Sex differences in the progression of salt-sensitive hypertension in aged Dahl salt-sensitive rats

Abstract

Salt-sensitive hypertension is a major cardiovascular risk factor that is exacerbated by aging. Emerging evidence suggests complex mechanistic pathways involving renal sodium handling, vascular dysfunction, and hormonal alterations that vary by sex and age. The Dahl salt-sensitive (SS) rat model provides an excellent platform for investigating age-related changes in blood pressure regulation under high-salt conditions. This study examined the development of salt-sensitive hypertension in aged Dahl SS rats (>30 weeks old; both males and females), focusing on sex differences in survival and blood pressure. Female animals were further categorized by breeding status (breeder vs. non-breeder) to evaluate the effects of reproductive history on cardiovascular outcomes. Male and female rats demonstrated similar overall survival rates (60%) during the high-salt challenge, although mortality occurred earlier in females. Males exhibited lower baseline mean arterial pressure compared with females. No significant differences were observed between breeder and non-breeder females in survival, blood pressure, or heart rate. Circadian rhythm analysis revealed that females had a higher mesor prior to high-salt exposure and a shifted acrophase during the first four days of the diet. Urinary cystatin C increased significantly in females but not in males following high-salt exposure. Overall, aged female rats exhibited higher baseline cardiovascular parameters (including blood pressure and heart-to-body weight ratio) and greater renal stress responses (cystatin C and electrolyte excretion) than males, despite similar survival rates. These findings provide new insights into sex-specific cardiovascular and renal responses to salt loading in aging and may help explain sex-dependent differences in salt-sensitive hypertension.

New & noteworthy

Using aged Dahl salt-sensitive rats, this study examines how aging, sex, and reproductive history influence salt-sensitive hypertension. Reproductive history did not affect cardiovascular outcomes in aged females. Integrated survival analysis, circadian blood pressure monitoring, and renal biomarkers reveal sex-specific responses to high-salt intake, providing new insights into mechanisms contributing to salt-sensitive hypertension in aging.

Graphical Abstract

INTRODUCTION

Salt-sensitive hypertension affects approximately 25% of normotensive and 50% of hypertensive individuals, representing a significant public health concern (1–3). The condition is characterized by blood pressure elevation in response to increased dietary sodium intake and is associated with increased cardiovascular morbidity and mortality (4). Understanding the mechanisms underlying salt sensitivity becomes particularly important in the context of aging, as both hypertension prevalence and salt sensitivity increase with age (3, 5–7) . Despite this, the mechanistic interplay between aging, sex, and salt sensitivity remains incompletely understood, and most preclinical studies have been conducted in young animals that may not adequately represent the aged phenotype.

Recent evidence has identified several interconnected pathways through which aging impairs renal sodium handling and promotes salt sensitivity. Aging attenuates afferent renal nerve sympathoinhibitory reflexes, permitting increased sympathetic tone that drives sodium reabsorption in the distal nephron. This is compounded by age-associated upregulation of the thiazide-sensitive sodium-chloride cotransporter (NCC) and altered WNK-SPAK signaling, which collectively reduce the natriuretic response to salt loading (5, 6). Together, these renal adaptations establish the mechanistic foundation for the well-documented increase in salt sensitivity observed across aging populations.

Sex profoundly modifies both the onset and severity of hypertension (3, 8, 9). In young animals and premenopausal women, females are substantially protected relative to males: male rodents develop hypertension earlier and with greater severity across multiple experimental models, including the Dahl salt-sensitive rat and the spontaneously hypertensive rat (10). This female advantage is driven in large part by the vasodilatory, natriuretic, and anti-inflammatory actions of estrogen, and is correspondingly lost following ovariectomy or natural reproductive senescence (8, 9). With aging and the transition through menopause, the sex difference in blood pressure narrows and eventually reverses: postmenopausal women show higher rates of hypertension than age-matched men, and their blood pressure becomes markedly more salt-sensitive (3, 9, 11). This reversal has important clinical implications, as hypertension in older women is more strongly associated with cardiovascular events and target organ damage than equivalent blood pressure elevations in men.

