Female Dahl Salt-sensitive Rats Research Articles

Establishment of a HFpEF model using female Dahl salt-sensitive rats: a valuable tool for elucidating the pathophysiology of HFpEF in women.

The pathogenesis of heart failure with preserved ejection fraction (HFpEF) remains unclear, and effective treatments are limited. HFpEF is more prevalent in females, indicating potential gender differences in its pathogenesis. However, no female HFpEF model animals have been established. Hypertension is a major contributor to HFpEF, and sympathetic activation is thought to play a role in both conditions. This study aimed to establish a female HFpEF model using hypertensive Dahl salt-sensitive rats and to assess the presence of sympathetic activation. Seven-week-old female Dahl salt-sensitive rats were fed an 8% high-salt diet (HS group, n = 6), while a low-salt diet group (LS group, n = 9) served as controls. The HS group exhibited increased systolic blood pressure and heart rate. Echocardiography revealed an increased left ventricular (LV) wall thickness, a decreased E/A ratio, and an increased E/e' ratio, all indicative of diastolic dysfunction without reduced LV ejection fraction. Additionally, the HS group showed elevated LV end-diastolic pressure, LV weight, and lung weight, along with histological cardiomyocyte hypertrophy and interstitial fibrosis. Gene expression markers for cardiac hypertrophy and fibrosis were also increased. Renal function was significantly impaired, and plasma norepinephrine levels were elevated, consistent with heightened pre-sympathetic neuronal activity in the brain. In conclusion, high salt loading from 7 weeks of age in female Dahl salt-sensitive rats induced hypertensive HFpEF phenotypes with LV hypertrophy and fibrosis, and sympathetic activation by 16 to 19 weeks of age. This model provides a valuable tool for studying HFpEF pathophysiology in women.

Abstract P226: Sex-Dependent Effects of Perivascular Adipose Tissue on Arterial Stiffness in Hypertensive

Dillon McClintock, Department of Mechanical Engineering, Michigan State University, East Lansing, MI Sydney Bush, Department of Mechanical Engineering, Michigan State University, East Lansing, MI Lisa Sather, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI Nathan Tykocki, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI Adam Lauver, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI Stephanie W Watts, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI Sara Roccabianca, Department of Mechanical Engineering, Michigan State University, East Lansing, MI Perivascular adipose tissue (PVAT) plays a crucial role in arterial mechanics by significantly reducing arterial stiffness in the thoracic aorta in normotensive rats. This study aims to test the hypothesis that impact of PVAT on arterial stiffness is sex-dependent in an adiposity-driven hypertension rat model. Thoracic aorta from male and female Dahl Salt-Sensitive rats were fed control (10% fat-CD) and high-fat (60% fat-HFD) diets over 16 weeks. Uniaxial mechanical tests were then conducted on aorta samples with PVAT (+PVAT) and without PVAT (-PVAT). In -PVAT samples, arterial wall stiffness at 20% stretch (mimicking in vivo conditions), was higher in males compared to females on a HFD but not on a CD. HFD rats also had higher systolic blood pressures, but the increase was significant only in females. +PVAT samples’ stiffness was reduced by a 95% factor in all groups as compared to -PVAT samples. Furthermore, when including PVAT, the sex difference in the HF diet group was absent. These results suggest that PVAT has a beneficial effect that is preserved in this adiposity-driven hypertension animal model after 16 weeks on diet. Furthermore, remodeling of the arterial wall is more pronounced in male versus female rats and the inclusion of PVAT dampens this sex difference.

Beneficial Effects of Mild Hyperuricemia in Salt-Sensitive Hypertension

Hyperuricemia (HrU) or increased plasma uric acid (UA) is associated with worsening hypertension (HTN) and kidney damage. Paradoxically, UA is also known to be an endogenous antioxidant. Some studies suggest that only HrU with crystals, but not asymptomatic HrU promotes the progression of these diseases. We hypothesized that mild asymptomatic HrU is beneficial in controlling the progression of salt-sensitive (SS) HTN. Due to sex differences in urate handling and SS HTN, we used both male and female Dahl SS rats in this study. Radiotelemetry was used to measure blood pressure and various approaches, including RNA-Seq, were employed to assess kidney injury and identify potential molecular mechanisms. Mild HrU was induced using 2% oxonic acid in the diet, a uricase inhibitor that prevents the breakdown of UA further into more soluble allantoin. Groups: 1-2) male and female 4% NaCl high salt (HS) diet; 3-4) male and female HS + 2% oxonic acid (HS/oxo) diet. After 3 weeks, in response to oxonic acid supplementation, both sexes showed a significant increase of UA in plasma compared to their respective HS-only controls (Males: 0.63 ±0.07 vs. 2.17 ±0.34; Females 0.78 ±0.15 vs. 2.04 ±0.35 mg/dl, HS vs. HS/oxo). Interestingly, only male HS/oxo rats showed a significant increase in uricosuria (Males: 0.23 ±0.03 vs. 0.45 ±0.06; Femaels: 0.26 ±0.06 vs. 0.26 ±0.001 UA/Cre, HS vs. HS/oxo). Moreover, the mild HrU was associated with a significant attenuation of the progression and magnitude of the mean arterial pressure in male but not female rats (Males: 157 ±3 vs. 136 ±3; Females: 155 ±6 vs. 154 ±5 mmHg, HS vs. HS/oxo). Xanthine oxidase (XO) is one of the enzymes that produce UA, and its activity has been shown to affect HTN as well. Therefore, we examined the level of XO activity in the plasma after the treatment. While there was no difference in the activity based on the diet within each sex, females had significantly lower levels of XO activity compared to males in each of the diets. To further investigate the beneficial phenotype seen in male rats, we evaluate changes in the progression of renal pathology. The HS/oxo group compared to the HS group had a lower kidney weight/body weight ratio and lower protein cast accumulation, indicating lower kidney damage. RNA-Seq analysis showed that the attenuated HTN phenotype was associated with an increased expression in Mas1 (MAS receptor), Klk-1 (Kallikrein-1), and Pcsk6 (PCSK6 enzyme) which can all lead to the activation of different vasodilatory pathways. Our study showed that in male but not female Dahl SS rats, asymptomatic mild HrU accompanied by hyperuricosuria ameliorates the progression of SS HTN and protects kidneys from further damage. This work is supported by the American Society of Nephrology Ben J. Lipps Research Fellowship Program (Dimitrios G. Oreopoulos Research Fellowship Award to L.V.D.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of vasoactive substances on biomechanics of small resistance arteries of male and female Dahl salt-sensitive rats.

