Aldosterone antagonism is more effective at reducing blood pressure and excessive renal ENaC activity in AngII-infused female rats than in males

Abstract

Background:

Angiotensin II dependent hypertension (AngII-HTN) causes comparable elevations of blood pressure (BP), aldosterone levels and renal epithelial Na⁺ channel (ENaC) activity in male and female rodents. Mineralocorticoid receptor (MR) antagonism has a limited antihypertensive effect associated with insufficient suppression of renal ENaC in male rodents with AngII-HTN. While MR blockade effectively reduces BP in female mice with salt-sensitive and leptin-induced hypertension, MR antagonism has not been studied in female rodents with AngII-HTN. We hypothesize that overstimulation of renal MR signaling drives redundant ENaC-mediated Na⁺ reabsorption and BP increase in female rats with AngII-HTN.

Methods:

We employ a combination of physiological, pharmacological, biochemical and biophysical approaches to compare the effect of MR inhibitors on BP and ENaC activity in AngII-infused male and female Sprague Dawley rats.

Results:

MR blockade markedly attenuates AngII-HTN in female rats but has only a marginal effect in males. Spironolactone increases urinary sodium excretion and urinary sodium-to-potassium ratio in AngII-infused female, but not male, rats. The expression of renal MR and 11β-hydroxysteroid dehydrogenase type 2 that determines the availability of MR to aldosterone is significantly higher in AngII-infused female rats than in males. ENaC activity is approximately two times lower in spironolactone-treated AngII-infused female rats than in males. Reduced ENaC activity in AngII-infused female rats on spironolactone correlates with increased interaction with ubiquitin ligase Nedd4-2, targeting ENaC for degradation.

Conclusions:

MR-ENaC axis is the primary determinant of excessive renal sodium reabsorption and an attractive antihypertensive target in female rats with AngII-HTN, but not in males.

Graphical Abstract

Introduction

Despite the abundance of available therapeutic options hypertension remains a global public health challenge and a major risk factor for cardiovascular and chronic kidney disease^1,2. Elevation of BP results from intricate interactions between various environmental and genetic factors³. Due to the complex nature of hypertension, there is no single effective treatment. Identification of the pathological mechanisms underlying hypertension in specific cohorts would allow optimizing treatment by matching the therapeutics to the disease mechanism. This strategy is imperative to reduce the burden of hypertension.

Biological sex is now recognized as a pivotal factor affecting BP control. Males and females employ the same systems to regulate BP, yet considerable sex-specific variations in the levels of volume regulatory hormones, expression and activity of renin-angiotensin-aldosterone system components and other determinants of cardiovascular homeostasis have been reported^4–7. A better understanding of sex differences pertaining to BP regulation would improve the effectiveness of antihypertensive regimen in both sexes.

The kidney plays a central role in chronic BP control through regulation of sodium reabsorption. Fine-tuning of renal Na⁺ transport takes place in the aldosterone-sensitive distal nephron (ASDN). ENaC is a key Na⁺ transporter in this nephron segment and the activity of the channel is under tight control by volume regulatory hormones, such as aldosterone^8,9. Excessive ENaC-dependent renal Na⁺ reabsorption has been clearly linked to volume expansion and hypertension^10–12. Sex disparities in renal ENaC expression, circulating aldosterone levels and aldosterone signaling have been reported^5,13–15. However, the interplay between biological sex, renal ENaC and aldosterone in hypertension remains to be fully understood.

Cumulative evidence suggests that aldosterone antagonism may be more effective at reducing hypertension in females than in males^4,7,16–18. However, the underlying mechanisms mediating this remain unclear. Inhibition of the aldosterone-MR axis is insufficient to effectively suppress ENaC activity and excessive Na⁺ reabsorption in male mice and rats with AngII-dependent hypertension¹⁹. L-NAME/AngII-induced hypertension in female rats, salt sensitive BP increase in Balb/C and obesity-induced hypertension in agouti yellow hyperleptinemic female mice are characterized by inappropriately elevated aldosterone levels^4,7,17. MR blockade reduces BP in hypertensive female mice^4,17 and prevents cardiovascular and renal damage in rats⁷. Nevertheless, the effect of MR blockade on ENaC-dependent Na⁺ reabsorption has never been tested in hypertensive female rodents.

