The current report establishes an important role for sex and sex steroids in regulating P2-mediated Na+ excretion. Our results showed that (i) infusion of the P2 antagonist, suramin, to the renal medulla attenuated urinary Na+ excretion in ovary-intact female rats, but not in male or OVX rats, (ii) OVX abolished the male-female difference in the mRNA expression of P2Y2, P2Y4 receptors and PLC-1d in the inner medulla of the kidney and (iii) the protein abundance of the P2Y2 receptor is higher in renal inner medulla from ovary-intact female rats, compared to males. (iv) We also provide in vitro evidence that E2 upregulates the mRNA expression of P2Y2 and P2Y4 receptors in mIMCD3. All together, these findings suggest an interaction between E2 and P2 signaling in the inner medulla to promote renal Na+ excretory function under basal physiological conditions in ovary-intact females.
Studies suggest that females have a more advanced capacity to excrete salt, compared to age-matched males [31, 32]. This female advantage appears in both experimental animals and humans [31-33]. Renal Na+ handling is a complex and highly regulated physiological process that involves multiple mechanistic pathways. The renal tubular P2 signaling plays important roles in regulating urinary Na+ excretion [10, 11], however, whether P2-dependent renal signaling is differentially regulated based on sex and sex hormones is not clear. We recently reported that infusion of the P2Y2 and P2Y4 agonist, UTP [34], into the renal medulla evokes natriuresis in male and OVX rats, but not ovary-intact females [20, 21]. This observation directed us to focus on the P2Y2/P2Y4 signaling cascade as a potential contributor to sex-related physiological differences in renal salt handling. This is particularly relevant to evidence demonstrating that sex hormones regulate extrarenal purinoceptor signaling [35-38].
To elucidate the role of endogenous activation of P2 receptors on urinary Na+ excretion under basal physiological conditions, we determined the effect of intramedullary infusion of the non-selective P2 antagonist, suramin, on basal urinary Na+ excretion in male rats and female rats with and without ovaries. We found that suramin attenuates urinary Na+ excretion in ovary-intact females, but not in males or OVX rats (Fig. 5), indicating that endogenous activation of P2 receptors inhibits tubular Na+ reabsorption in ovary-intact females, but not males or OVX females. The impact of antagonism of P2 receptors on renal P2Y2 and P2Y4 expression level and localization is not clear. Chronic treatment with suramin for 3 months reduces myocardial P2Y2 receptor abundance in a mouse model of muscular dystrophy [39]. We presume that short-term infusion of suramin for 30 min does not impact P2Y2 and P2Y4 expression, however, it is possible that acute effects of suramin may involve changes in the localization/activation of P2Y2 and/or P2Y4 receptors.
Given that suramin is a non-selective blocker for P2 receptors, our results do not provide us with definite clues regarding which P2 receptor subtype(s) enhance(s) urinary Na+ excretion in ovary-intact females. Evidence primarily points to P2Y2 and P2Y4 as important players in evoking natriuresis and regulating blood pressure [10, 11]. Thus, in the present study, we focused on studying aspect of the P2Y2 and P2Y4-mediated signaling cascades as potential natriuretic pathways that may contribute to sex-related differences in Na+ excretion. Additional studies are needed to fully understand the impact of sex and sex steroids on the control of Na+ excretory function by P2Y and P2X receptors.
The signaling mechanisms by which P2 receptor activation evokes natriuresis involves inhibition of ENaC activity via a PLC-dependent pathway [10, 11, 15, 16]. It has been shown that inhibiting P2 receptors rapidly enhances ENaC activity [16]. Experimental evidence documents the expression and functionality of P2Y2 and P2Y4 receptors in the collecting duct [11, 13, 16, 40]. P2Y2 knockout mice are hypertensive, possibly due to ENaC hyperactivity leading to enhanced renal Na+ reabsorption [14, 16]. The current study demonstrates that the renal inner medulla from ovary-intact female rats have higher mRNA expression of P2Y2 and P2Y4 receptors and PLC, compared to males. Importantly, this male-female difference is eliminated by ovariectomy. Consist with increased activity of P2 receptors in females, we found that ENaC mRNA expression was markedly lower within inner medulla from female rats compared to males. However, we did not observe OVX-related differences in inner medullary ENaCα mRNA expression. It is possible that there are sex and sex hormonal-related differences in the activity, rather than the expression, of this ion channel. It has been demonstrated that the distal nephrons of female rats have a higher abundance of cleaved forms of ENaCα and γ, compared to males [31]. Given that PLC signaling couples P2 receptor to ENaC [16], sex-dependent regulation of PLC may present an indirect regulatory role for sex on ENaC activity. Further functional studies are necessary to identify sex and sex hormonal-dependent modulation of ENaC activity.
