Ovariectomy uncovers purinergic receptor activation of endothelin-dependent natriuresis

Abstract

We recently reported that natriuresis produced by renal medullary salt loading is dependent on endothelin (ET)-1 and purinergic (P2) receptors in male rats. Because sex differences in ET-1 and P2 signaling have been reported, we decided to test whether ovarian sex hormones regulate renal medullary ET-1 and P2-dependent natriuresis. The effect of medullary NaCl loading on Na⁺ excretion was determined in intact and ovariectomized (OVX) female Sprague-Dawley rats with and without ET-1 or P2 receptor antagonism. Isosmotic saline (284 mosmol/kgH₂O) was infused in the renal medullary interstitium of anesthetized rats during a baseline urine collection period, followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH₂O) infusion. Medullary NaCl loading significantly enhanced Na⁺ excretion in intact and OVX female rats. ET_A+B or P2 receptor blockade did not attenuate the natriuretic effect of medullary NaCl loading in intact females, whereas ET_A+B or P2 receptor blockade attenuated the natriuretic response to NaCl loading in OVX rats. Activation of medullary P2Y₂ and P2Y₄ receptors by UTP infusion had no significant effect in intact females but enhanced Na⁺ excretion in OVX rats. Combined ET_A+B receptor blockade significantly inhibited the natriuretic response to UTP observed in OVX rats. These data demonstrate that medullary NaCl loading induces ET-1 and P2-independent natriuresis in intact females. In OVX, activation of medullary P2 receptors promotes ET-dependent natriuresis, suggesting that ovarian hormones may regulate the interplay between the renal ET-1 and P2 signaling systems to facilitate Na⁺ excretion.

the prevalence of cardiovascular and chronic kidney diseases differs significantly between men and women of a similar age (49, 52, 70). Ovarian hormones play a central role in the cardiovascular and renal protection observed within the premenopausal female population (21, 26, 60, 63). There is an increased risk of different cardiovascular diseases, including salt-sensitive hypertension, after loss of ovarian function (8, 30, 53, 72).

Renal medullary endothelin-1 (ET-1) plays a central role in the maintenance of Na⁺ homeostasis and appears to contribute to sex differences in blood pressure control and kidney function (40, 51, 66). Acute increases of renal medullary NaCl stimulate ET-1 release/production (6, 23, 64), which in turn inhibits the tubular reabsorption of Na⁺, resulting in enhanced urinary Na⁺ excretion (U_NaV) mediated mainly through inhibiting epithelial Na⁺ channel (ENaC) activity (42).

Renal medullary purinergic (P2) signaling is another cornerstone in the regulation of blood pressure and Na⁺ homeostasis (50, 68, 69). Increased tubular flow stimulates ATP release from renal tubular cells and inhibits Na⁺ reabsorption (32, 50). Previous studies have highlighted an important role for P2Y₂ receptor in the regulation of Na⁺ excretion (44, 56). Taken together, the physiological effects of medullary ET-1 and P2 receptors are complementary. However, the physiological relationship between ET-1 and P2 signaling within the renal medulla is not yet clearly defined. Recently, it has been shown that ET-1 production in cultured inner medullary collecting duct (IMCD) cells is regulated by P2Y₂ and P2X₇ receptors (54). This in vitro evidence is further supported by recent data from our laboratory demonstrating that activation of renal medullary P2 receptors promotes ET-1-dependent natriuresis (23). We demonstrated this interplay between ET-1 and P2 in the renal medulla of male rats; however, whether female rats use this natriuretic pathway to efficiently excrete Na⁺ is not yet clear.

To date, most of the published work on the ET-1 and P2 systems has been conducted in male animals or in pooled samples from both sexes. However, a number of studies have demonstrated sexual dimorphism in the ET-1 (22, 24, 33, 38–40, 51, 65, 66) and purinergic (5, 13, 15, 43) systems. The current study was designed to study the interplay between ET-1 and P2 signaling systems on renal Na⁺ excretory function in intact female rats. Taking into consideration the increased incidence of salt sensitivity after depletion of ovarian hormones, we tested this potential interplay in ovariectomized (OVX) rats as well.