In this study, we test the hypothesis that MR activation is the primary determinant of ENaC-dependent renal Na⁺ reabsorption and a dominant BP regulatory mechanism in AngII-infused female, but not male, Sprague Dawley (SD) rats. To test this hypothesis at the molecular, cellular and systemic levels, we combined patch-clamp electrophysiology, immunoblotting, quantitative PCR with metabolic cage studies and BP assessment. Our findings indicate that MR blockade is more effective at reducing ENaC-mediated sodium retention and lowering BP in female rats with AngII-HTN, when compared to males. Our study reconciles the existing data on poor efficacy of MR antagonism in male rodent models with the available reports showing that females can benefit from MR inhibition. This suggests that MR antagonism may be the preferred therapeutic intervention to optimize treatment of hypertension in females.

Methods

Experimental animals

Male and female SD rats were used in all studies. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Medical College of Georgia at Augusta University (AUP 2017-0844) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

BP monitoring

BP telemetry was performed, as described earlier²⁰, in 4 groups of SD rats: 1) AngII-infused males (MA), 2) AngII-infused males on spironolactone (MAS), 3) AngII-infused females (FA), 4) AngII-infused females on spironolactone (FAS). To exclude off-target effects of spironolactone, the antihypertensive effect of MR blockade was validated by treatment with a more specific MR antagonist, eplerenone. BP was measured on a weekly basis by tail-cuff plethysmography in the rats randomized into 4 groups: 1) AngII-infused males (MA), 2) AngII-infused males on eplerenone (MAE), 3) AngII-infused females (FA), 4) AngII-infused females on eplerenone (FAE).

Statistical analysis

Results are presented as mean ± SEM. Following the published guidelines for reporting statistics²¹, longitudinal BP data were analyzed by a two-way mixed-design ANOVA with the Greenhouse-Geisser correction and the endpoint data – by a two-way ANOVA. Pairwise comparisons to identify significant differences between specific group means were performed by the Holm-Sidak multiple comparison test. P<0.05 was considered significant. Statistical analysis was performed using OriginPro 2022 (OriginLab Corp., Northampton, MA, USA).

Further details pertaining to animal studies, experimental procedures and statistical analysis are provided in the Online Supplement.

Data supporting the findings of this study are available from the corresponding author upon a reasonable request.

Results

MR blockade results in a pronounced antihypertensive effect in female AngII-infused rats, but not in males

Consistent with the existing data⁶, infusion of AngII at 400 ng·kg⁻¹·min⁻¹ for 2 weeks resulted in a significant elevation of mean arterial pressure (MAP) in male SD rats from 111±1 to 173±7 mmHg (Fig. 1A). Males, administered with an aldosterone antagonist, spironolactone (30 mg·kg⁻¹·day⁻¹ in drinking water), exhibited a comparable increase of MAP (111±2 to 165±7 mmHg). MAP was not significantly different between the two groups. In contrast, the magnitude of AngII-driven MAP elevation was approximately 2 times lower in female SD rats supplemented with spironolactone (from 104±2 to 128±8 mmHg), when compared to the vehicle-treated AngII-infused females (from 104±1 to 156±4 mmHg, Fig. 1B). Significant differences in MAP between vehicle- and spironolactone-treated AngII-infused females were observed after 6 days of treatment. AngII infusion blunted the diurnal rhythmicity of MAP in females and administration of spironolactone resulted in a noticeable improvement of the diurnal MAP rhythm, reaching statistical significance at the end of the 14-day observation period (Figure S1 C, D). In contrast, spironolactone did not affect the amplitude of the diurnal MAP rhythm in AngII-infused males (Figure S1 A, B). Thus, spironolactone markedly attenuated AngII-induced hypertension in female rats, but not in males.

Figure 1. MR blockade attenuates hypertension in AngII-infused female rats, but not in males.

(A, B) Daily mean arterial pressure (MAP) averages recorded by telemetry at baseline and after AngII infusion in male and female SD rats treated with vehicle (MA, FA) or spironolactone (MAS, FAS). N=9 per group. Data were analyzed by a two-way mixed-design ANOVA. * – denotes P<0.05 for pairwise comparisons using the Holm-Sidak multiple comparisons test. (C, D) Daily mean arterial pressure (MAP) averages recorded by tail-cuff plethysmography at baseline, day 7 and day 14 of AngII infusion in male and female SD rats treated with vehicle (MA, FA) or eplerenone (MAE, FAE). N=8 per group. Data were analyzed by a two-way mixed-design ANOVA. * – denotes P<0.05 for pairwise comparisons using the Holm-Sidak post hoc test.

