Role for ovarian hormones in purinoceptor-dependent natriuresis

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.


Introduction 67
Women are largely protected from hypertension during their premenopausal age, 68 compared to age-matched men [1]. The risk of hypertension is increased after menopause which 69 is a state characterized by the cessation of ovarian production of the female sex steroid, estradiol 70 (E2) [2,3]. Data suggest that E2 exerts protective effects on cardiovascular and renal health in 71 7 paired with a CETAC ASX-520 AutoSampler, ThermoFisher Scientific) in the flame photometry 131 mode. Western blotting. Renal inner medullary tissues were processed as previously described 144 [24]. Briefly, inner medulla protein lysates were transferred and incubated with rabbit anti-P2Y2 145 or anti-P2Y4 receptor primary antibody (APR-010, APR-006, respectively, Alomone Labs) at 146 1:1500 dilution at 4°C overnight. The blots were then incubated for 1 hour at room temperature 147 with anti-rabbit IgG, HRP-Linked secondary antibody (7074, Cell Signaling Technology) at 1:7000 148 dilution. Images were developed after exposure to X-ray film. The blots were then re-probed with 149 anti-β-actin (A2228, Sigma-Aldrich Co.) at 1:10000 dilution as a loading control. Relative band 150 densities were quantified using AlphaEaseFC™ software version 3.1.2 (Genetic Technologies Inc.). legend. This includes analysis by one-way ANOVA followed by assessment of differences 164 between the means of the groups using Bonferroni's or Dunnett's multiple comparison tests. 165 Two-way ANOVA followed by assessment of differences between the means of the groups using 166 Sidak's post-hoc tests was used for analysis of figure 2 data (data are presented in different panels 167 for clarity) . Data are presented as means ± SEM, with a probability of p<0.05 considered 168 significant. Statistical analysis was performed using GraphPad Prism version 8. 169

RESULTS 171
Natriuretic role for P2 receptors in the renal medulla. To determine the contribution of 172 renal medullary P2 receptors, we infused the P2 antagonist, suramin, into the renal medulla of 9 male, ovary-intact female and OVX rats (Fig. 2). Suramin significantly decreased urinary Na + 174 excretion and urine flow relative to vehicle-infused values (Fig 2B, E) only in ovary-intact females. 175 Urine flow and Na + excretion did not significantly change during medullary blockade of P2 176 receptors in male or OVX rats ( Fig. 2A were not significantly altered by suramin in males, ovary-intact females or OVX. 178 Renal inner medullary P2Y2 and P2Y4 receptor mRNA expression. We determined P2Y2 179 and P2Y4 receptor expression in inner medullary tissues from kidneys obtained from male, ovary-180 intact female and OVX rats. We found that P2Y2 receptor mRNA expression is higher in the inner 181 medulla of ovary-intact female rats compared to males (Fig. 3A). This sex difference was 182 eliminated by ovariectomy (Fig. 3A). Renal inner medullary P2Y4 receptor mRNA expression 183 followed the same pattern as P2Y2 receptor mRNA expression (Fig. 3B). This 36-kD band was significantly higher in ovary-intact females, in comparison with males 193 (p=0.03), however, ovariectomy did not impact this 36-kD band. Overall, the combined mean 194 densities of the two bands for P2Y2 receptor were higher in inner medulla from kidneys obtained 195 from ovary-intact females, compared to males, consistent with the mRNA data (Fig. 3C). 196 Ovariectomy did not change the combined mean densities of the two bands for P2Y2 receptor 197 (Fig. 3C). 198 Western blots of renal inner medullary lysates for P2Y4 receptor also showed two 199 apparently distinct bands (Fig. 3D). The lower molecular weight band is consistent with the 200 expected molecular weight for P2Y4 receptor protein (approximately 42-kD). The other more 201 intense band had a slightly higher molecular weight, which may represent the glycosylated form 202 of P2Y4 receptor [26]. Importantly, preincubation with P2Y4 blocking peptide resulted in complete 203 ablation of these two bands (Supplemental Fig. 1B). The 42-kD band for P2Y4 receptor had a 204 slightly higher abundance in ovary-intact females relative to males, however, this trend did not 205 reach statistical significance. Notably, the relative abundance of the 42-kD band for P2Y4 receptor 206 in OVX rats was significantly lower, compared to ovary intact females (p=0.02), consistent with 207 the mRNA data. As quantified in figure 3D, the combined relative intensity of the both bands was 208 not different between male, ovary-intact or OVX female rats. The higher prevalence of the higher 209 molecular weight band for P2Y4 receptors completely masked the effect of OVX observed in the 210 42-kD band. 211

Renal inner medullary PLC-1 and ENaC mRNA expression.
Downstream of 212 purinoceptor activation, PLC-1-dependent inhibition of ENaC activity was shown to promote 213 natriuresis [10, 11, 15, 16]. We determined the mRNA expression levels of PLC-1 and ENaC 214 (SCNN1A) in kidneys from male, ovary-intact female and OVX rats. We found that mRNA 215 expression of PLC-1 was higher in the renal inner medulla of ovary-intact female rats compared 216 to males (Fig. 4A). Ovariectomy abolished this male-female difference in PLC-1 mRNA 217 expression (Fig. 4A). In contrast, no significant differences were detected in the mRNA expression 218 of inner medullary ENaC between groups (Fig. 4B). 219 E2 increases P2Y2 and P2Y4 receptor mRNA. To identify the impact of the female sex 220 steroid, E2, on P2Y2 and P2Y4 receptor mRNA expression in the inner medullary collecting ducts, 221 we treated mIMCD3 cells with different doses of E2 (10, 100, 1000 nM) or vehicle (0.1% ethanol) 222 for 24 hours. We observed that E2 dose-dependently increases the mRNA expression of P2Y2 and 223 P2Y4 receptors in mIMCD3 cells (Fig. 5). 224 225