We hypothesize that ovarian sex hormones regulate renal medullary ET and P2-dependent natriuresis. To test our hypothesis, we determined the effect of medullary NaCl loading on U_NaV in intact female and OVX Sprague-Dawley rats in the presence or absence of ET (ET_A and ET_B) or P2 receptor antagonism. Additionally, we studied the effect of intramedullary infusion of the P2Y₂ and P2Y₄ agonist, UTP, in the presence and absence of ET_A+B receptor antagonists.

METHODS

General methods.

Female Sprague-Dawley rats [n = 6–9/group; Envigo (Harlan Laboratories, Indianapolis, IN)] were used in the current study. Rats were maintained on 7917 Irradiated NIH-31 Mouse/Rat diet from Envigo (0.8% NaCl) with free access to water. During the entire experimental period, animals were conventionally housed (2 animals/cage, without environmental enrichment) in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle. Animals were randomized into different groups. Experiments involving intact females were conducted in a periodic manner by performing experiments over four consecutive days to ensure the presentation of different estrous cycle phases. All protocols were in accordance with the Guide for the Care and Use of Laboratory Animals and were approved in advance by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

Ovariectomy.

Female rats (13–15 wk old) were subjected to bilateral ovariectomy through dorsal bilateral incisions as described in our previous studies (24). Ovaries were exposed, tied off with sutures, and removed. After ovariectomy (3 wk), rats were prepared for acute intramedullary infusion experiments as detailed below.

Acute intramedullary infusion.

Female rats (16–18 wk old) were anesthetized using thiobutabarbitone (Inactin, hydrate 100 mg/kg ip; Sigma-Aldrich, St. Louis, MO) and surgically prepared for acute intramedullary infusion as described in detail in previous studies by our laboratory (6, 7). In brief, rats were placed on a heated surgical table to maintain body temperature at 37°C throughout the experiment, and a tracheotomy was performed (PE-205) to facilitate breathing. A catheter (PE-50) was inserted in the femoral vein for infusing 3% bovine serum albumin in phosphate-buffered saline at a rate of 1.2 ml/h to maintain euvolemia. Another catheter (PE-50) was inserted in the femoral artery and connected to a pressure transducer and a PowerLab data acquisition system to monitor blood pressure over the course of the experiment (ADInstruments, Colorado Springs, CO). A midline incision was then performed, and a catheter (PE-10) was placed in the left ureter to allow urine collection. The left renal artery was isolated and fitted with an ultrasonic perivascular flow probe (1PRB probe; Transonic Systems, Ithaca, NY) to measure the total renal blood flow (RBF) continuously during each experiment. RBF was normalized to left kidney weight. Finally, a stretched PE-10 catheter was inserted in the left kidney as deep as the outer/inner medullary junction to allow infusion of isosmotic NaCl (0.9%) directly in the renal medulla (0.5 ml/h). This catheter was fixed to the outer renal capsule with superglue to maintain the catheter stable.

Following surgical preparation, a 60- to 80-min equilibration period was allowed, followed by a 30-min baseline urine collection during intramedullary infusion of isosmotic saline (284 mosmol/kgH₂O, 0.9% NaCl, 154 mM NaCl). This was followed by isosmotic or hyperosmotic saline (1,800 mosmol/kgH₂O, 5.7% NaCl, 976 mM NaCl) infusion for two further 30-min periods (Fig. 1).

Fig. 1.

Experimental timeline. OVX, ovariectomized.