To circumvent potential progestational and antiandrogenic effects of spironolactone, we verified our findings in AngII-infused rats treated with eplerenone, a more selective MR antagonist. After 2 weeks of treatment, MAP increase was significantly blunted in AngII-infused females receiving eplerenone, when compared to vehicle-treated AngII-infused females (110±3 vs 130±3 mmHg, respectively, after 2 weeks of treatment). This difference became significant after 1 week of treatment (102±3 vs 119±2 mmHg, Fig. 1D). MAP was only marginally lower in AngII-infused males on eplerenone than in vehicle-treated male rats (125±4 vs 130±3 mmHg, respectively, after 2 weeks, Fig. 1C). Thus, the antihypertensive effect of MR blockade is considerably more pronounced in AngII-infused female rats, when compared to males.

Spironolactone increases urinary Na:K ratio in AngII-infused female rats, but not in males

Distal nephron, responsible for final adjustments to Na⁺ reabsorption and K⁺ secretion, is a universally recognized site affected by aldosterone antagonism. Thus, next we assessed if spironolactone alters urinary Na⁺ and K⁺ excretion in AngII-infused SD rats of both sexes. MR blockade did not appreciably alter renal Na⁺ excretion in AngII-infused males. Urinary Na⁺ excretion was significantly higher in spironolactone-treated AngII-infused females when compared to vehicle-treated females or males on spironolactone (Fig. 2A). Importantly, AngII-infused females on spironolactone maintain urinary Na⁺ excretion at significantly lower BP, indicative of improved pressure-natriuresis response. Consistent with the recognized potassium-sparing effect of MR inhibitors, K⁺ excretion was moderately, but significantly lower in AngII-infused rats receiving spironolactone (P_spir=0.047). AngII-infused females excreted more K⁺ in urine when compared to males, regardless of spironolactone intervenion (Fig. 2B). The effect of spironolactone on urinary Na⁺ to K⁺ ratio, indicative of electrolyte transport in the distal nephron, was significant (P_spir=0.013) and strongly dependent on the animal’s sex (P_interaction=0.001; Fig. 2C). Na⁺:K⁺ ratio was significantly higher in AngII-infused females on spironolactone than in vehicle-treated females (P<0.001). Administration of spironolactone resulted in a marginal elevation of serum K⁺ levels in AngII-infused rats of both sexes that did not reach statistical significance and did not appreciably affect serum Na⁺ and Cl^- in either sex (Table 1). MR blockade did not change body weight or gross readouts of renal function, such as kidney to body weight ratio and serum creatinine. While spironolactone did not improve microalbuminuria, we found that urinary albumin levels were significantly lower in AngII-infused females than in males (Table 1). Overall, a stronger antihypertensive effect of MR antagonism in AngII-infused females is, at least in part, determined by improved electrolyte handling by the kidney.

Figure 2. Spironolactone is more effective at improving renal electrolyte handling in AngII-infused females than in males.

Endpoint (day 14) urinary sodium excretion, U_NaV (A); urinary potassium excretion, U_KV (B), and urinary Na⁺ to K⁺ ratio (C) in AngII-infused male and female SD rats treated with vehicle (MA and FA) or spironolactone (MAS and FAS). N=8 per group. The results of a two-way ANOVA are summarized below the graphs. * – indicates a significant difference (P<0.05) between respective group means determined by the Holm-Sidak test.

Table 1. Endpoint physiological readouts for the experimental rats of both sexes.

A two-way ANOVA was used for statistical comparisons. Data are presented as mean values ± the standard error of mean (SEM). AngII – AngII-infused rats receiving vehicle for 14 days, AngII + Spir – AngII-infused rats administered with spironolactone for 14 days. N≥8 for each group.