The Dahl salt-sensitive (SS) rat is one of the most widely used and well-characterized models of salt-sensitive hypertension, recapitulating the genetic predisposition, renal sodium retention, and end-organ damage observed in human salt-sensitive disease (12). Despite the extensive literature in young Dahl SS rats, only a limited number of studies have examined this model in aged animals (13, 14), and the intersection of aging, sex, and salt loading in this strain remains poorly characterized. Specifically, it remains unclear whether the age-related reversal of female cardiovascular protection observed in humans and other rodents also occurs in aged female Dahl SS rats, and whether previous reproductive history influences these results.

The present study aimed to characterize the hemodynamic, cardiac, renal, and circadian responses to high-salt diet challenge in aged male and female Dahl SS rats, with the primary objective of defining sex differences in survival and blood pressure trajectories in this underexplored age group. As an exploratory secondary objective, we examined whether prior breeding history modifies cardiovascular outcomes in aged females, given evidence that reproductive experience may have lasting effects on hormonal status and cardiometabolic physiology.

METHODS

Animals

Animal experiments and procedures adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and the protocols were reviewed and approved by the University of South Florida (USF) Institutional Animal Care and Use Committee. Male and female aged Dahl salt-sensitive rats (SS; SS/JrHsdMcwi; RRID: RGD_61499) were maintained on a normal salt (NS) diet (0.4% NaCl; #113755; Dyets Inc., Bethlehem, PA) followed by a high-salt (HS) diet (4% NaCl diet, #113756) for 21 days. Breeder females were maintained under a standard institutional breeding protocol. Each female was paired with a single male and typically underwent 3–4 pregnancies. Re-pairing was performed only if the male partner died. Females were retired from the breeding colony at approximately 30–35 weeks of age. Aged rats were 30–40 weeks old at the start of the high-salt challenge. For the Cystatin C comparison shown in Figure 4, a separate cohort of young Dahl SS rats (8 weeks old, both sexes) was used under the same protocol. In aged rats, both retired breeders and non-breeders were present in both groups. Breeding status was used as a stratification variable only for the aged female survival, blood pressure, and heart rate analyses, for all other analyses data represent the pooled aged cohort of each sex.

Figure 4. Renal function in young and aged Dahl SS rats.

Urinary cystatin C levels in young and aged male and female rats were measured on Days 0 and 21 of HS exposure. Data are presented as mean ± SEM with individual data points shown. In aged rats, statistical analysis was performed by two-way repeated-measures ANOVA (time × sex) followed by Fisher's LSD post-hoc tests. Time effect: p = 0.007; sex effect: p = 0.217; interaction: p = 0.386. Within-sex comparisons (Day 0 vs. Day 21): aged males p = 0.155 (ns); aged females p = 0.009 (**). Between-sex comparisons (males vs. females): Day 0 p = 0.134 (mean difference 72.3 μg/24h); Day 21 p = 0.412 (mean difference 39.0 μg/24h). Data represent the pooled aged cohort of each sex, including both retired breeders and non-breeders. Young vs. aged comparisons at Day 0 were performed by unpaired two-tailed t-tests: males p = 0.014 (#); females p < 0.001 (###).

Surgical procedures

For blood pressure measurements, rats were anesthetized on a temperature-controlled platform via inhalation of 2.5% isoflurane in 0.5 L/min (O₂/N₂: 30%/70%), and a blood pressure transmitter (HD-S10; Data Sciences International (DSI)) was implanted subcutaneously. The catheter tip was secured in the abdominal aorta via the femoral artery as described (15, 16). Rats were allowed to recover for 4–5 days, and blood pressure and heart rate were recorded using DSI Ponemah software. At the end of the experimental timeline, rats were anesthetized with 5% isoflurane, and kidneys were flushed with phosphate-buffered saline via aortic catheterization (17, 18). The left kidney was snap-frozen, and the right kidney was placed in 10% formalin.