Separate groups of rats (2 groups of intact females and 2 groups of OVX) were employed to determine the role of ET_A and ET_B receptors in response to medullary NaCl loading. Rats received an intravenous bolus injection (0.5 ml/kg via femoral vein catheter) of vehicle or a combination of the selective ET_A receptor antagonist ABT-627 (5 mg/kg; AbbVie, Abbott Park, IL) simultaneously with the selective ET_B receptor antagonist A-192621 (10 mg/kg; AbbVie, Abbott Park, IL) 30 min before the end of the equilibration period. These doses are known to maintain efficient blockade of both receptor subtypes for the duration of our experimental protocol as previously demonstrated (3, 23). After the equilibration period and the 30-min baseline urine collection during which isosmotic saline was infused, hyperosmotic saline was infused for two subsequent 30-min urine collection periods.

To determine the role of P2 signaling in response to medullary NaCl loading, the nonselective P2 receptor antagonist suramin [750 μg·kg⁻¹·min⁻¹; Sigma-Aldrich (23)] was included in the intramedullary infusate to additional groups of intact female and OVX rats.

Separate groups of rats (2 intact female and 2 OVX groups) were employed to determine the role of the ET-1 system in response to P2 receptor activation. Vehicle or ABT-627 and A-192621 were administered in combination as described above. Baseline urine was collected while isosmotic saline was infused to the renal medulla. The P2Y₂ and P2Y₄ receptor agonist UTP (200 pmol·kg⁻¹·min⁻¹; Sigma) was then infused in the renal medullary interstitium for two subsequent 30-min urine collection periods. We have recently reported that this intramedullary dose of UTP stimulates water and Na⁺ excretion in male Sprague-Dawley rats (23). At the end of each experiment, the proper positioning of the catheter tip at the outer-inner medullary junction was confirmed by dissection of the kidney. The inner medulla was then isolated, snap-frozen in liquid nitrogen, and stored at −80°C until analysis. Euthanasia was confirmed by bilateral thoracotomy.

Tissue analysis.

Total RNA was isolated from renal inner medullary tissues using the Purelink Mini RNA extraction kit (Ambion, Austin, TX) according to the manufacturer’s instructions. Next, the isolated RNA was reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Finally, the resulting cDNA was used to quantify ET-1 mRNA by Real Time-PCR (CFX96 Real-Time System; Bio-Rad, Hercules, CA) using TaqMan primer gene expression assays with ET-1 (assay no. Rn01775763_g1) and GAPDH (assay no. Rn01775763_g1) primers. ET-1 gene expression was quantified relative to GAPDH using the 2^−ΔΔCt method.

Urine analysis.

Urinary Na⁺ and K⁺ concentrations were measured with an atomic absorption spectrometer in the flame photometry mode (model 3100; PerkinElmer, Rodgau, Germany). Osmolality of infused solutions and urine samples was determined by a vapor pressure osmometer (VAPRO 5600; ELITechGroup, Logan, UT).

Statistics.

Based on the SD for physiological parameters from earlier preliminary results, sample size was determined by power analysis at a power of 0.8 to achieve statistical significance at P < 0.05. Data are presented as means ± SE. Statistical comparisons from baseline to first and second 30 min of intramedullary infusions were performed by repeated-measures two-way ANOVA followed by Dunnett’s multiple-comparisons test. mRNA data were compared using an unpaired Student’s t-test. A probability of P < 0.05 was considered significant. The primary and secondary experimental outcomes assessed in our experiments are U_NaV and urine flow, respectively.

RESULTS

Ovarian hormones regulate the natriuretic response to renal medullary NaCl loading.

Baseline urine flow was significantly higher in intact females compared with OVX rats (Table 1). OVX rats had significantly higher basal urine osmolality and mean arterial pressure (MAP) compared with intact females (Table 1). No significant differences were detected in U_NaV, urinary K⁺ excretion (U_KV), or RBF between intact females and OVX at baseline (Table 1). During infusion of isosmotic saline in the renal medulla of intact female or OVX rats, urinary flow, electrolytes, and osmolality remained unchanged (Fig. 2, A–H). MAP (Fig. 2, I and J) and the total RBF (Fig. 2, K and L) remained unchanged during infusions of isosmotic or hyperosmotic saline to intact female and OVX rats.