Parameter	Males		Females		Significance
	AngII	AngII+spir	AngII	AngII+spir	Psex	Pspir	Pinteraction
Body weight, g	323±7.8	322±5.8	228±4.2	232±2.2	<0.001	0.826	0.665
Total kidney to body weight, %	0.72±0.01	0.74±0.01	0.71±0.02	0.71±0.02	0.225	0.560	0.710
Serum creatinine, mg/dL	0.26±0.03	0.26±0.03	0.25±0.03	0.24±0.03	0.574	0.800	0.912
Urinary albumin, mg/dL·24h	20.6±4.1	24.0±4.0	4.28±1.94	4.19±1.12	<0.001	0.648	0.518
Serum Na+, mmol/L	138±0.7	139±0.6	136±0.5	138±0.6	0.005	0.115	0.409
Serum K+, mmol/L	5.1±0.23	5.4±0.24	5.2±0.2	5.3±0.24	0.159	0.787	0.900
Serum Cl-, mmol/L	96±0.7	96±0.7	96±0.8	98±0.95	0.220	0.178	0.174

Renal expression of MR and 11β-hydroxysteroid dehydrogenase type 2 is higher in AngII-infused females than in males

Cumulative experimental evidence identifies excessive circulating aldosterone levels and elevated MR expression in the tissues as critical sex-specific determinants of hypertension. Consistent with the previous report⁶, we found that serum aldosterone concentrations are significantly higher in AngII-infused male and female SD rats (~3000 pg/ml) than those typically observed at baseline without AngII-infusion (<100 pg/ml). After 2 weeks of AngII infusion circulating aldosterone levels are comparable in AngII-infused rats of both sexes (Fig. 3A). Interestingly, quantitative RT-PCR demonstrated that expression of mRNA encoding MR (Nr3c2) was significantly higher in the kidneys isolated from AngII-infused females, when compared to males (P_sex=0.001; Fig. 3B), regardless of spironolactone intervention. The availability of MR to aldosterone in the ASDN is controlled by an enzyme, 11β-hydroxysteroid dehydrogenase type 2 (HSD11β2), that inactivates highly abundant corticosterone and protects MR from binding to glucocorticoids. We found that Hsd11b2 expression is significantly higher in AngII-infused female rats than in males (P_sex<0.001; Fig. 3C). This points to a higher sensitivity of renal epithelium to stimulation by aldosterone or MR antagonism in female rats with AngII-dependent hypertension, when compared to males.

Figure 3. Renal expression of mRNA encoding MR and hydroxysteroid 11β-hydroxysteroid dehydrogenase type 2 is higher in females with AngII-induced hypertension, while circulating aldosterone levels are comparable in both sexes.

Endpoint serum aldosterone concentrations (A), relative expression of Nr3c2 mRNA, encoding MR (B), and Hsd11b2 mRNA (C) in the renal tissue isolated from male and female AngII-infused rats receiving vehicle (MA and FA) or spironolactone (MAS and FAS) for 2 weeks. Relative mRNA expresion values were normalized to that in AngII-infused vehicle-treated males. The results of a two-way ANOVA are summarized below the graphs. * – indicates a significant difference (P<0.05) between respective group means determined by a post-hoc Holm-Sidak test. N≥6 per group.

Spironolactone is more effective at reducing ENaC activity in AngII-infused females, when compared to males

We have previously reported that ENaC activity was similar in SD males and females at baseline conditions, and almost tripled after AngII infusion without any apparent sex differences⁶. Our patch-clamp experiments in freshly isolated split-open ASDNs revealed that spironolactone inhibited ENaC-mediated currents in AngII-infused female rats to a significantly larger extent than in males (Fig. 4). ENaC activity was significantly lower in AngII-infused female rats on spironolactone, when compared to the respective male group (fNP_o of 0.37±0.06 vs 0.75±0.08, P=0.002, Fig. 4A). This difference was associated with significantly reduced levels of functional channels on the membrane (fN of 1.11±0.10 vs 1.76±0.15, P=0.007, Fig. 4B) and ENaC open probability (P_o of 0.27±0.02 vs 0.41±0.03, P<0.001, Fig. 4C) in spironolactone-treated females when compared to males. Consistently, α-ENaC subunit reporting signal is markedly lower in ASDNs isolated from spironolactone-treated AngII-infused females than in males (Fig. 4E). Moreover, in spironolactone-treated females α-ENaC is retained inside the cells, further away from the luminal surface, strongly indicative of decreased channel functionality. Overall, we conclude that renal ENaC is more sensitive to MR blockade in AngII-infused females than in males.

