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. 2020 Sep 14;11(1):52.
doi: 10.1186/s13293-020-00329-0.

Role for ovarian hormones in purinoceptor-dependent natriuresis

Eman Y Gohar  1 , Malgorzata Kasztan  2 , Shali Zhang  2 , Edward W Inscho  2 , David M Pollock  2
Affiliations

Role for ovarian hormones in purinoceptor-dependent natriuresis

Eman Y Gohar et al. Biol Sex Differ. .

Abstract

Background: Premenopausal women have a lower risk of hypertension compared to age-matched men and postmenopausal women. P2Y2 and P2Y4 purinoceptor can be considered potential contributors to hypertension due to their emerging roles in regulating renal tubular Na+ transport. Activation of these receptors inhibits epithelial Na+ channel activity (ENaC) via a phospholipase C (PLC)-dependent pathway resulting in natriuresis. We recently reported that activation of P2Y2 and P2Y4 receptors in the renal medulla by UTP promotes natriuresis in male and ovariectomized (OVX) rats, but not in ovary-intact females. This led us to hypothesize that ovary-intact females have greater basal renal medullary activity of P2 (P2Y2 and P2Y4) receptors regulating Na+ excretion compared to male and OVX rats.

Methods: To test our hypothesis, we determined (i) the effect of inhibiting medullary P2 receptors by suramin (750 μg/kg/min) on urinary Na+ excretion in anesthetized male, ovary-intact female, and OVX Sprague Dawley rats, (ii) mRNA expression and protein abundance of P2Y2 and P2Y4 receptors, and (iii) mRNA expression of their downstream effectors (PLC-1δ and ENaCα) in renal inner medullary tissues obtained from these three groups. We also subjected cultured mouse inner medullary collecting duct cells (segment 3, mIMCD3) to different concentrations of 17ß-estradiol (E2, 0, 10, 100, and 1000 nM) to test whether E2 increases mRNA expression of P2Y2 and P2Y4 receptors.

Results: Acute P2 inhibition attenuated urinary Na+ excretion in ovary-intact females, but not in male or OVX rats. We found that P2Y2 and P2Y4 mRNA expression was higher in the inner medulla from females compared to males or OVX. Inner medullary lysates showed that ovary-intact females have higher P2Y2 receptor protein abundance, compared to males; however, OVX did not eliminate this sex difference. We also found that E2 dose-dependently upregulated P2Y2 and P2Y4 mRNA expression in mIMCD3.

Conclusion: These data suggest that ovary-intact females have enhanced P2Y2 and P2Y4-dependent regulation of Na+ handling in the renal medulla, compared to male and OVX rats. We speculate that the P2 pathway contributes to facilitated renal Na+ handling in premenopausal females.

Keywords: Ovariectomy; Purinoceptors; Renal medulla; Sodium excretion.

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Conflict of interest statement

The authors declare that there are no conflicts of interest. Dr. Gohar is also affiliated with the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Egypt.

Figures

Fig. 1
Fig. 1
Schematic presentation of the experimental timeline employed in intramedullary infusion experiments
Fig. 2
Fig. 2
Anti-natriuretic response to renal medullary P2 blockade in ovary-intact female rats only. Urinary Na+ excretion (UNaV) (a-c), urine flow (UV) (d-f), urinary K+ excretion (UKV) (g-i), and mean arterial blood pressure (MAP) (j-l) in anesthetized male, ovary-intact female, and OVX Sprague Dawley rats receiving renal medullary interstitial infusions of suramin (P2 antagonist, 750 μg/kg/min) or vehicle. n = 6-9 in each group. Statistical comparisons performed by two-way ANOVA followed by Sidak’s post hoc tests. ANOVA results: UNaV: Pinteraction = 0.1, Psuramin = 0.04, Psex = 0.9, UV: Pinteraction = 0.2, Psuramin = 0.02, Psex = 0.4; UKV: Pinteraction = 0.9, Psuramin = 0.7, Psex = 0.2; MAP: Pinteraction = 0.8, Psuramin = 0.7, Psex = 0.2
Fig. 3
Fig. 3
Inner medullary P2Y2 and P2Y4 receptor mRNA expression and protein abundance. Relative mRNA expression and total protein abundance of P2Y2 (a, c) and P2Y4 (b, d) receptor in the inner medulla from male, ovary-intact female, and OVX Sprague Dawley rats (representative Western blots are presented). Gene expression and protein abundance values represent fold change from ovary-intact female levels. n = 4-8 rats in each group. Statistical comparisons performed using one-way ANOVA followed by Sidak’s post hoc tests. ANOVA results: P2Y2 mRNA: P = 0.004, F = 7.1; P2Y4 mRNA: P = 0.007, F = 8.0; P2Y2 protein: P = 0.02, F = 4.9; P2Y4 protein: P = 0.5, F = 0.7
Fig. 4
Fig. 4
Inner medullary PLC-1δ and SCNN1A mRNA expression. Relative mRNA expression of PLC-1δ (a) and SCNN1A (ENaCα) (b) in the inner medulla from male, ovary-intact female, and OVX Sprague Dawley rats. Gene expression values represent fold change from ovary-intact female levels. n = 5-8 rats in each group. Statistical comparisons performed using one-way ANOVA followed by Sidak’s post hoc tests. ANOVA results: PLC-1δ: P = 0.001, F = 13.0; SCNN1A: P = 0.02, F = 4.7
Fig. 5
Fig. 5
E2 promotes P2Y2 and P2Y4 receptor mRNA expression in the inner medullary collecting duct cells (IMCD-3). P2Y2 (a) and P2Y4 (b) receptor mRNA expression in IMCD-3 cells incubated with E2 (1, 10, 100, 1000 nM) or vehicle for 24 h. n = 3-6 in each group. Statistical comparisons performed using one-way ANOVA followed by Dunnett’s post hoc tests. ANOVA results: P2Y2 mRNA: P = 0.04, F = 3.7; P2Y4 mRNA: P = 0.01, F = 6.1
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