Electrolyte concentration measurements

Urine was collected for 24 hrs in metabolic cages (no. 37000M071, Tecniplast, West Chester, PA) at baseline and every 7 days of the HS protocol. Prior to euthanasia, blood samples were collected by aortic catheterization in anesthetized animals. Glucose, creatinine, and electrolytes (Na⁺, K⁺, Ca²⁺, Cl⁻) in plasma and urine were measured with a blood gas analyzer (ABL system 800 Flex, Radiometer, Copenhagen, Denmark).

Survival analysis

Animal survival was monitored daily, and survival curves were constructed using Kaplan-Meier analysis. Animals were monitored for up to 21 days on a high-salt diet, with humane endpoints defined in accordance with institutional guidelines.

Cystatine C measurements

Urinary cystatin C levels were measured using a commercially available Mouse/Rat Cystatin C Quantikine ELISA Kit (# MSCTC0, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Samples were diluted as needed to fall within the assay's linear range, and measurements were performed in duplicate. Results are expressed as μg/24 hrs.

Circadian analysis

Arterial blood pressure was continuously recorded and sampled at 2-hour intervals throughout the study period. Circadian rhythmicity of blood pressure was analyzed using a custom-developed software tool implementing standard and widely accepted circadian analysis formulas, consistent with classical cosinor methodology. For each animal, blood pressure data were analyzed on a day-by-day basis using a single-component cosinor model with a fixed period of 24 hours. From each fitted curve, the following circadian parameters were calculated: mesor, defined as the 24-hour rhythm-adjusted mean blood pressure value; amplitude, defined as half the peak-to-trough difference of the fitted cosine curve; acrophase, defined as the time (in hours) at which the maximum value of the fitted rhythm occurred. Circadian parameters were computed independently for each experimental day, allowing assessment of dynamic changes in circadian blood pressure regulation during the high-salt diet intervention. Group-level data are presented as mean ± SE for each circadian parameter across days on a high-salt diet.

Statistics

Data are presented as mean ± standard error of the mean (SE). Data were tested for normality (Shapiro-Wilk) and equal variance (Levene's homogeneity test). Statistical analysis consisted of one- or two-way ANOVA, Student's t-test, or Kolmogorov-Smirnov test (GraphPad Prism 10.0), with a p-value of <0.05 considered significant. In addition, when an ANOVA test was significant, post hoc Holm-Sidak's or Dunnett’s multiple-comparison test was performed.

RESULTS

Sex Differences in Survival, Blood Pressure, and Heart Rate

Overall survival rates were similar between male and female aged Dahl SS rats during the high-salt diet challenge (Fig. 1A). Both groups demonstrated 60% survival at day 21 (9 out of 15 rats survived in each group). The survival curves showed that mortality began earlier in females (around day 5) compared to males (around day 7–8). This earlier onset of mortality in females may be related to their higher baseline blood pressure.

Figure 1.

Sex differences in survival, arterial pressure, circadian blood pressure rhythm parameters, heart rate, and Aged male and female rats were maintained on a 4% high-salt (HS) diet for 3 weeks. (A) Kaplan-Meier survival curves showing percent survival over the course of HS exposure in male and female rats. (B) Mean arterial pressure (MAP) was measured longitudinally during HS exposure. (C) Heart rate (HR) measured longitudinally during HS exposure. (D) Mesor (rhythm-adjusted mean arterial pressure). (E) Amplitude of the circadian arterial pressure rhythm. (F) Acrophase (timing of peak arterial pressure) is expressed in hours. Data in panels B, C, D-F are presented as mean ± SEM. Statistical comparisons between groups at individual time points were performed using multiple unpaired t-tests with or without correction for multiple comparisons. Asterisks indicate significant differences between groups at the indicated time points (*p < 0.05, **p < 0.01).