Figure 4. ENaC activity and α-ENaC subunit abundance are significantly lower in the collecting ducts isolated from AngII-infused females, when compared to males.

Summary graphs of ENaC activity (fNP_o; A), functional channels (fN; B) and open probability (P_o; C) in AngII-infused male and female SD rats receiving vehicle (MA and FA) or spironolactone (MAS and FAS) for 2 weeks. The results of a two-way ANOVA are summarized below the graphs. * – indicates a significant difference (P<0.05) between the respective groups determined by the post-hoc Holm-Sidak test. Each group included at least 8 rats and 66 tested cells. (D) Representative single-channel ENaC current traces recorded in the ASDN principal cells from AngII-infused SD rats on spironolactone. Patches were held at a test potential of V_h=−V_p=−60 mV. Inward currents are downward. Dashed lines indicate the respective open (o_i) and closed (c) states of the channel. Areas under the traces marked in blue (male) or pink (female) visualize ENaC activity. (E) Representative immunofluorescent micrographs of rat kidney sections showing the abundance and localization of α-ENaC subunit (pseudocolor green) in the AQP2-positive (pseudocolor red) collecting duct principal cells. Nuclear DAPI staining is shown in pseudocolor blue.

Sodium-chloride cotransporter, NCC, localized to the distal convoluted tubule, is another important determinant of AngII-induced hypertension²². NCC can be activated via MR, although, likely, independently of aldosterone^23,24. We found that spironolactone did not affect the abundance of total or phosphorylated (active) forms of NCC in the renal tissue isolated from AngII-infused rats of both sexes (Figure S2). Thus, the effect of spironolactone appears to be mediated through aldosterone-MR cascade.

Nedd4-2 abundance is significantly higher in spironolactone-treated females than in males

Ubiquitination of ENaC subunits by the E3 ubiquitin ligase Nedd4-2 results in subsequent retrieval of the channel from the plasma membrane and is a key converging point for the regulation of ENaC surface density by aldosterone. Aldosterone-induced phosphorylation of Nedd4-2 prevents its binding to ENaC, consequently, increasing the abundance of the channel on the membrane. Here, we used a ratio of total to phosphorylated Nedd4-2 protein in kidney homogenates to gauge the interaction of Nedd4-2 with ENaC (Fig. 5). The ratio was significantly higher in the renal tissue homogenates isolated from spironolactone-treated AngII-infused female rats, when compared to AngII-infused males on spironolactone (1.6 times, P<0.001) or AngII-infused females receiving vehicle (1.4 times, P=0.002). This is indicative of a higher Nedd4-2 activity, greater ENaC internalization and reduced ENaC-dependent renal sodium reabsorption in spironolactone-treated AngII-infused females, when compared to males.

Figure 5. Nedd4-2 activity is remarkably higher in the kidneys of spironolactone-treated AngII-infused females than in males.

(A) Representative western blots from whole kidney homogenates of AngII-infused male and female SD rats receiving vehicle (MA and FA) or spironolactone (MAS and FAS), showing the total Nedd4-2 protein and its inactive form phosphorylated at serine 328. (B) A summary graph comparing Nedd4-2 to pS³²⁸Nedd4-2 ratio (indicative of Nedd4-2 interaction with ENaC) in AngII-infused male and female rats. The relative Nedd4-2 to pS³²⁸Nedd4-2 ratio, normalized to vehicle-treated AngII-infused males = 1, is shown. The results of a two-way ANOVA are summarized below the graph. * – indicates a significant difference (P<0.05) between respective group means determined by the post-hoc Holm-Sidak test. N=6 per group.

Discussion

This study allowed us to make several novel observations: 1) BP in female rats with AngII-dependent hypertension is more responsive to MR antagonism than in males; 2) spironolactone increases urinary Na⁺ excretion and urinary Na⁺ to K⁺ ratio specifically in AngII-infused females; 3) renal expression of mRNA encoding MR and HSD11β2 is significantly higher in AngII-infused female rats than in males; 4) MR blockade is markedly more potent at decreasing ENaC activity in hypertensive female rats than in males. Our findings indicate that elevated expression of the mineralocorticoid receptor in the kidney determines higher sensitivity of renal ENaC and BP to aldosterone antagonism in female rats with AngII-dependent hypertension, when compared to males. Overstimulation of MR signaling drives BP increase via excessive ENaC-dependent renal Na⁺ reabsorption in females with AngII-dependent hypertension, but not in males. The data reveal that ENaC sensitivity to MR antagonism is an unrecognized sex-specific approach to combat hypertension and provides pre-clinical evidence supporting the use of MR antagonists in hypertensive women to improve BP control rates.