Changes in vascular biomechanics leading to increase in arterial stiffness play a pivotal role in circulatory dysfunction. Our objectives were to examine sex-specific pharmacological changes related to the biomechanics and any structural modifications in small resistance arteries of Dahl salt-sensitive male and female rats. The composite Young modulus (CYM) was determined using pressure myograph recordings, and immunohistochemistry was used for the evaluation of any structural changes in the third-order mesenteric arteries (n = 6). Animals on high-salt diet developed hypertension with significant elevation in central and peripheral blood pressures and pulse wave velocity compared to those on regular diet. There were no significant differences observed in the CYM between any of the groups (i.e., males and females) in vehicle-treated time-control studies. The presence of verapamil (0.3 μM) significantly reduced CYM in hypertensive males without changes within females compared to vehicle. This effect was abolished by phenylephrine (0.3 μM). BaCl2 (100 μM), ouabain (100 μM), and L-NAME (0.3 μM) combined significantly increased CYM in vessels from in normotensive males and females but not in hypertensive males compared to vehicle. The increase in CYM was abolished in the presence of phenylephrine. Sodium nitroprusside (0.3 μM), in the presence of phenylephrine, significantly reduced CYM in male normotensive versus hypertensive, with no differences within females. Significant differences were observed in immunohistochemical assessment of biomechanical markers of arterial stiffness between males and females. Our findings suggest sex possibly due to pressure differences to be responsible for adaptive changes in biomechanics, and varied pharmacological responses in hypertensive state.

Effect of SGLT2 inhibition on salt-induced hypertension in female Dahl SS rats

Sodium-glucose co-transporters (SGLTs) in the kidneys play a pivotal role in glucose reabsorption. Several clinical and population-based studies revealed the beneficial effects of SGLT2 inhibition on hypertension. Recent work from our lab provided significant new insight into the role of SGLT2 inhibition in a non-diabetic model of salt-sensitive hypertension, Dahl salt-sensitive (SS) rats. Dapagliflozin (Dapa) blunted the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in the Renin–Angiotensin–Aldosterone System. However, our initial study used male SS rats only, and the effect of SGLT2 inhibitors on hypertension in females has not been studied. Therefore, the goal of this study was to determine whether SGLT2 inhibition alters blood pressure and kidney function in female Dahl SS rats. The result showed that administration of Dapa for 3 weeks prevented the progression of salt-induced hypertension in female rats, similar to its effects in male SS rats. Diuresis and glucose excretion were significantly increased in Dapa-treated rats. SGLT2 inhibition also significantly attenuated kidney but not heart fibrosis. Despite significant effects on blood pressure, Dapa treatment caused minor changes to electrolyte balance and no effects on kidney and heart weights were observed. Our data suggest that SGLT2 inhibition in a non-diabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension independent of sex.

Abstract P385: Sex-specific Long Non-coding Gene Expression In Rat Nephron Segments

Transcriptomic and proteomic approaches have identified electrolyte transporters, channels and claudins with sex-specific expression along the nephron which adapt to physiological stressors in a sex-specific manner to maintain proper fluid and electrolyte homeostasis. Yet, less is known how long non-coding gene expression in specific nephron segments differs between sexes. We performed polyA-independent RNA-seq on manually isolated proximal tubule (PT) and medullary thick ascending limb (mTAL) from 6-7-week-old male and female Dahl salt sensitive rats. Sex specific gene expression differences [log2(fold change) ≥ 0.5 or ≤ -0.5; p adj ≤ 0.05] are summarized in the table. More sex-specific differences of both protein coding and lncRNA genes were detected in PT compared to mTAL. lncRNAs were less likely to demonstrate sex-specific differences than protein coding genes (p=2.59e-12 for PT and p=5.68e-3 for mTAL). Rare lncRNAs were expressed in one sex or the other (sex-exclusive). An interesting example is ENSRNOG00000069167 and ENSRNOG00000063718 which are transcribed on opposite strands from an overlapping sequence in male or female mTAL, respectively. Multiple sex-exclusive lncRNAs were colocalized with protein coding genes significantly enriched in the same sex, suggesting they may be co-regulated within a specific chromatin domain. For example, ENSRNOG00000068738 was exclusively expressed in male PT and is colocalized in a ~560-kbp region with Pcnx1 and Sipa1l1, both of which were also enriched in male PT. Collectively, these results extend our understanding of sex-specific gene expression differences in these nephron segments to include long non-coding genes.