Mean arterial pressure (MAP) was elevated compared to younger animals, as reported previously (19–21). Both male and female rats exhibited increases in MAP following initiation of a high-salt diet (Fig. 1B), consistent with the established salt-sensitive phenotype of Dahl SS rats (10). In males, baseline MAP was approximately 175 mmHg and increased progressively to over 200 mmHg during the high-salt diet. In females, baseline MAP was higher (~190 mmHg) than in males, with statistically significant sex differences observed on days 1 and 3 of the recording period. Both groups demonstrated progressive blood pressure elevation, with values converging at later time points as male MAP reached levels comparable to the female baseline. Heart rate (HR) analysis revealed that mean HR was consistently higher in females prior to and during the first 7 days following initiation of the high-salt diet (Fig. 1C). After day 7, HR increased in males, resulting in comparable HR between sexes, with no significant differences thereafter.

Circadian Rhythm Changes

Circadian blood pressure regulation plays a critical role in cardiovascular health, and disruption of normal circadian patterns has been associated with increased cardiovascular risk (22, 23). To assess whether high-salt diet exposure affects circadian blood pressure rhythms in aged Dahl SS rats, we analyzed three key circadian parameters – mesor, amplitude, and acrophase – derived from continuous arterial pressure recordings over the high-salt diet period (Figs. 1D–F). The mesor was significantly higher in females compared to males before switching to the high-salt diet, consistent with the elevated baseline MAP observed in females (Fig. 1D). During the high-salt diet period, mesor values were generally comparable between sexes, as expected given that both groups exhibited progressive blood pressure elevation that narrowed the initial sex difference.

The amplitude of the circadian blood pressure rhythm, which reflects the magnitude of oscillation between peak and trough blood pressure values, was generally similar between males and females throughout the observation period (Fig. 1E). However, on the day immediately prior to switching to the high-salt diet, amplitude was significantly lower in females compared to males. Notably, variability in amplitude values increased in both groups after approximately 7 days on the high-salt diet, suggesting that prolonged high-salt exposure may introduce greater day-to-day fluctuation in circadian blood pressure oscillation strength. Acrophase was significantly shifted in females compared to males during the first 4 days of high-salt diet exposure, indicating a transient sex-dependent alteration in the timing of peak blood pressure (Fig. 1F). After this initial period, acrophase values normalized and became comparable between groups.

Effect of Breeding Status on Survival, Blood Pressure, and Heart Rate in Females

To assess whether reproductive history influenced cardiovascular outcomes, breeder and non-breeder females were compared for survival, blood pressure, and heart rate (Fig. 2). Survival was similar between breeder and non-breeder females, though non-breeders showed a slightly earlier mortality onset (approximately day 5) compared to breeders (approximately day 7) (Fig. 2A). No significant differences in MAP were observed between breeder and non-breeder females throughout the high-salt diet period (Fig. 2B). Similarly, heart rate did not differ significantly between groups, although non-breeder females exhibited greater variability in heart rate after 7 days on the high-salt diet (Fig. 2C). Overall, no detectable effect of breeding status onsurvival, blood pressure, or heart rate responses to a high-salt diet was observed in aged female Dahl SS rats.

Figure 2. The effect of breeding status in aged rats exposed to a high-salt diet.

(A) Kaplan–Meier survival curves comparing breeder and non-breeder females. (B) Mean arterial pressure in breeder vs. non-breeder females. (C) Heart rate in breeder vs. non-breeder females. Data in panels B and C are presented as mean ± SEM.

Body and Organ Weights and Urinary Electrolyte Excretion

Body weight was assessed before and after 21 days of high-salt diet exposure (Fig. 3A). Males showed a trend toward body weight decline after 21 days on the high-salt diet. Kidney weight normalized to body weight (2KBW) was similar between male and female groups (Fig. 3B). In contrast, the heart-to-body weight ratio (HBW) was significantly higher in females than in males (Fig. 3C, p < 0.05), consistent with the elevated heart rate and higher baseline blood pressure observed in the female group. Diuresis was comparable between males and females at baseline and throughout the high-salt diet period (Fig. 3D).