Table 1.

Baseline characteristics of anesthetized intact female and ovariectomized (OVX) rats prior to intramedullary infusion experiment

	Intact Females	OVX
Urine flow, μl/min	8.3 ± 0.7	6.0 ± 0.6*
UNaV, μmol/min	0.87 ± 0.10	0.66 ± 0.07
UKV, μmol/min	0.42 ± 0.06	0.55 ± 0.05
Urine osmolality, mosmol/kgH2O	711 ± 50	941 ± 74*
Body wt, g	264 ± 4	312 ± 3*
MAP, mmHg	105 ± 2	114 ± 3*
RBF, ml·min−1·kg−1	8.3 ± 0.9	8.8 ± 0.8

Data are means ± SE of 12 rats in each group. Baseline measurements of urine flow, urinary excretion rate of Na⁺ (U_NaV) and K⁺ (U_KV), urine osmolality, mean arterial pressure (MAP), and total renal blood flow (RBF) at baseline in anesthetized intact female and ovariectomized (OVX) rats receiving renal medullary infusions of isosmotic saline (284 mosmol/kgH₂O).

^*P < 0.05 vs. corresponding intact female values.

Fig. 2.

Urine flow (A and B), urinary Na⁺ excretion (U_NaV) (C and D), urinary K⁺ excretion (U_KV) (E and F), urine osmolality (G and H), mean arterial blood pressure (MAP) (I and J), and total renal blood flow (RBF) (K and L) in anesthetized intact female and OVX rats receiving renal medullary interstitial infusions of isosmotic (284 mosmol/kgH₂O) or hyperosmotic (1,800 mosmol/kgH₂O) saline. Data are means ± SE. Repeated-measures two-way ANOVA with Dunnett’s post hoc test for multiple comparisons. P < 0.05 vs. corresponding baseline (*) and isosmotic saline (†) values; n = 6 in intact female isosmotic and hyperosmotic saline groups, n = 7 in OVX isosmotic saline group, and n = 9 in OVX hyperosmotic saline group.

In intact females, intramedullary infusion of hyperosmotic saline stimulated U_NaV and urine flow in the second 30-min time period of increased medullary NaCl (1,114 ± 375 and 111 ± 18% change vs. baseline, respectively; Fig. 2, A and C). In OVX rats, U_NaV increased markedly within the first 30 min of commencing intramedullary infusions of hyperosmotic saline (Fig. 2D); the natriuretic and diuretic responses in OVX rats were also evident during the second 30-min time interval of hyperosmotic saline infusion (740 ± 149 and 201 ± 48% change in U_NaV and urine flow, respectively, vs. baseline; Fig. 2, B and D). Urine flow in OVX rats was increased significantly during the second 30 min of intramedullary infusion of hyperosmotic saline (Fig. 2B). Medullary NaCl loading did not change U_KV in intact females or OVX (Fig. 2, E and F). Urine osmolality was enhanced in OVX rats only compared with corresponding baseline (Fig. 2H). An increase in urine osmolality was detected in intact females during the first 30 min of intramedullary infusion of hyperosmotic saline compared with the corresponding time point in the isosmotic saline group (Fig. 2G).

ET-1 expression in intact and OVX rats.

Infusion of hyperosmotic saline in the renal medulla for 60 min did not cause any significant changes in the expression of inner medullary ET-1 mRNA compared with intact females and OVX rats infused with isosmotic saline; fold changes in intact females were 0.8 ± 0.1 vs. 1.0 ± 0.1 for hyperosmotic vs. isosmotic infusion, respectively, and 1.2 ± 0.3 vs. 1.0 ± 0.3 for hyperosmotic vs. isosmotic saline infusion, respectively, in OVX rats.

Blockade of ET_A+B receptors inhibited the natriuretic response to medullary NaCl loading in OVX rats.