Corn Bedding Mediates Protection from the Dahl Salt‐Sensitive Hypertensive and Renal Damage Phenotype

The Dahl Salt‐Sensitive (SS) rat is a well‐established translational model of human salt sensitive hypertension. When challenged with a high salt (4.0% NaCl) diet, Dahl SS develop an increase in mean arterial pressure (MAP) and renal damage, as assessed by an increase in albumin excretion and infiltration of immune cells in the kidneys. Our colony of Dahl SS recently moved to a new animal facility, and we observed that the SS rats challenged on a high salt diet showed an attenuation of the high salt‐induced increase in MAP and albumin excretion. Upon further investigation, differences in the animal bedding were noted between the locations. In the present study, the salt‐sensitive disease phenotype was examined in age‐matched, genetically‐identical male Dahl SS rats fed the same diets (AIN‐76A with 0.4% or 4.0% NaCl) maintained on either an aspen‐based wood bedding or a corn‐cob based bedding. Based upon the known protective effects of a plant‐based diet and observations that the rats were eating the corn‐cob bedding, we hypothesized that the change in bedding from wood to corn was providing a protective phenotype to the SS rats. We tested this hypothesis by comparing the disease phenotypes in Dahl SS maintained on the corn‐cob or wood bedding from birth until the end of the study. SS rats were instrumented at 7 weeks of age with radiotelemeters for continuous blood pressure monitoring and were allowed about a week to recover. While the rats were maintained on a low salt (0.4% NaCl) diet, there were no significant differences in MAP (123.2±1.1 vs 126.8±0.8 mmHg, n=13, 16) or albumin excretion rate (7.5±1.8 vs 18.8±3.5 mg/day, n=10, 17) in Dahl SS maintained on the corn‐cob vs the wood bedding, respectively. While MAP and albumin excretion significantly increased in all Dahl SS following a 21‐day high salt challenge, there was significantly reduced albumin excretion in the corn‐cob bedding rats (128±27 mg/day, n=10) when compared to the wood bedding rats (332±50 mg/day, n=17, p˂0.005). The SS rats maintained on the corn‐cob bedding also had significantly attenuated MAP at the end of the 21 day challenge when compared to the rats on the wood bedding (corn‐cob bedding: 141.1±4.2 mmHg vs wood bedding: 159.5±3.6 mmHg, p˂0.0001, n=12, 16 respectively). This was further characterized with a significant reduction in CD45+ leukocytes infiltrating in the SS rat kidneys on corn‐cob bedding by ~48% (p<0.003). That occurred by a significant reduction observed in the CD11b/c+ macrophages/monocytes by ~49% (p<0.003) and in CD45R+ B‐cells by ~64% (p<0.004). There was no difference between the body weights of the rats between the different beddings (corn‐cob bedding: 336±6 g vs wood bedding: 339±5 g, n=13, 17) respectively but kidney weight was a significantly reduced by ~17% (p<0.0001) in corn‐cob bedding compared to the SS rats on the wood bedding. This attenuated phenotype was also mirrored in female Dahl SS rats when maintained on the corn‐cob bedding compared to age‐matched females on the wood bedding. These data suggest that the change in bedding from wood to corn had a significant protective effect in the Dahl SS rat. Further work will need to be performed to determine the exact cause in the reduction of the salt‐sensitive phenotype when on a corn‐cob bedding. These data highlight the importance of controlling all environmental parameters in experimental work, and to consider environmental effects that may influence other disease phenotypes.

Cardiovascular, Cerebral and Behavioral Response to Anti‐Hypertensive Treatment in Female Dahl Salt Sensitive Rats