Figure 3. Sex differences in body weight, organ indices, and electrolyte handling in rats exposed to a high-salt diet.

(A) Body weight (BW) was measured before HS exposure and at day 21. (B) Kidney weight normalized to body weight (2KBW; sum of both kidneys). (C) Heart weight normalized to body weight (HBW). (D) Diuresis was measured at baseline (Day 0) and during HS exposure (Days 7, 14, and 21). (E–H) Urinary electrolyte excretion normalized to creatinine at Day 0 and Day 21: (E) potassium (K⁺/Cre), (F) sodium (Na⁺/Cre), (G) calcium (Ca²⁺/Cre), (H) chloride (Cl⁻/Cre). Data are presented as mean ± SEM with individual data points shown. Statistical comparisons between males and females at individual time points were performed using unpaired t-tests without correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data represent the pooled aged cohort of each sex, including both retired breeders and non-breeders.

Dietary salt intake is a major determinant of renal electrolyte handling, particularly in salt-sensitive models such as the Dahl SS rats, which exhibit impaired sodium handling, enhanced pressure natriuresis, and progressive renal injury in response to high-salt intake. Both sexes exhibited comparable K⁺/Cre levels at baseline and following 3 weeks of high-salt diet (Fig. 3E), with values slightly elevated in females compared to males at both time points, indicating that potassium handling was not markedly affected by dietary salt intake or sex. In contrast, urinary sodium excretion (Na⁺/Cre) increased substantially following high-salt feeding in both sexes (Fig. 3F). Notably, the increase in sodium excretion was greater in females compared to males, indicating effective renal sodium excretion in response to salt loading despite known sex-specific differences in salt-sensitive hypertension. Urinary chloride excretion (Cl⁻/Cre) followed a pattern similar to sodium, increasing significantly at Day 21 in both sexes (Fig. 3H). Female rats exhibited a greater increase in Cl⁻/Cre compared to males, consistent with the enhanced natriuretic response observed in females and reflecting the coupled handling of sodium and chloride in renal tubular transport.

Overall, both male and female rats demonstrated robust natriuretic responses to a high-salt diet, with females exhibiting greater increases in urinary sodium, calcium, and chloride excretion compared to males. Potassium excretion remained unchanged across dietary conditions and sexes. Together, these data align with established characteristics of the Dahl SS rat model and support sex-specific differences in renal electrolyte handling during salt loading.

Renal Function

To assess the effect of aging on renal stress responses, urinary cystatin C levels were measured in both young and aged male and female rats before (Day 0) and after 3 weeks of a high-salt diet (Day 21) (Fig. 4). Cystatin C is a low-molecular-weight (13 kDa) cysteine protease inhibitor produced by all nucleated cells and freely filtered by the glomerulus. Unlike creatinine, its serum and urinary levels are less affected by muscle mass, making it a sensitive biomarker of glomerular filtration rate and early renal dysfunction (24, 25). Because of its stability and responsiveness to renal stress, it serves as a valuable marker for studying salt-sensitive nephropathy and sex-specific renal adaptations. In young rats, baseline cystatin C excretion was low in both sexes (approximately 5–10 μg/24h). Following 21 days of a high-salt diet, cystatin C increased significantly in both male and female young rats, indicating that salt-induced renal stress occurs regardless of sex in young animals. In aged rats, baseline cystatin C levels were significantlyhigher than in young rats, with values ranging from approximately 50–100 μg/24h – roughly 10-fold greater than in young animals (young vs. aged at Day 0, unpaired two-tailed t-test: males p = 0.014; females p < 0.001), indicating age-related increases in baseline renal stress or tubular handling of cystatin C. In aged males, cystatin C showed a modest increase from baseline following 3 weeks of high-salt intake, but this change did not reach statistical significance (p = 0.15). In contrast, aged female rats exhibited a significant elevation in urinary Cystatin C following high-salt feeding. Aged females showed a pronounced increase in cystatin C excretion from Day 0 to Day 21, with this difference reaching statistical significance (p < 0.01). Direct between-sex comparisons in aged rats showed no statistically significant differences at either time point (Day 0: p = 0.134; Day 21: p = 0.412; two-way RM ANOVA with Fisher's LSD post-hoc), although aged males demonstrated a trend toward higher baseline cystatin C (mean difference 72.3 μg/24h, 95% CI: −23.7 to 168.3) that narrowed by Day 21 (mean difference 39.0 μg/24h).