Discussion 226
The current report establishes an important role for sex and sex steroids in regulating P2-227 mediated Na + excretion. Our results showed that (i) infusion of the P2 antagonist, suramin, to the 228 renal medulla attenuated urinary Na + excretion in ovary-intact female rats, but not in male or 229 OVX rats, (ii) OVX abolished the male-female difference in the mRNA expression of P2Y2, P2Y4 230 receptors and PLC-1 in the inner medulla of the kidney and (iii) the protein abundance of the 231 P2Y2 receptor is higher in renal inner medulla from ovary-intact female rats, compared to males. 232 (iv) We also provide in vitro evidence that E2 upregulates the mRNA expression of P2Y2 and P2Y4 233 receptors in mIMCD3. All together, these findings suggest an interaction between E2 and P2 234 signaling in the inner medulla to promote renal Na + excretory function under basal physiological 235 To elucidate the role of endogenous activation of P2 receptors on urinary Na + excretion 248 under basal physiological conditions, we determined the effect of intramedullary infusion of the 249 non-selective P2 antagonist, suramin, on basal urinary Na + excretion in male rats and female rats 250 with and without ovaries. We found that suramin attenuates urinary Na + excretion in ovary-intact 251 females, but not in males or OVX rats (Fig. 5), indicating that endogenous activation of P2 252 receptors inhibits tubular Na + reabsorption in ovary-intact females, but not males or OVX 253 females. Given that suramin is a non-selective blocker for P2 receptors, this experiment does not 254 provide us with definite clues regarding which P2 receptor subtype(s) enhance(s) urinary Na + 255 excretion in ovary-intact females. Evidence primarily points to P2Y2 and P2Y4 as important players 256 in evoking natriuresis and regulating blood pressure [10, 11]. Thus, in the present study, we 257 focused on studying aspect of the P2Y2 and P2Y4-mediated signaling cascades as potential 258 natriuretic pathways that may contribute to sex-related differences in Na + excretion. Additional 259 studies are needed to fully understand the impact of sex and sex steroids on the control of Na + 260 excretory function by P2Y and P2X receptors.  Similar to our mRNA data, the protein abundance of P2Y2 receptor was higher in ovary-277 intact females, compared to males. This sex difference at the protein level was not abolished by 278 OVX. Despite our observation that ovary-intact female rats exhibit higher P2Y 4 receptor mRNA 279 expression in their renal inner medulla compared to males or OVX rats, no differences were 280 observed at the total protein level. The disconnect between the level of mRNA expression and 281 the receptor protein levels for P2Y4 receptor may reflect differences in programmed receptor 282 destruction or post-translational modifications, rather than differences in transcription. Further 283 studies are needed to address potential sex and sex hormone-dependent differences in the 284 processing of mRNA to translation, modification, localization and protein degradation. 285 E2 is pivotal for maintenance of cardiovascular and renal health in females [4,5]. We provide 286 in vitro evidence that E2 dose-dependently increases mRNA expression of P2Y2 and P2Y4 receptor 287 in mIMCD3 cells. These data are consistent with our finding that ovary-intact female rats have an 288 enhanced renal P2Y2/P2Y4 signaling system, compared to males. Thus, we propose that E2 289 regulates the renal medullary P2 system. Notably, renal estrogen receptor, ER, expression has 290 been shown in multiple studies [36][37][38]. Binding studies using radiolabeled E2 revealed that 291 radioactivity localizes to the proximal tubule and the inner medullary collecting duct [39], which 292 is relevant to our findings in mIMCD3 cells. Data showed that classical ER, ERα and ERβ, and 293 membrane-associated ER, G protein-coupled ER, are expressed in the collecting ducts [40]. 294 However, the exact relationship between the ER and P2 signaling systems in the kidney is not 295

clear. 296
Overall, studies in recent years generally reinforce the importance of ovarian hormones in 297 determining quality of life and prognosis of cardiovascular and renal diseases in female patients. 298 When results of needed studies of the modulatory role of sex hormones on critically important 299 systems involved in the control of Na + homeostasis and blood pressure are available, developing 300 new clinical practice guidelines will be applicable. 301 302

Study limitations 303
Despite that it is established that OVX is the standard approach for studying the impact 304 of ovarian hormones on female health in preclinical research [41], it is important to note that 305 there are limitations for OVX as a model for the study of postmenopausal females. Aging is a 306 confounding factor that contributes to postmenopausal physiological changes, however OVX 307 surgery was conducted in the current study in relatively young animals. In addition, OVX results 308 in an abrupt decline in the plasma concentration of ovarian hormones, which is different from 309 the slow nature of the human menopause transition, which typically spans over few (4-6) years 310 [41]. Due to the sudden nature combined with the age of the animals employed in the current 311 study, OVX accurately models surgical, rather than natural, menopause in women. 312 In addition to the sex-related differences in the signaling pathway downstream to P2Y2 313 and P2Y4 activation in the renal medulla that we identified in the current study, it is possible that 314 there are differences in P2Y2 and P2Y4 receptor upstream signaling that may contribute to sex  Statistical comparisons performed by two-way ANOVA followed by Sidak's post-hoc tests. 367