Similar to our mRNA data, the protein abundance of P2Y2 receptor was higher in ovary-intact females, compared to males. This sex difference at the protein level was not abolished by OVX. Despite our observation that ovary-intact female rats exhibit higher P2Y4 receptor mRNA expression in their renal inner medulla compared to males or OVX rats, no differences were observed at the total protein level. The disconnect between the level of mRNA expression and the receptor protein levels for P2Y4 receptor may reflect differences in programmed receptor destruction or post-translational modifications, rather than differences in transcription. Further studies are needed to address potential sex and sex hormone-dependent differences in the processing of mRNA to translation, modification, localization and protein degradation.
E2 is pivotal for maintenance of cardiovascular and renal health in females [4, 5]. We provide in vitro evidence that E2 dose-dependently increases mRNA expression of P2Y2 and P2Y4 receptor in mIMCD3 cells. These data are consistent with our finding that ovary-intact female rats have an enhanced renal P2Y2/P2Y4 signaling system, compared to males. In contrast, ovariectomy did not alter the protein abundance of P2Y2 and P2Y4 receptors. Thus, we propose that other sex-specific factors, besides E2, regulate the renal medullary P2 system. Notably, the gene coding for the P2Y4 receptor is located on chromosome X [41]. Whether the chromosomal complement plays a role in regulating renal P2Y2/P2Y4 signaling system remains to be determined. Importantly, renal estrogen receptor, ER, expression has been shown in multiple studies [42-44]. Binding studies using radiolabeled E2 revealed that radioactivity localizes to the proximal tubule and the inner medullary collecting duct [45], which is relevant to our findings in mIMCD3 cells. Data showed that classical ER, ERα and ERβ, and membrane-associated ER, G protein-coupled ER, are expressed in the collecting ducts [46]. However, the exact relationship between the ER and P2 signaling systems in the kidney is not clear.
Sex-specific discrepancies in the expression of renal ER have been previously reported [43]. Hutson et al. demonstrated that GPER mRNA expression was higher in kidneys from female Sprague Dawley rats, compared to males [43]. Consistently, we recently showed that the mRNA expression and protein abundance of GPER within the renal inner medulla from female Sprague Dawley rats is higher than males [47]. Whereas, renal ERα mRNA expression is greater in male, compared to female Sprague-Dawley rats [43]. Of note, renal ERα mRNA expression was markedly diminished after OVX in Wistar rats [48]. Other investigators within our group have verified that mIMCD3 cells were originally derived from male mice. Whether the expression of ER in IMCD cells is sexually dimprohic remains to be determined. Given that renal ER expression appears to be regulated by sex and sex hormones [43, 47, 48], we speculate that the sex of cells may impact the effect of E2 treatment on P2 receptor expression. Future studies are needed to determine the effect of E2 on P2Y2 and P2Y4 in freshly isolated IMCD from male, ovary-intact female and OVX rats.
Overall, studies in recent years generally reinforce the importance of ovarian hormones in determining quality of life and prognosis of cardiovascular and renal diseases in female patients. When results of needed studies of the modulatory role of sex hormones on critically important systems involved in the control of Na+ homeostasis and blood pressure are available, developing new clinical practice guidelines will be applicable.
Study limitations
Despite that it is established that OVX is the standard approach for studying the impact of ovarian hormones on female health in preclinical research [49], it is important to note that there are limitations for OVX as a model for the study of postmenopausal females. Aging is a confounding factor that contributes to postmenopausal physiological changes, however OVX surgery was conducted in the current study in relatively young animals. In addition, OVX results in an abrupt decline in the plasma concentration of ovarian hormones, which is different from the slow nature of the human menopause transition, which typically spans over few (4-6) years [49]. Due to the sudden nature combined with the age of the animals employed in the current study, OVX accurately models surgical, rather than natural, menopause in women.
In addition to the sex-related differences in the signaling pathway downstream to P2Y2 and P2Y4 activation in the renal medulla that we identified in the current study, it is possible that there are differences in P2Y2 and P2Y4 receptor upstream signaling that may contribute to sex differences in P2-mediated natriuresis. Future studies are needed to determine whether there are discrepancies between males and females in renal medullary ATP levels, ecto-ATPases and Connexin 30 channel expression and function.
Perspective and significance
To our knowledge, this is the first study showing sex and sex-hormonal related differences in P2-dependent regulation of urinary Na+ excretion. This finding may contribute to the lower prevalence of hypertension and enhanced ability to handle salt challenges evident in premenopausal females. Additional studies are necessary to identify the contribution of ATP/P2Y2/P2Y4/PLC/ENaC signaling cascade to salt sensitivity in postmenopausal female population.