Overall, while aged male rats displayed a trend toward increased Cystatin C excretion after high-salt exposure, the change was not significant, whereas aged females demonstrated a pronounced and significant rise, indicating a potential sex-dependent response to high-salt diet in renal Cystatin C handling. The markedly higher cystatin C levels in aged compared to young rats (approximately 10-fold difference) further underscore the contribution of aging to baseline renal vulnerability, with a high-salt diet unmasking sex-specific differences in renal injury susceptibility that are amplified with age.

DISCUSSION

The present study confirms and extends previous observations of sex differences in salt-sensitive hypertension in the Dahl SS model (3, 26). Aged female Dahl SS rats demonstrated higher baseline blood pressure compared to males, with both groups showing progressive blood pressure elevation following high-salt diet challenge. Clinical data support this trajectory, demonstrating that women are more salt-sensitive than men at all ages, with a marked increase in salt sensitivity in the postmenopausal period (6, 8), suggesting that the aged female SS rat may more closely recapitulate the human female phenotype than younger counterparts. Despite higher baseline pressure and earlier mortality onset in females, overall survival rates were identical between sexes at 60%. This can be explained by the subsequent trajectory of male blood pressure. As males progressed through the high-salt challenge, their blood pressure rose to levels comparable to those of females.

The significantly higher heart-to-body weight ratio in aged females, together with their elevated baseline heart rate, indicates that substantial cardiac remodeling had already occurred prior to the high-salt challenge. This is consistent with the established concept that females develop proportionally greater myocardial hypertrophy relative to body size under sustained pressure overload. In the Dahl SS model, female animals on high-salt diets have been shown to exhibit greater relative wall thickening and diastolic dysfunction than age-matched males, with ovarian function modulating the magnitude of this hypertrophic response (27).

Dahl SS rats exhibit marked age-dependent differences in their blood pressure response to salt. Zicha et al. (13) summarized 50 years of Dahl SS rat studies and noted that interventions which prevent severe hypertension in young (~ 6–8 weeks) Dahl SS (e.g. high potassium or calcium intake) are much less effective in adult Dahl rats (~12 −16 weeks), and vice versa for other treatments, however, only a limited number of studies have examined the effect of high-salt challenge in aged (>30 weeks) Dahl SS rats (13, 14). Recent comparative studies have directly addressed this gap, demonstrating that aging reduces hemodynamic responsiveness to salt but paradoxically increases susceptibility to salt-induced renal injury in Dahl SS rats, with aged animals showing greater oxidative stress and renal fibrosis despite blunted blood pressure rises (14). But authors showed that in the aged group, MAP was similar to that of young rats at the beginning of the experiment, around 120 mmHg. In our study, aged animals of both sexes had already elevated MAP even before HS challenge. This discrepancy may reflect differences in animal age, housing conditions, or the duration of normal-salt feeding prior to the experiment, and highlights the importance of characterizing baseline hemodynamics in aged cohorts before salt challenge. In our study, the elevated baseline MAP in rats >30 weeks suggests that age-related vascular and renal changes had advanced to the point that resting blood pressure was significantly elevated, potentially limiting the dynamic range of further salt-induced pressure responses.

