Clinical studies indicate that salt-sensitive hypertension is more prevalent in women than men. However, animal models of salt-sensitivity have primarily focused on the mechanisms of salt-sensitivity in male animals, therefore, elucidation of these mechanisms in female animal models are needed. We have previously shown that female Balb/C mice have higher aldosterone synthase expression and aldosterone production than males. We hypothesized that female Balb/C mice develop salt-sensitive increases in blood pressure (BP). Seven day feeding of a 4% NaCl high-salt diet (HSD) increased BP in female mice without altering BP in males. Females on a HSD displayed no apparent increases in sodium retention as assessed by 24-hour urine collection, sodium balance measure and saline loading excretion analysis. Females on HSD exhibited lower renin-angiotensin system activity (plasma angiotensin II, plasma renin activity and angiotensin converting enzyme activity) compared to males but developed a salt-induced elevation in adrenal aldosterone synthase expression and retained higher aldosterone levels than males on high-salt. This resulted in a higher aldosterone/plasma renin activity ratio in females compared to males on high-salt feeding. Adrenal mRNA expression of angiotensinogen and leptin receptor were increased in female mice on HSD. HSD impaired endothelium-dependent relaxation in female mice only. Mineralocorticoid receptor inhibition (eplerenone) restored BP and endothelial function in HSD females. Collectively, these data indicate that Balb/C mice develop sex-discrepant salt-sensitive hypertension likely via aldosterone-mineralocorticoid receptor-mediated mechanisms involving impaired endothelium-dependent relaxation in females only. This study presents the first model of spontaneous sex-specific salt-sensitivity which mimics the human pathology.

Recent findings from our group demonstrated that females exhibit higher endothelial mineralocorticoid receptor (MR) expression than males which predisposes them to aldosterone-mediated endothelial dysfunction in the context of metabolic disorders. However, whether the endothelium of female mice presents a higher propensity to MR-mediated dysfunction than that of males in the absence of comorbidities remains unknown. We therefore sought to investigate whether increasing aldosterone production endogenously with sodium restriction impairs endothelial function in otherwise healthy female mice. We fed male and female Balb/C mice a normal (0.4% NaCl, NSD) or sodium restricted diet (0.05% NaCl, SRD) for 4 weeks. Females exhibited higher baseline endothelial function (relaxation to acetylcholine) and lower vascular contractility (constriction to phenylephrine, serotonin and KCl). However, SRD impaired endothelial-dependent relaxation and increased vascular contractility in female mice, effectively ablating the baseline sex-difference. Female sex also increased baseline adrenal CYP11B2 expression, however, SRD significantly enhanced CYP11B2 expression in male and female mice and ablated the sex difference. NOS inhibition with LNAME eliminated both sex as well as diet-induced differences in endothelial dysfunction. In accordance, females demonstrated higher vascular eNOS expression at baseline, which SRD significantly decreased. In addition, SRD diminished vascular NOX4 expression in female mice only. MR blockade with spironolactone protected female mice from decreases in endothelial-dependent relaxation, but not increases in vascular contractility. Utilizing sodium restriction as a method to increase plasma aldosterone levels in healthy female mice we demonstrated that female mice are more susceptible to vascular damage via MR-activation in the vascular endothelium only.

