Aldosterone Antagonism Is More Effective at Reducing Blood Pressure and Excessive Renal ENaC Activity in AngII-Infused Female Rats Than in Males

Abstract

Background: AngII (angiotensin II)-dependent hypertension causes comparable elevations of blood pressure (BP), aldosterone levels, and renal ENaC (epithelial Na⁺ channel) 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-hypertension. 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-hypertension. We hypothesize that overstimulation of renal MR signaling drives redundant ENaC-mediated Na⁺ reabsorption and BP increase in female rats with AngII-hypertension.

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-hypertension 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 HSD11β2 (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 ≈2× 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 (neural precursor cell expressed developmentally down-regulated protein 4-2), 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-hypertension, but not in males.

Keywords: aldosterone; angiotensin II; hypertension; mineralocorticoid receptor antagonists; sex differences.

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.

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.

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.

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.

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.