Consistent with this interpretation, urinary cystatin C levels were approximately 10-fold higher at baseline in aged males and females compared to young rats, a striking age-dependent elevation that has not been extensively characterized in the Dahl SS model. This likely reflects cumulative renal tubular damage and declining glomerular filtration over the extended period of aging on a normal salt diet, before any experimental intervention. Following the high-salt challenge, cystatin C increased significantly in both young males and young females, and in aged females, but not in aged males. This sex-specific pattern in the aged cohort may reflect two non-exclusive possibilities: aged males may have reached a functional ceiling of measurable tubular injury before the challenge began, with limited capacity for further detectable increases. The trend toward higher baseline urinary cystatin C in aged males relative to females, although not reaching statistical significance in the present cohort, is directionally consistent with reported sex-dependent differences in kidney biomarkers (28). Also, Tsuji et al. showed that male rats consistently exhibit higher urinary cystatin C excretion than females in young rats (29), and a follow-up study identified testosterone as a key driver, with subcutaneous testosterone administration to female rats elevating urinary cystatin C levels (30).

The greater increases in urinary sodium, calcium, and chloride excretion observed in females compared to males following high-salt diet are consistent with known sex-dependent differences in renal tubular transport mechanisms in the Dahl SS model (11). High-salt diets promote natriuresis as an adaptive mechanism to maintain sodium balance and extracellular fluid volume, while simultaneously increasing urinary calcium excretion through coupled transport processes in the proximal tubule and loop of Henle (1, 4). The enhanced electrolyte excretion in females, taken together with the sex-specific cystatin C responses described above, suggests that aged females mount a more pronounced overall renal stress response to salt loading than aged males, one that extends beyond simple pressure-mediated glomerular injury to encompass differential tubular dysfunction.

Circadian analysis of blood pressure revealed that the fundamental architecture of cardiovascular rhythmicity was largely preserved across both sexes and both diet conditions, but with informative sex-dependent perturbations. The elevated mesor in females before high-salt exposure directly reflects their higher baseline blood pressure and is therefore consistent with the hemodynamic findings described above. A transient but significant shift in acrophase was observed exclusively in females during the first four days of high-salt exposure, which represents a novel finding in this model. This early phase shift may indicate sex-dependent differences in acute autonomic or hormonal responses to sodium loading.

Previous reproductive activity may have lasting effects on the aldosterone-mineralocorticoid receptor axis, particularly in females (31, 32). Breeding females may have an altered baseline hormonal status that influences their response to salt loading. However, no detectable effect of breeding status on survival, blood pressure, or heart rate was observed in aged female Dahl SS rats, suggesting that any hormonal legacy effects of reproduction may be insufficient to modify cardiovascular outcomes in the context of advanced age and severe salt-sensitive hypertension, but adequately powered studies incorporating longitudinal hormonal profiling will be required to definitively address this question.

Several limitations should be considered when interpreting these findings. The relatively small sample sizes may limit statistical power, particularly for comparisons within the breeding status subgroup, and replication in larger cohorts is warranted. Also, daily food intake was not weighed individually in this study. While our findings align with mechanistic predictions from the literature, direct measurement of the proposed pathways, including NCC expression, aldosterone, and mineralocorticoid receptor levels, as well as molecular markers of cellular senescence (e.g., p16, p21), would substantially strengthen mechanistic conclusions and is a priority for follow-up studies. Additionally, the absence of longitudinal hormonal profiling (estrogen, aldosterone) across the aging period limits our ability to attribute the sex-dependent renal and hemodynamic differences to specific endocrine mechanisms.

Taken together, these findings demonstrate that aged female SS rats show higher baseline blood pressure, increased cardiac hypertrophy relative to body size, and greater renal susceptibility to salt-induced injury, a combination that more closely mirrors postmenopausal women. These findings establish a strong foundation for future mechanistic studies focusing on sex-specific renal transport and circadian regulation of cardiovascular function in aged salt-sensitive hypertension. They also support including sex and reproductive stage as explicit variables in both preclinical research design and cardiovascular risk assessment.

DATA AVAILABILITY

All data generated or analyzed during this study are included in this published article.