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Up to half of essential hypertension cases in women are associated with salt sensitive blood pressure (BP) increases, however, the sex-specific mechanisms of salt sensitivity in women are unknown. Our lab has shown that female mice are more sensitive to the hypertensive effects of aldosterone than male mice but it is unknown if aldosterone plays a role in salt sensitivity in female mice. We hypothesized that increasing dietary sodium via a high salt diet would increase blood pressure in female mice which would be abrogated by mineralocorticoid receptor (MR) antagonism. To determine salt sensitivity male and female Balb/C mice were implanted with radiotelemeters for continuous recording of BP. BP was recorded during baseline (7 days) and throughout the administration of a high salt (4% NaCl, HS) diet for 7 days with or without concurrent eplerenone supplementation (daily 200 mg/kg/day). Plasma and kidneys were then harvested. Systolic (SBP) and diastolic (DBP) BP were increased in female mice, but not in males, on HS (7.8±3.3 SBP and 7.8±4.0 DBP Δ change in mmHg in female (P<0.05) vs -3.7±3.1 SBP and 3.1±2.1 DBP in male, respectively, n=7-8). Plasma aldosterone levels were decreased in HS male mice compared to control (224±57 vs 151±19 pg/ml, n=3-5), however, increased modestly in HS females (254±56 vs 394±158 pg/ml, n=3-6). Preliminary data indicated that MR antagonism with eplerenone ablated increases in SBP and DBP in HS female mice, while having no effect on blood pressure in HS males (-22.8 SBP and -23.9 DBP Δ change in mmHg in female vs 1.0 SBP and -1.5 DBP in male, n=2). In addition, and in association with an absence of aldosterone suppression with HS, renal mRNA expression of renin (1.4±0.1-fold, P<0.05, n=5), angiotensinogen (4.4±0.2-fold, P<0.05, n=5), AT1 receptor (52.9±0.9-fold, P<0.05, n=5), MR (1.6±0.2-fold, P<0.05, n=5) and α-ENAC (1.3±0.0-fold, P<0.05, n=5) were increased in HS female mice compared to control females. These data indicate that BP increases in HS female mice are associated with unexpected increases in plasma aldosterone as well as mRNA expression of proteins associated with renin angiotensin aldosterone system activation, which may be novel mechanisms via which females have increased risk for salt sensitive hypertension.

Our previous work indicates that high salt diet fails to suppress plasma aldosterone in female Balb/C mice, in contrast to males, which reflects clinical data in women. Balb/C female mice also develop sex‐specific, high salt‐induced endothelial dysfunction, which is ablated by mineralocorticoid receptor (MR) antagonism. In contrast, other reports indicate C57Bl6 female mice suppress aldosterone production and do not develop sex‐specific endothelial impairment on high salt, indicating strain‐dependent effects of salt on aldosterone production and endothelial function in female mice. We hypothesized that low salt diet will more pronouncedly increase plasma aldosterone and induce endothelial impairment in female Balb/C mice compared to male Balb/C or C57BL6 female mice. Male and female Balb/C (n=5) and C57Bl6 mice (n=5–12) were fed a normal (NS, 0.4% NaCl) or low salt (LS, 0.05% NaCl) diet for 28 days. LS increased plasma aldosterone level in female Balb/C (1435±52 LSBalbC vs 375±104 NSBalbC pg/ml, 3‐way ANOVA, *P<0.05) and C57BL6 mice (677±124 LSC57 vs 204±43 NSC57, *P<0.05). LS increased aldosterone levels in Balb/C male mice (913±176 LSBalbC vs 265±96 NSBalbC, *P<0.05), however, not in C57BL6 mice (397±95 LSBalbC vs 220±55 NSBalbC). Aldosterone levels of both male and female Balb/C mice on LS were significantly higher than those of their same‐sex C57BL6 counterparts (*P<0.05). These data indicate an additive effect of both female sex and Balb/C strain to potentiate aldosterone production in response to LS stimulus. Endothelial‐dependent aorta relaxation responses to acetylcholine were impaired by LS in Balb/C female mice, but not in Balb/C male mice (2‐way ANOVA with repeated measures, *P<0.05). In contrast, neither male nor female C57BL6 mice developed endothelial impairment in response to LS. Relaxation responses to acetylcholine in the presence of LNAME indicated that reduced nitric oxide activity mediates endothelial impairment in Balb/C female mice. Neither constriction responses to phenylephrine nor endothelial‐independent relaxation to sodium nitroprusside were altered between any groups. Therefore, LS induces endothelial impairment only in female Balb/C mice. We and others have previously shown a crucial role for endothelial MR in endothelial dysfunction in females. Our qRT‐PCR analysis demonstrated a sex‐difference in endothelial MR mRNA expression favoring a higher level in females of both Balb/C and C57Bl6 mice. However, both male (5.2±1.3‐fold change from NSC57, 1‐way ANOVA, *P<0.05) and female (12.8±3.7, *P<0.05) Balb/C mice demonstrated a higher endothelial MR expression level than their same‐sex C57BL6 counterparts. Collectively, these data indicate that female Balb/C strain potentiates sex‐specific increases in aldosterone levels and endothelial impairment on LS, the latter of which may be mediated by increased endothelial MR expression. These data add evidence that the Balb/C mouse is a clinically relevent model of aldosterone‐induced endothelial impairment in females, which reflects the associat...