In intact female rats, the natriuretic and diuretic responses to increased medullary NaCl were still evident after combined antagonism of ET_A and ET_B receptors (727 ± 93 and 114 ± 35% change in U_NaV and urine flow, respectively, vs. baseline; Fig. 3, A and C). Unexpectedly, ET receptor blockade increased the natriuretic response during the initial 30 min of increasing medullary NaCl (Fig. 3C). In contrast, the natriuretic and diuretic responses to hyperosmotic saline in OVX rats were significantly attenuated during combined blockade of ET_A+B receptors (368 ± 174 and 1 ± 17% change in urine flow and U_NaV, respectively, vs. baseline; Fig. 3, B and D). U_KV was not significantly changed during these experiments compared with baseline values. In OVX rats receiving ET_A+B blockers, U_KV was decreased during the second 30-min time interval compared with the corresponding time point in the hyperosmotic saline group (Fig. 3, E and F). Increased medullary NaCl enhanced urine osmolality in both intact females and OVX rats treated with ET_A+B blockers (Fig. 3, G and H). MAP was slightly, but not significantly, decreased in OVX rats receiving intravenous ET_A and ET_B receptor antagonists during the second 30-min time period (Fig. 3, I and J). Total RBF was unchanged by intramedullary infusions and was not different between all four groups (Fig. 3, K and L).

Fig. 3.

Urine flow (A and B), U_NaV (C and D), U_KV (E and F), urine osmolality (G and H), MAP (I and J), and total RBF (K and L) in anesthetized intact female and OVX rats receiving renal medullary interstitial infusions of hyperosmotic saline (1,800 mosmol/kgH₂O) with or without the selective endothelin (ET) receptor A blocker (ABT-627) plus the selective ET_B receptor blocker (A-192621). Data are means ± SE. Repeated-measures two-way ANOVA with Dunnett’s post hoc test for multiple comparisons. P < 0.05 vs. corresponding baseline (*) and isosmotic saline (†) values; n = 6 rats in each group.

Blockade of P2 receptors inhibited the natriuretic response to medullary NaCl loading in OVX rats.

In intact females, natriuresis and diuresis were not changed by suramin (1,076 ± 354 and 169 ± 37% change in U_NaV and urine flow, respectively, vs. baseline; Fig. 4, A and C). On the other hand, blockade of medullary P2 receptors by suramin abolished the natriuretic and diuretic responses to medullary NaCl loading in OVX rats (119 ± 84 and 12 ± 36% change in U_NaV and urine flow, respectively, vs. baseline; Fig. 4, B and D). Additionally, suramin inhibited the increase in urine osmolality in OVX rats in response to intramedullary infusion of hyperosmotic saline (Fig. 4H). U_KV, MAP, and total RBF were not significantly altered in response to hyperosmotic saline infusion in the presence or absence of suramin (Fig. 4, E, F, and I–L).

Fig. 4.

Urine flow (A and B), U_NaV (C and D), U_KV (E and F), urine osmolality (G and H), MAP (I and J), and total RBF (K and L) in anesthetized intact female and OVX rats receiving renal medullary interstitial infusions of hyperosmotic saline (1,800 mosmol/kgH₂O) with or without the nonselective antagonist of purinergic (P2) receptors, suramin. Data are means ± SE. Repeated-measures two-way ANOVA with Dunnett’s post hoc test for multiple comparisons. P < 0.05 vs. corresponding baseline (*) and isosmotic saline (†) values; n = 6 rats in each group.

Blockade of ET_A+B receptors inhibited the natriuretic response to medullary P2 receptor activation in OVX rats.