We found that MR blockade is significantly more effective at attenuating hypertension in AngII-infused female SD rats than in males. This observation is consistent with the emerging clinical evidence and basic science studies showing that females may receive additional antihypertensive and cardiovascular benefits from MR inhibition, when compared to males. MR blockade effectively attenuates salt-sensitive hypertension in female Balb/C mice and decreases BP in agouti yellow obese female mice with leptin-induced hypertension^4,17. In contrast, only a marginal hypotensive effect was detected in male AngII-infused rats and mice administered with MR blockers^25–28. Clinically, aldosterone antagonism may be more effective at reducing BP in African American women, women with salt sensitive and resistant hypertension, when compared to men^7,16,29. To the best of our knowledge, our study is the first to directly compare the antihypertensive effects of MR antagonism in male and female rats with AngII-induced hypertension.

Our study primarily employs spironolactone to explore sex-specific effects of MR blockade in AngII-infused rats. Spironolactone is a potent antimineralocorticoid and its action in the body is enhanced by long-lived therapeutically active metabolites³⁰. Unfortunately, spironolactone is a nonselective MR antagonist with discernable antiandrogenic and progestogenic properties^30,31. We confirmed that AngII-infused females derive their antihypertensive benefits specifically from inhibition of MR, using a highly selective second-generation MR antagonist, eplerenone. However, eplerenone is extensively metabolized to inactive derivatives and has a 2-4 times shorter elimination half-life than spironolactone^31,32. Due to its pharmacokinetic properties and low cost to the patient, spironolactone is still widely used in clinic, despite its off-target side effects^33,34. Moreover, studies in rodents showed that spironolactone and its active derivatives are accumulated in the kidney with remarkably lower levels found in the cardiac tissue^{30,31,35–38}. Thus, upon careful consideration, we chose to pursue our experimental strategy using spironolactone.

In line with the potassium-sparing effect of MR antagonists, we observed a mild, although statistically insignificant, increase in serum potassium levels in spironolactone-treated AngII-infused rats. Hyperkalemia is, perhaps, the most alarming side effect of MR blockade in the clinical setting^31,39. Often, the risk-to-benefit ratio for this intervention is defined by the magnitude of serum potassium change. Nevertheless, the incidence and severity of hyperkalemia strongly depends on the dose of the MR antagonist and the level of renal function³⁹. Spironolactone concentrations that we used in this study are in the lower dosage range for rodents and the duration of intervention was only 2 weeks. Our findings and previously published data indicate that renal function is largely preserved in SD rats after 2 weeks of AngII infusion. It is not, therefore, surprising that spironolactone treatment does not cause major disruptions in K⁺ homeostasis in AngII-infused rats in our experimental setting. Perhaps, future long-term experiments could better inform of potential cumulative side-effects of MR antagonism related to potassium balance.

Our data indicate that treatment with spironolactone improves the diurnal rhythmicity of BP in AngII-infused female rats, but not in males. Blunted diurnal BP variation is a known risk factor for deterioration in renal function, cardiovascular morbidity and mortality, worsening the long-term prognosis for hypertensives with a non-dipping BP pattern^40–44. Nocturnal dipping has been associated with the ability to excrete sodium during the day in individuals of African descent⁴⁵. A mounting body of evidence coming from rodent models and cell lines suggests that a crosstalk between circadian clock, aldosterone and ENaC is an important determinant of systemic Na⁺ balance, circulating volume and BP^46–48. Recent studies reveal sex differences in the circadian gating of Na⁺ homeostasis and BP control^49,50. Yet, the interplay between sex, circadian clock, aldosterone-MR axis, renal Na⁺ handling and BP regulation is far from being fully understood. Our findings open an interesting avenue for future investigations exploring the contribution of sexual and temporal variability in aldosterone-MR signaling to the pathophysiology of AngII-dependent hypertension and efficacy of BP control interventions.