Background Cerebrovascular disease is associated with central arterial stiffness (CAS), which can lead to reduced brain blood supply, neuronal density loss and cognitive impairment. Dahl salt sensitive (DSS) rats represent a model of vascular dementia and have a compromised renin-angiotensin system, develop CAS, hypertension, and hippocampal memory decline with age on a normal salt (NS) diet. Lisinopril (LIS), an angiotensin converting enzyme inhibitor, effectively treated this pathology in aged male DSS rats. We investigated the relationship between systolic blood pressure (SBP), pulse wave velocity (PWV), a marker of CAS, hippocampal blood flow (CBF), neuronal density and anxiety in female DSS rats treated with LIS. Methods Female DSS rats (n=23) were fed a NS diet (0.5% NaCl) for the duration of the study. Rats were administered LIS (15mg/kg BW/day in drinking water, n=12) or control treatment (CTRL; n=11) for 7-mo beginning at 6-mo of age. The following measurements were assessed at 6-mo (baseline; BL) and at 13-mo of age: SBP (by plethysmography), PWV (by echocardiography), CBF (by continuous arterial spin labeling), and hippocampal N-acetyl aspartate concentration (NAA; by proton magnetic resonance spectroscopy). In open field test (OFT; a metric of anxiety, a precursor to symptoms of dementia) travel activity (distance) and rearing durations (vertical activity; VA) in the center of the field were collected as measures of exploratory behavior. Pearson's correlation matrix and 2-way ANOVA mixed effects model analyses were performed. Results At BL, the treatment groups were not different in any parameters except heart rate (HR). At 13-mo CTRL, SBP and PWV increased, CBF, total distance, total VA and distance in center of the OFT decreased compared to BL. In 13-mo LIS group, SBP and PWV were lower compared to 13-mo CTRL; 13-mo LIS group had higher HR and lower SBP, CBF and total VA in OFT compared to BL (Table 1). At 6-mo, NAA and VA were marginally correlated. HR and central distance were significantly correlated (Table 2A). For 13-mo CTRL, SBP positively correlated with PWV and NAA. NAA negatively correlated with distance in center. HR negatively correlated with distance in center and VA in center (Table 2B). This relationship with HR was reversed for 13-mo LIS group, for which PWV and CBF were negatively associated with central distance and central VA, while NAA was negatively correlated with central VA only (Table 2C). Conclusion In both CTRL and LIS groups, advancing age adversely affected hippocampal perfusion, but not neuronal density. ChronicLIS treatment reduced hypertension and PWV but did not affect cerebral parameters in aged female DSS rats. Increased level of anxiety, known to predate cognitive decline, was associated with PWV for LIS group, suggesting that CAS can adversely affect brain structures and function. CTRL and LIS groups showed differences in associations between cerebral, behavioral, and cardiovascular parameters. Further analyses will be conducted to investigate the role of sex hormones and their associations with age in the patterns observed.

Activation of G protein-coupled estrogen receptor 1 ameliorates proximal tubular injury and proteinuria in Dahl salt-sensitive female rats.

Recent evidence indicates a crucial role for G protein-coupled estrogen receptor 1 (GPER1) in the maintenance of cardiovascular and kidney health in females. The current study tested whether GPER1 activation ameliorates hypertension and kidney damage in female Dahl salt-sensitive (SS) rats fed a high-salt (HS) diet. Adult female rats were implanted with telemetry transmitters for monitoring blood pressure and osmotic minipumps releasing G1 (selective GPER1 agonist, 400 μg/kg/day ip) or vehicle. Two weeks after pump implantation, rats were shifted from a normal-salt (NS) diet (0.4% NaCl) to a matched HS diet (4.0% NaCl) for 2 wk. Twenty-four hour urine samples were collected during both diet periods and urinary markers of kidney injury were assessed. Histological assessment of kidney injury was conducted after the 2-wk HS diet period. Compared with values during the NS diet, 24-h mean arterial pressure markedly increased in response to HS, reaching similar values in vehicle-treated and G1-treated rats. HS also significantly increased urinary excretion of protein, albumin, nephrin (podocyte damage marker), and KIM-1 (proximal tubule injury marker) in vehicle-treated rats. Importantly, G1 treatment prevented the HS-induced proteinuria, albuminuria, and increase in KIM-1 excretion but not nephrinuria. Histological analysis revealed that HS-induced glomerular damage did not differ between groups. However, G1 treatment preserved proximal tubule brush-border integrity in HS-fed rats. Collectively, our data suggest that GPER1 activation protects against HS-induced proteinuria and albuminuria in female Dahl SS rats by preserving proximal tubule brush-border integrity in a blood pressure-independent manner.

Non-invasive vagus nerve stimulation attenuates proinflammatory cytokines and augments antioxidant levels in the brainstem and forebrain regions\xa0of Dahl salt sensitive rats

The anti-inflammatory effects of vagus nerve stimulation are well known. It has recently been shown that low-level, transcutaneous stimulation of vagus nerve at the tragus (LLTS) reduces cardiac inflammation in a rat model of heart failure with preserved ejection fraction (HFpEF). The mechanisms by which LLTS affect the central neural circuits within the brain regions that are important for the regulation of cardiac vagal tone are not clear. Female Dahl salt-sensitive rats were initially fed with either low salt (LS) or high salt (HS) diet for a period of 6 weeks, followed by sham or active stimulation (LLTS) for 30 min daily for 4 weeks. To study the central effects of LLTS, four brainstem (SP5, NAb, NTS, and RVLM) and two forebrain sites (PVN and SFO) were examined. HS diet significantly increased the gene expression of proinflammatory cytokines in the SP5 and SFO. LLTS reversed HS diet-induced changes at both these sites. Furthermore, LLTS augmented the levels of antioxidant Nrf2 in the SP5 and SFO. Taken together, these findings suggest that LLTS has central anti-inflammatory and antioxidant properties that could mediate the neuromodulation of cardiac vagal tone in the rat model of HFpEF.