To provide additional pharmacological evidence for the interplay between the renal medullary ET-1 and P2 systems, separate groups of intact female and OVX rats received intramedullary infusions of UTP, the P2Y₂ and P2Y₄ receptor agonist, with or without combined ET_A+B receptor blockade. In intact females, 60 min of intramedullary infusion of UTP had no significant impact on urine flow or U_NaV in the presence or absence of ET receptor antagonism (Fig. 5, A and C). In contrast, UTP significantly enhanced U_NaV and urine flow in OVX rats (424 ± 130 and 223 ± 55% change vs. baseline, respectively; Fig. 5, B and D). The UTP-induced natriuresis and diuresis observed in OVX rats were completely abolished by dual antagonism of ET_A and ET_B receptors (10 ± 21 and 15 ± 35% change in U_NaV and urine flow, respectively, vs. baseline; Fig. 5, B and D). In intact females, U_KV was not significantly changed during these experiments (Fig. 5E). Urine osmolality was increased during the second 30 min of intramedullary infusion of UTP in intact females receiving dual ET receptor blockers (Fig. 5G). Coadministration of ET_A+B blockers to OVX rats decreased U_KV (Fig. 5F) and had no effect on urine osmolality (Fig. 5H).

Fig. 5.

Urine flow (A and B), U_NaV (C and D), U_KV (E and F), urine osmolality (G and H), MAP (I and J), and total RBF (K and L) in anesthetized intact female and OVX rats receiving renal medullary interstitial infusions of UTP with or without the selective ET_A receptor blocker (ABT-627) plus the selective ET_B receptor blocker (A-192621). Data are means ± SE *P < 0.05 vs. corresponding baseline values. Repeated-measures two-way ANOVA with Dunnett’s post hoc test for multiple comparisons. †P < 0.05 vs. corresponding UTP values; n = 6 in intact female UTP, ET_A/B blockers + UTP, and OVX UTP groups; n = 7 in OVX ET_A/B blockers + UTP group.

MAP remained unchanged during intramedullary infusion of UTP in intact females and was slightly, but significantly, decreased in OVX rats receiving intravenous ET_A+B receptor blockers during the second 30-min time period compared with the corresponding time point in the UTP group (Fig. 5, I and J). However, there were no statistically significant differences in the MAP between the second 30-min time point in OVX rats and its corresponding baseline values (Fig. 5J). In both intact female and OVX rats, total RBF remained unchanged during these experiments (Fig. 5, K and L).

DISCUSSION

The current study highlights that antagonism of ET_A+B or P2 receptors inhibits the natriuretic response to increased medullary NaCl in OVX but not in intact females. Additionally, blockade of ET_A+B receptors inhibited the UTP-induced natriuretic response in OVX rats. Thus, the renal ET-1 and P2 systems work cooperatively to help the efficient excretion of medullary NaCl in OVX rats, similar to what we have recently reported in male rats (23).

There is growing evidence for a potential role for ovarian hormones in regulating renal Na⁺ excretion (18, 29, 37, 67). More specifically, recent studies have shown that ENaC, which facilitates renal Na⁺ absorption in the collecting duct, is regulated by estradiol (2, 11, 20, 58). We found that increased medullary NaCl enhances U_NaV in both intact female and OVX rats. Compared with intact females, OVX displayed faster changes in U_NaV in response to intramedullary infusion of hyperosmotic saline. Furthermore, these results showed that infusion of hyperosmotic saline in the renal medulla of OVX resulted in the same time-dependent natriuretic pattern as we recently reported in male rats (23). Earlier evidence suggests male-female differences in response to increased inner medullary osmolality (59). Increasing the papillary osmolality decreased the water content and increased the total amount of solutes in the renal medulla of rats. Females showed greater changes than males in the amount of urea in the inner medulla in response to increasing urine osmolality (59).

At baseline, anesthetized OVX rats had lower urine flow and higher urine osmolality compared with intact female rats. These results are consistent with those of Cheema et al. who showed that estradiol inhibited the expression of collecting duct aquaporin 2 (12). This effect is likely due to differences in vasopressin that are modulated by the estrous cycle, ovariectomy, and estradiol replacement (19, 27, 36, 55, 62). In the current study we did not observe differences between groups in relation to RBF, nor were MAP or RBF affected by intramedullary infusion. Furthermore, prior studies demonstrate that the intramedullary infusion technique does not have observable effects on hemodynamic or renal function outside the renal medulla (46, 61). Therefore, we suggest that our data are not the result of pressure natriuresis or changes in RBF. Nonetheless, it is possible that renal interstitial pressure could account for some of these differences in urine flow and/or U_NaV; this will need to be explored in future studies.