An important observation of this study is that MR expression is significantly higher in the kidneys of AngII-infused female rats, when compared to males. For many years, studies in the field of hypertension have been focused on circulating or tissue-specific levels and production of aldosterone, a primary salt-retaining hormone, while the implications of renal sensitivity to mineralocorticoid remained largely unexplored. Plasma aldosterone concentration is not always sufficient to reliably predict adverse cardiovascular or prohypertensive outcomes. High circulating aldosterone levels during chronic sodium deficiency (Yanomami population) or pregnancy allow for efficient sodium reabsorption in the kidney, but do not result in any cardiovascular morbidities or hypertension^51–53. Animal data and clinical guidelines on normal circulating aldosterone levels vary considerably, often by orders of magnitude^54,55. Studies exploring sex, race and age differences add to the existing controversy, and raise a question of what should be regarded as a normal or elevated aldosterone level in a given group^7,56–59. It appears that this question cannot be answered without considering both the ligand and its receptor. Inordinate renal Na⁺ reabsorption is a recognized determinant of hypertension^10,12. The existing evidence indicates that elevated sensitivity to aldosterone might have a detrimental effect on sodium balance and BP^56,60. There is a gap in knowledge between aldosterone-MR signaling in the distal nephron and differential BP sensitivity to aldosterone or its antagonists in hypertensive patients⁶¹. Our study bridges this gap and provides a mechanistic explanation of how increased renal sensitivity to MR antagonists in AngII-infused females translates into higher BP responsiveness to aldosterone antagonism.

Our study reveals that a stronger antihypertensive effect of MR blockade is paralleled by a higher expression of Hsd11b2 in the renal tissue of AngII-infused female rats, when compared to males. This observation is particularly interesting in light of several clinical and basic science studies showing that the effects of MR inhibition on BP, renal Na⁺ and K⁺ handling, cardiovascular and renal health do not always result from aldosterone antagonism^24,62–64. Aldosterone synthase-deficient mice generally exhibit a milder phenotype than kidney-specific MR knockouts²⁴. While substantial MR effects are reported to be mediated independently of aldosterone in the distal convoluted tubule, the cortical collecting duct is much more reliant upon aldosterone-MR signaling, largely, due to a higher activity of HSD11β2^24,64. Higher Hsd11b2 expression that we observed in the kidneys of AngII-infused female rats points to a more prominent role of aldosterone in stimulation of renal Na⁺ reabsorption in this model of hypertension. Interestingly, spironolactone did not affect the abundance of NCC transporter, that is localized to the DCT and is much more likely to be regulated independently of aldosterone. Thus, our findings indicate that higher sensitivity of BP and renal ENaC activity to MR inhibitors in females with AngII-dependent hypertension stems from the blockade of aldosterone actions on MR.

Here, we demonstrate that ENaC activity is significantly lower in the ASDNs isolated from spironolactone-treated AngII-infused female rats than in males. MR inhibition reduces ENaC activity in AngII-infused females not only through lowering the number of functional channels on the membrane, but also through decreasing ENaC open probability. The former is likely a genomic effect of MR inhibition that involves canonical aldosterone signaling pathways. A downstream convergent element of these cascades is Nedd4-2 – a ubiquitin ligase, facilitating the clearance of ENaC from the membrane. Nedd4-2 activity is inhibited by phosphorylation, thereby increasing ENaC surface expression⁶⁵. Loss of Need4-2 in mice causes salt-sensitive hypertension and enhanced renal ENaC expression⁶⁶ resistant to eplerenone⁶⁷. We observed that spironolactone markedly increased the ratio of total over phosphorylated form of Nedd4-2 in the kidney, indicative of higher protein activity and greater ENaC internalization in AngII-infused females, but not in males. This suggests lower ENaC activity due to higher protein degradation in spironolactone-treated females. In addition to increasing ENaC expression and abundance on the membrane, aldosterone was shown to stimulate the activity of the channel^68–71, an effect that can be blocked by spironolactone. The exact mechanisms mediating aldosterone-induced increase in ENaC activity remain uncertain, but may employ non-canonical signaling cascades involving membrane phospholipids^72,73, methylation of the channel^71,74 or generation of reactive oxygen species⁷⁵. Overall, our results suggest that spironolactone-driven inhibition of ENaC activity in AngII-infused females employs both canonical and noncanonical MR-dependent pathways. This provides a strong impetus for further studies addressing the effect of non-canonical aldosterone signaling pathways on renal sodium handling and BP control.