High fat diet in female Dahl salt sensitive rats causes non‐neurogenic hypertension and minimal renal injury

There is evidence in human subjects and animal models that obesity‐associated hypertension has a smaller neurogenic component in females than males. In addition, females typically show less severe hypertension, immune cell infiltration in the kidney and renal injury than males. The mechanistic links between these phenomena, however, are not clear. We previously found that feeding Dahl salt‐sensitive (SS) rats a high fat diet (HFD) from weaning through 6 months caused equivalent hypertension in male and female animals when compared to SS rats fed control diet (CD). Yet female rats exhibited significantly less renal injury than males. Therefore, in the current study, we quantified the contribution of neurogenic and other blood pressure control mechanisms to HFD associated hypertension in female Dahl SS rats. We also reexamined the impact of long‐term hypertension on histological indices of renal injury in these rats. After 24 weeks on diet feeding starting at weaning, all rats were hypertensive (via telemetric measurements), but HFD females (60% kcal from fat, 0.3% NaCl n=10) had significantly higher mean arterial pressure (MAP) than females fed the CD (10% kcal from fat, 0.3% NaCl, n=7) (MAP 165±4mmHg vs 141±3mmHg, p<0.05). Thereafter, all rats were treated sequentially with prazosin (150mg/L, 5days), atenolol (500mg/L, 7days), prazosin+atenolol (7days), trichlormethiazide (250mg/L, 5 days), and enalapril (250mg/L, 7 days) via the drinking water. Each treatment was followed by washout for 7 days. We found that MAP was moderately and similarly reduced by treatment with prazosin (−14±1mmHg vs −17±1mmHg), atenolol (−31±2mmHg vs −27±2mmHg), prazosin+atenolol (−26±1mmHg vs −29±2mmHg) and trichlormethiazide (−16±2mmHg vs −17±2mmHg) in CD versus HFD fed rats. Enalapril treatment caused more reduction of blood pressure than any other treatment, but the decreases of MAP were similar in CD and HFD fed rats (−36±5 mmHg vs −49±9 mmHg) (P>0.05). Renal histological alterations and immune cell infiltration were determined at 34–36 weeks on diet feeding. Overall CD and HFD fed rats showed similar, and low grade, renal injury scores and renal T‐cell (CD3+) and macrophage (CD68+) infiltration. However, a few HFD rats did show severe renal injury and inflammation, and we noted that in each of these animals MAP levels were consistently > 170 mmHg. Splenic immune cell analysis by flow‐cytometry did not support HFD associated alternations in immune cell population/distributions (CD4+, CD8+, NK cells, B cells et al). Taken together the data indicate that in the Dahl SS rats, HFD causes hypertension via mechanisms that do not include increased neurogenic support of blood pressure or volume expansion. Increased activity of the renin‐angiotensin system may play a part but is not wholly responsible. Female Dahl SS rats on HFD are strongly protected against hypertension‐associated renal inflammation and injury, but the mechanisms responsible remain unknown.Support or Funding InformationNHLBI 2P01HL070687 for G.D.F, J.J.G. and H.X

Contribution of T‐lymphocytes to the Sex Differences Observed in Dahl Salt‐sensitive Hypertension and Renal Damage

Previous studies in our laboratory have established a clear role for the immune system in the development of hypertension and renal damage in male Dahl salt‐sensitive (SS) rats. Far less is known about the salt‐sensitive phenotype in female Dahl SS rats and how it compares to males, especially in regard to the contribution of immune‐related mechanisms. Using radiotelemetry to measure blood pressure, urine and renal histological analysis to assess kidney damage, and flow cytometry to assess renal immune cell infiltration, the current study examined the hypertensive and kidney injury phenotype in response to a 3 week high salt challenge (HS, 4.0% NaCl, AIN‐76A) in female versus male Dahl SS rats (n>10/group). During the low‐salt (LS, 0.4% NaCl) period, there was no difference in baseline mean arterial pressure (MAP) between females and males (129.4±2.0 vs 123.7±1.9 mmHg, respectively). However, after 3 weeks of HS, females were significantly protected from developing the same extent of hypertension observed in the males (161.5±3.1 vs 176.7±6.7 mmHg). This protection in the females coincided with significantly less renal damage, evident by markedly reduced albuminuria (105.0±15.7 vs 183.2±15.6 mg/day) and medullary protein cast formation (6.96±0.72 vs 14.62±1.14%) compared to the males. There was also a significant 48.5% reduction in the number of CD45+ total leukocytes in the female versus male kidneys (3.3±.3 vs 6.3±0.5 ×106 cells/kidney), which was true across all immune subsets – CD3+ T‐lymphocytes (50.8% reduction), CD45R+ B‐lymphocytes (73.5% reduction), and CD11b/c+ monocytes/macrophages (46.7% reduction). To interrogate the potential contribution of adaptive immunity, specifically T‐lymphocytes, to the development of this sex difference, the same parameters were investigated in both female and male SSCD247−/− rats. The lack of functional CD3+ T‐lymphocytes in SSCD247−/− rats significantly reduced blood pressure in both females (147.1±5.9 vs 161.5±3.1 mmHg; SSCD247−/− vs SS) and males (157.2±8.1 vs 176.7±6.7 mmHg) after a 3 week salt challenge, where there was no statistical difference between female and male SSCD247−/− rats. There was a corresponding decrease in kidney damage, with reduced albuminuria in both female (37.2±12.1 vs 105.0±15.7 mg/day) and male (88.4±12.7 vs 183.2±15.6 mg/day) SSCD247−/− rats when compared to SS rats with the full complement of immune cells. Flow cytometric analysis confirmed the lack of T‐lymphocytes in all SSCD247−/− rats, and additionally, there was significantly less medullary protein cast formation in the SSCD247−/− rats compared to the SS, regardless of sex (females: 1.82±0.37 vs 6.96±0.72%; males: 9.10±0.75 vs 14.62±1.14%; SSCD247−/− vs SS). Together, our data indicate that although female SS rats are significantly protected from progressing to the same severity of disease compared to male SS rats, female SS rats do still develop salt‐induced hypertension and renal damage that is amplified by T‐lymphocytes.Support or Funding Information19CDA34660184, HL116264, HL137748

Sex Differences in Renal Inflammation and Injury in High-Fat Diet-Fed Dahl Salt-Sensitive Rats.