Increased NaCl delivery in the medullary interstitium is considered an important stimulus for renal ET-1 synthesis. Evidence derived from experiments in male rats suggests that intramedullary infusion of hyperosmotic saline promotes ET-1-dependent diuresis and natriuresis (6, 23), but whether this occurs in female rats has not previously been determined. Our results show that natriuresis in response to intramedullary hypertonic saline in intact female rats was not changed by dual blockade of ET_A+B receptors. Consistently, female ET_B-deficient rats have a more efficient natriuresis than male ET_B-deficient rats, suggesting that females have a more robust mechanism for handling NaCl that is independent of the ET-1 system (34). Taking into consideration that many redundant systems facilitate Na⁺ excretion (17, 57), current findings pinpoint that natriuretic factors other than ET-1 are responsible for the natriuretic response to medullary NaCl loading in intact females. Further studies are needed to identify the signaling mechanism underlying the renal Na⁺ handling in intact female rats more clearly.

Surprisingly, ET_A+B receptor blockade in intact females enhanced the natriuretic response demonstrated during the first 30 min of increasing medullary NaCl. Similarly, our laboratory has previously reported that ET_B receptor blockade alone did not inhibit the U_NaV in response to intramedullary infusion of hyperosmotic saline (7). We think that the unexpected increase in U_NaV that we observed with dual receptor blockade in the current study might be related to activation of other natriuretic pathways as a compensatory response to pharmacological blockade of the ET-1 pathway. It is also possible that inhibition of ET_A-mediated vasoconstrictor tone caused an increase in medullary blood flow, resulting in enhanced natriuresis.

In contrast to intact female rats, the dual blockade of ET_A+B receptors inhibited the natriuretic response to intramedullary infusion of hyperosmotic saline in OVX rats, suggesting that the increased NaCl to the renal medulla of OVX rats promotes ET-1-dependent natriuresis. Furthermore, we recently reported that ET_A and ET_B receptor expression within the renal inner medulla was increased 3 wk after ovariectomy, and this increase was prevented by supplementation of OVX rats with estradiol and progesterone (24). Thus, the medullary ET-1 system may have a greater impact on natriuresis in OVX rats compared with intact females.

Notably, our finding that intramedullary infusion of hyperosmotic saline for 60 min did not affect the ET-1 mRNA expression within the renal inner medulla in OVX rats does not conflict with our finding that blockade of ET receptors inhibited the natriuresis in OVX rats. One possible explanation is related to differences in ET receptor abundance (24, 33). Differences in the medullary ET receptor function and localization are also possible contributors to sex- and OVX-related differences in the role of the medullary ET-1 system in response to enhanced medullary NaCl loading.

P2 receptors play a fundamental role in controlling Na⁺ excretion (50, 68, 69). In the collecting duct, P2Y₂ receptor activation by nucleotides or ET_B receptor activation by ET-1 decreases ENaC activity (9, 56). Recent evidence revealed that P2 receptors mediate the flow-stimulated ET-1 release in IMCD (54). Additionally, activation of the medullary P2 receptors is necessary for the ET-1-dependent natriuresis in male rats (23). Our current data show that the natriuretic response to intramedullary hyperosmotic infusion was not affected by suramin in intact females, minimizing a role for activation of medullary P2 receptors in mediating natriuresis in intact females. In contrast, suramin inhibited the natriuretic response to increased medullary NaCl in OVX rats, similar to our previous findings in male rats. Given that suramin is a broad P2 receptor antagonist, further experiments will be needed to help clarify the potential contribution of different subtypes of medullary P2Y and P2X receptors to the ET-1-dependent natriuresis. Nonetheless, we can conclude that ovariectomy uncovers an important role for medullary P2 receptors in natriuresis in response to increased NaCl to renal medulla.

