased plasma levels of estradiol (<i>p</i>=0.0063), but not progesterone (<i>p</i>=0.6748) in GPER1 KO mice. Of note, GPER1 deletion increased mean arterial pressure in virgin mice (KO: 126±3; WT: 107±2 mmHg; <i>p</i>&lt;0.0001). Interestingly, prior pregnancies eliminated this genotypic difference in blood pressure. Cosinor analysis revealed augmented mean arterial blood pressure amplitude in virgin GPER1 KO mice, that was attenuated in previously-pregnant GPER1 KO mice. We also measured urinary excretion of endothelin and aldosterone which are major contributors to blood pressure regulation. Previous pregnancies lowered urinary levels of endothelin-1 (<i>p</i>=0.0049) and aldosterone (<i>p</i>=0.0099) in GPER1 KO mice. Overall, our data demonstrate an elevated blood pressure in GPER1 KO virgin mice compared to WT virgin and previously-pregnant GPER1 KO aged mice. The current data suggest that GPER1 and former pregnancies provide protection against hypertension in aged females. Funded by R00DK119413 to EG.

G protein-coupled estrogen receptor 1 mediates estrogen effect in red common carp (Cyprinus carpio)

Hypertension prevalence is lower in women than in 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 coexpression 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 pharmacological 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 coexpression of aquaporin 2 (AQP2) and GPER1 confirmed the knockout (KO) of GPER1 from the principal cell (PC) in the kidney. Thus, renal GPER1 functionally interacts with ENaC in a sex-specific manner to promote natriuresis.NEW & NOTEWORTHY This study identified GPER1 as a sex-specific upstream regulator of ENaC. We found that GPER1 and ENaC were coexpressed in the rat renal tubules in a sex and region-specific manner. Activation of GPER1 inhibited ENaC activity in isolated mouse collecting ducts, whereas 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.