We examined the impact of sex on high-fat diet (HFD)-induced renal alterations in Dahl salt-sensitive and Sprague Dawley rats. In Dahl rats, HFD (60% kcal from fat for 24-26 weeks starting at weaning) significantly and equally increased blood pressure in males and females when compared with rats fed a control diet (10% kcal from fat). Male Dahl rats on HFD exhibited progressive renal histological injury and moderately increased renal macrophage infiltration at 10 and 24 weeks of feeding when compared with males on control diet. Female Dahl rats had lower grade renal injury and less macrophage infiltration (except at 17 weeks) than males regardless of diet. Male Dahl rats on both diets showed progressively increasing numbers of renal T-cells, a pattern not observed in females. HFD per se did not significantly affect renal T-cell number. Male Dahl rats had lower renal regulatory T-cells cell ratio than females at 24 weeks. Renal macrophage and T-cell infiltrations were highly correlated to final mean arterial pressure levels in males but not in females. Sprague Dawley rats fed HFD were normotensive without significant renal injury/inflammation after 24 weeks of feeding. In summary, HFD feeding fails to increase arterial blood pressure in Sprague Dawley rats but strongly promotes hypertension in both male and female Dahl salt-sensitive rats. Only Dahl males, however, exhibited blood pressure-associated renal inflammation and injury. Maintenance of regulatory T-cells ratio may protect against hypertension-associated renal injury/inflammation but not HFD-induced hypertension.

High-fat diet-induced hypertension is associated with a proinflammatory T cell profile in male and female Dahl salt-sensitive rats.

Evidence supports a sex difference in the impact of a high-fat diet (HFD) on cardiovascular outcomes, with male experimental animals exhibiting greater increases in blood pressure (BP) than female experimental animals. The immune system has been implicated in HFD-induced increases in BP, and there is a sex difference in T-cell activation in hypertension. The goal of this study was to determine the impact of HFD on BP and aortic and renal T cell profiles in male and female Dahl salt-sensitive (DSS) rats. We hypothesized that male DSS rats would have greater increases in BP and T cell infiltration in response to a HFD compared with female DSS rats. BP was measured by tail-cuff plethysmography, and aortic and renal T cells were assessed by flow cytometric analysis in male and female DSS rats on a normal-fat diet (NFD) or HFD from 12 to 16 wk of age. Four weeks of HFD increased BP in male and female DSS rats to a similar degree. Increases in BP were accompanied by increased percentages of CD4+ T cells and T helper (Th)17 cells in both sexes, although male rats had more proinflammatory T cells. Percentages of renal CD3+ and CD4+ T cells as well as Th17 cells were increased in both sexes by the HFD, although the increase in CD3+ T cells was greater in male rats. HFD also decreased the percentage of aortic and renal regulatory T cells in both sexes, although female rats maintained more regulatory T cells than male rats regardless of diet. In conclusion, both male and female DSS rats exhibit BP sensitivity to a HFD; however, the mechanisms mediating HFD-induced increases in BP may be distinct as male rats exhibit greater increases in the percentage of proinflammatory T cells than female rats.NEW & NOTEWORTHY Our study demonstrates that male and female Dahl salt-sensitive rats exhibit similar increases in blood pressure to a high-fat diet and an increase in aortic and renal T cells. These results are in contrast to studies showing that female rats remain normotensive and/or upregulate regulatory T cells in response to hypertensive stimuli compared with male rats. Our data suggest that a 4-wk high-fat diet has sex-specific effects on the T cell profile in Dahl salt-sensitive rats.

Abstract 453: Young Adult Female Dahl Salt-Sensitive Rats with Heart Failure with Preserved Ejection Fraction have a Distinct Survival Advantage over Age-Matched Afflicted Males