Unlike our recently published findings in male rats (23), intramedullary infusion of UTP for 60 min did not alter urine flow or U_NaV in intact females, highlighting a sex-related difference in the P2-mediated control of Na⁺ excretion. Ovariectomy revealed the natriuretic response to UTP. It is unclear to the authors why UTP did not cause natriuresis in intact females; however, this observation might be linked to sex differences in basal levels of ATP, the distribution and the relative roles of P2 receptor subtypes, and/or basal levels of ecto-ATPases that hydrolyze nucleoside triphosphates and diphosphates and regulate P2 receptor-dependent signaling (69). Previous studies show that estrogen interacts with P2 signaling in different tissue systems, including sensory neurons (13, 14, 43, 47, 48, 71), microglia (15, 16), breast cancer (45), uterine cervical cells (25), uterine vasculature (28), urinary bladder (41), and kidney cells (10). Thus, more studies are needed to address possible regulation of renal medullary P2 signaling by sex and sex steroids, which is currently poorly understood.

Our studies also revealed an inhibitory effect of dual blockade of ET_A+B receptors on the diuretic and natriuretic effect of activation of medullary P2Y₂ and P2Y₄ receptors in OVX rats. This finding further supports the possibility of P2 receptor control of renal medullary ET-1 activity within the renal medulla of OVX rats, similar to males. Overall, our findings that pharmacological blockade of P2 or ET_A+B receptors attenuated the diuretic and natriuretic response to intramedullary infusion of a NaCl load or UTP, respectively, in OVX rats highlight an important connection between the medullary P2 and ET-1 signaling systems in controlling Na⁺ excretion in OVX rats, similar to our recent findings in males (23).

Finally, both renal ET-1 and P2 signaling systems have been shown to produce dramatic effects on Na⁺ excretion and regulating blood pressure. Despite their overlapping spheres of influence, information on potential interplay between renal P2 and ET receptors is rare. The interaction between ET-1 and P2 signaling systems has been also demonstrated in extrarenal tissues (1, 4, 31, 35). Our current findings demonstrate that activation of medullary P2 receptors promotes ET-1-dependent natriuresis in OVX rats, similar to male rats (23). However, NaCl loading to the renal medulla of intact females induced P2- and ET-1-independent natriuresis, highlighting an important role for sex steroids in regulating medullary P2 and ET-1 natriuretic pathways, which play a central role in Na⁺ homeostasis and blood pressure regulation.

Study limitation.

Although ovariectomy is a validated surgical approach representing features of estrogen deficiency, it does not reflect age-related postmenopausal changes that occur regarding receptor expression. Thus, in the current study, we showed the effect of the absence of sex hormones on the P2- and ET-1-dependent natriuresis. Whether this occurs in menopausal and postmenopausal females needs to be explored in the future.

GRANTS

This study was supported by grants from the National Heart Lung and Blood Institute (P01-HL-095499 to D. M. Pollock, K99-HL-127178 to J. S. Speed, and T32-HL-007457 to B. K. Becker), a postdoctoral grant from the American Heart Association (15POST25090329 to E. Y. Gohar), and an American Society of Nephrology (Ben J. Lipps Research Fellowship Program, Joseph A. Carlucci Research) Fellowship to M. Kasztan.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors. Dr. Gohar is also affiliated with Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt.

AUTHOR CONTRIBUTIONS

E.Y.G. and D.M.P. conceived and designed research; E.Y.G. performed experiments; E.Y.G., M.K., B.K.B., and J.S.S. analyzed data; E.Y.G., M.K., B.K.B., and D.M.P. interpreted results of experiments; E.Y.G. prepared figures; E.Y.G. drafted manuscript; E.Y.G., M.K., B.K.B., J.S.S., and D.M.P. edited and revised manuscript; E.Y.G., M.K., B.K.B., J.S.S., and D.M.P. approved final version of manuscript.