In our study a stronger BP lowering effect of MR blockade in AngII-infused females is attributed to a more pronounced reduction of renal ENaC activity, when compared to males. A mounting body of evidence indicates that MR antagonism can reduce BP in hypertensive female rodents through mechanisms that are extrinsic to the kidney⁷⁶. Increased endothelial cell MR activity reportedly contributes to endothelial dysfunction and BP increase in salt sensitive Balb/C and agouti yellow obese hyperleptinemic female mice^4,17,77. Activation of MR in endothelial cells increases membrane abundance of endothelial cell sodium channel resulting in reduced endothelium nitric oxide (NO) synthase 3 activity, NO levels and excessive arterial stiffness^78,79. ENaC activity in dendritic cells is an important determinant of the sodium-induced proinflammatory response and hypertension in male C57BL/6 mice⁸⁰. MR in T cells appear to be critical for development of AngII-induced hypertension in C57Bl/6 mice, although the sex of the animals used in this study is not specified⁸¹. The relevance of these mechanisms in hypertensive female animals remains to be elucidated, given that the research performed in female rodents mostly points to an anti-inflammatory role of immune system resulting in attenuation of hypertension. Interestingly, inflammatory cytokines, such as interleukin 6 or interferon-γ, can affect MR and ENaC-dependent sodium transport in ASDN cross-linking the inflammatory immune response and renal sodium transport^82–84. Overall, the non-epithelial effects of MR observed in vasculature, immune cells and other tissues are undoubtedly important in the pathophysiology of hypertension. Further studies are necessary to integrate the renal and extrarenal effects of MR antagonism pertaining to BP control and achieve a more complete understanding of MR biology in both sexes.

In summary, our study uncovers a sex-divergence in the sensitivity of renal ENaC to MR blockade in hypertensive rats. We and others have previously reported that overstimulation of ENaC is largely independent of aldosterone, primarily driven by intrarenal AngII generation and effectively inhibited by angiotensin converting enzyme blockade in AngII-infused males. Here we show that activation of MR is a primary factor driving excessive ENaC-dependent renal Na⁺ reabsorption in hypertensive females. Our findings provide the mechanistic rationale to optimize the use of MR antagonists in hypertensive women to improve BP control rates.

Perspectives.

Biological sex has become increasingly recognized as a pivotal factor affecting the development and pathophysiological outcomes of hypertension, however, limited understanding of the sex-specific mechanisms regulating BP poses a critical barrier to the optimization of antihypertensive therapies. Our study differentiates the pathophysiological mechanisms governing Na⁺ retention in hypertensive males and females and improves our understanding of BP control mechanisms in both sexes. Our findings provide the missing pre-clinical evidence to optimize the use of MR antagonists in hypertensive patients of both sexes.

Novelty and Relevance.

What Is New?

Female rats with AngII-dependent hypertension benefit from MR antagonism, exhibiting a stronger BP reduction, when compared to males. MR blockade is also much more potent at decreasing renal ENaC activity in AngII-infused female rats than in males. Higher sensitivity of renal ENaC activity and BP to MR antagonism in female rats with AngII-dependent hypertension is determined by the elevated expression of mRNA encoding MR and HSD11β2 in the kidney.

What Is Relevant?

Identification of the mechanisms responsible for elevation of BP in specific patient cohorts is critical to effectively combat hypertension and related diseases. Our study provides mechanistic evidence for a beneficial therapeutic effect of MR inhibitors in females with AngII-dependent hypertension.

Clinical/Pathophysiological Implications?

Our findings facilitate the development of more effective stratified treatment strategies based on biological sex as a critical covariate.

Abbreviations list:

ASDN

aldosterone-sensitive distal nephron

AngII

Angiotensin II

AngII-HTN

Angiotensin II dependent hypertension

BP

blood pressure

DAPI

4’,6-diamidino-2-phenylindole

ENaC

epithelial Na⁺ channel

HSD11β2

11β-hydroxysteroid dehydrogenase type 2

MR

mineralocorticoid receptor

NO

nitric oxide

SD

Sprague Dawley