Introduction: Cardiovascular disease is the #1 killer of women. Heart failure with preserved ejection fraction (HFpEF) represents ≥50% of HF patients, and affects women ~2x more than men. With no treatment strategies showing decreased mortality, this indicates a critical unmet medical need. Objectives: To identify sex difference in HFpEF, we are establishing the female model of HFpEF in the Dahl salt-sensitive (DS) rat. We aim to compare male (M) and female (F) DS rats with HFpEF to identify sex-related differences in underlying mechanisms of HFpEF, for the first time. Methods: 7-week-old DS rats of both sexes are randomly assigned to low-salt (LS; control) or high-salt (HS; 8%) diets. At 7, 14 and 18-weeks of age, rats undergo echocardiography. The endpoint for LS and HS groups is 18-weeks, when HS diet groups have developed diastolic dysfunction and HFpEF. Invasive hemodynamics are performed prior to euthanasia. Results: HS (M) and (F) DS rats show a significant increase in systolic (M: 233±20; F: 243±15 bpm vs control; p<0.001) and diastolic (M: 186±18; F: 183±25 bpm vs control; p<0.001) blood pressure at 18-weeks, with no change in ejection fraction. Normalized LV wall thickness is differentially increased between sexes, with anterior wall thickness (systole) greater in (M) (M:F = 1.6-fold; p<0.001), but posterior wall thickness (diastole) greater in (F) (F:M = 1.5-fold; p<0.001), indicating structural differences. Normalized heart weight/tibia length in HS vs LS diets is increased in (F) (F: 0.39 vs. M: 0.23; p<0.001), indicating increased cardiac hypertrophy in (F). Hemodynamics show increased normalized end-diastolic pressure volume relationship in (F) (F: 1.01 vs. M: 0.64; p<0.01). Although (F) DS rats with HFpEF show increased cardiac hypertrophy and worse diastolic dysfunction compared with age-matched (M) DS rats with HFpEF, Kaplan-Meier survival analysis shows an 18-week mortality rate significantly greater in (M) (~50%) vs. (F) (~16%). Conclusion: This study characterizes the female DS model of HFpEF, and elucidates worsening (F) cardiac dysfunction vs. (M) at 18-weeks of age, with provocative results showing significantly decreased morbidity and mortality among females at the same time-point, indicating cardioprotective effects.

Sex differences in renal inflammation and injury in high fat diet induced hypertension in Dahl salt sensitive rats

The progression of hypertension and renal failure are normally faster in males than females. Renal inflammation is a common feature of experimental and clinical salt sensitive hypertension. The role of renal inflammation in high fat (HF) diet associated hypertension and renal injury has been studied using animal models in males, but the underlying mechanisms in females are less clear. We found that HF with normal salt (60% kcal from fat with 0.3% NaCl) feeding significantly and equally increases mean arterial blood pressure (MAP) in male and female Dahl salt sensitive rats compared to normal fat (NF) normal salt feeding (10% kcal from fat with 0.3% NaCl) for 24–26 weeks starting at weaning (3 weeks of age). MAP in HF rats was similar to MAP in NF rats at 10 weeks of feeding. At 17 weeks, MAP was significantly higher in HF than in NF rats. MAP was dramatically higher (~35 mmHg) in HF rats after 24 weeks of feeding. We determined if the mechanisms responsible for renal inflammation/injuries are similar in males and females. Renal tissues were taken from rats at 10, 17 and 24 weeks of feeding. Renal injury was histologically characterized. Renal inflammation was identified by the infiltration of macrophages (CD68) and T cells (CD3) in cortex and medulla. At 10 weeks, females did not show renal injuries, all males showed low grade renal injury with higher CD68 cells than females. CD3 cells were similar in males and females at lower levels. At 17 weeks, HF males displayed more severe renal injury compared to NF males and all females. CD68 and CD3 cells in males were unchanged compared to their 10 week values, females had similar CD68 and higher CD3 cells compared to males. At 24 weeks, HF males displayed much more severe renal injury with higher CD68 cells than NF males and all females. All males had significantly increased CD3 cells compared with their 10 and 17 weeks values. HF females had lower grade renal injury and lower CD68 cells than HF males, and CD3 cells were similar to males. The expression of renal proliferating cell nuclear antigen (PCNA, a cellular proliferation marker) was similar in all males and females at 17 weeks, and significantly and equally increased in all HF rats at 24 weeks. Expression of renal phospho‐S6 ribosomal protein (pS6, a surrogate marker of mTORC1 activity) was significantly higher in all females compared with all males at 17 weeks, but similar in all rats at 24 weeks. All females had higher expression of phospho‐Akt (pAkt, the canonical readout of mTORC2 activity) than males at 17 and 24 weeks. HF feeding increased pAkt expression only in females at 24 weeks. At 24 weeks, neither HF males nor females had larger final body weights than their respective NF groups. No differences in plasma levels of fasting glucose, insulin, leptin, cholesterol, triglycerides, creatinine or BUN were found between HF versus NF males or females. All females had higher plasma albumin levels than males at 17 weeks, but not at 24 weeks. Our studies show that HF diet promotes hypertension equally in Dahl salt sensitive males and females without causing overt metabolic dysfunction in either sex. In this setting, females are less prone to develop hypertension associated renal injury than males. Females do develop renal inflammation, however, the time course and types of immune cell infiltration are different from what is observed in males. Higher activities of renal mTORC2 and other immune regulatory mechanisms may protect against renal injury in females by increasing cell survival and improving the injured tissue repair.Support or Funding InformationNIH NHLBI 2P01HL070687This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiac adaptation to hypertension in adult female Dahl salt-sensitive rats is dependent on ovarian function, but loss of ovarian function does not predict early maladaptation.

Aim of study was to examine experimentally the adult female hypertensive heart in order to determine the role of ovary function in the response of the heart to salt‐dependent hypertension. Dahl salt‐sensitive rats, age 12 weeks, with/without ovariectomy were fed a standard (0.3% NaCl) or high‐salt diet (8%) for 16 weeks. Mean arterial blood pressure monitored noninvasively in conscious state increased significantly by high salt. Echocardiography was performed at baseline and endpoint. Heart function and molecular changes were evaluated at endpoint by left ventricle catheterization, by sirius red staining for collagen and by gene expression using quantitative RT‐PCR for selected genes. At endpoint, significant concentric hypertrophy was present with high salt. Increase in relative wall thickening with high salt compared to normal diet was more pronounced with intact ovaries (0.33 ± 0.02 and 0.57 ± 0.04 vs. 0.29 ± 0.00 and 0.46 ± 0.03) as was the reduction in midwall fractional shortening (20 ± 0.6 and 14 ± 2 vs. 19 ± 0.9 and 18 ± 1). Ovariectomy increased stroke volume and decreased the ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (E′) (E/E' ratio) when compared to hearts from intact rats. High salt increased expression of collagen I and III genes and perivascular collagen in the heart slightly, but % interstitial collagen by sirius red staining remained unchanged in intact rats and decreased significantly by ovariectomy. Added volume load but not deterioration of function or structure characterized the nonfailing hypertensive heart of salt‐sensitive females ovariectomized at mature age when compared to corresponding intact females.

Sex-specific genetic determinants for arterial stiffness in Dahl salt-sensitive hypertensive rats.

BackgroundArterial stiffness is an independent predictor of cardiovascular outcomes in hypertensive patients including myocardial infarction, fatal stroke, cerebral micro-bleeds which predicts cerebral hemorrhage in hypertensive patients, as well as progression to hypertension in non-hypertensive subjects. The association between arterial stiffness and various cardiovascular outcomes (coronary heart disease, stroke) remains after adjusting for age, sex, blood pressure, body mass index and other known predictors of cardiovascular disease, suggesting that arterial stiffness, measured via carotid-femoral pulse wave velocity, has a better predictive value than each of these factors. Recent evidence shows that arterial stiffening precedes the onset of high blood pressure; however their molecular genetic relationship (s) and sex-specific determinants remain uncertain. We investigated whether distinct or shared genetic determinants might underlie susceptibility to arterial stiffening in male and female Dahl salt-sensitive rats. Thus, we performed a genome-wide scan for quantitative trait loci (QTLs) affecting arterial stiffness in six-week old F2 (Dahl S x R)-intercross male and female rats characterized for abdominal aortic pulse wave velocity and aortic strain by high-resolution ultrasonography.ResultsWe detected five highly significant QTLs affecting aortic stiffness: two interacting QTLs (AS-m1 on chromosome 4 and AS-m2 on chromosome16, LOD 8.8) in males and two distinct interacting QTLs (AS-f1 on chromosome 9 and AS-f2 on chromosome11, LOD 8.9) in females affecting pulse wave velocity. One QTL (AS-1 on chromosome 3, LOD 4.3) was found to influence aortic strain in a sex-independent manner. None of these arterial stiffness QTLs co-localized with previously reported blood pressure QTLs detected in equivalent genetic intercrosses.ConclusionsThese data reveal sex-specific genetic determinants for aortic pulse wave velocity and suggest distinct polygenic susceptibility for arterial stiffness and salt-sensitive hypertension in Dahl rats based upon reported blood pressure QTLs in equivalent (Dahl S x R)-intercrosses.

Synthesis of an Endogenous Steroidal Na Pump Inhibitor Marinobufagenin, Implicated in Human Cardiovascular Diseases, Is Initiated by CYP27A1 via Bile Acid Pathway.

The bioactive steroid, marinobufagenin, is an endogenous Na/K-ATPase bufadienolide inhibitor that is synthesized by adrenocortical and placental cells. Marinobufagenin binding to Na/K-ATPase initiates profibrotic cell signaling, and heightened marinobufagenin levels are implicated in the pathogenesis of hypertension, preeclampsia, and chronic kidney disease. Steroids are derived from cholesterol through the traditional steroidogenesis pathway initiated by enzyme CYP11A1, and via the acidic bile acid pathway, which is controlled by enzyme CYP27A1. The mechanism of marinobufagenin biosynthesis in mammals, however, remains unknown. Here, we show that post-transcriptional silencing of the CYP27A1 gene in human trophoblast and rat adrenocortical cells reduced the expression of CYP27A1 mRNA by 70%, reduced total bile acids 2-fold, and marinobufagenin levels by 67% when compared with nontreated cells or cells transfected with nontargeting siRNA. In contrast, silencing of the CYP11A1 gene did not affect marinobufagenin production in either cell culture, but suppressed production of progesterone 2-fold in human trophoblast cells and of corticosterone by 90% in rat adrenocortical cells when compared with cells transfected with nontargeting siRNA. In vivo, in a high-salt administration experiment, male and female Dahl salt-sensitive rats became hypertensive after 4 weeks on a high-NaCl diet, their plasma marinobufagenin levels doubled, and adrenocortical CYP27A1 mRNA and protein increased 1.6-fold and 2.0-fold. Therefore, the endogenous steroidal Na/K-ATPase inhibitor, marinobufagenin, is synthesized in mammalian placenta and adrenal cortex from cholesterol through the novel acidic bile acid pathway. These findings will help to understand the role of marinobufagenin in highly prevalent human cardiovascular diseases.

High-intensity interval training attenuates endothelial dysfunction in a Dahl salt-sensitive rat model of heart failure with preserved ejection fraction.

Heart failure patients with preserved left ventricular ejection fraction (HFpEF) have endothelial dysfunction, but the underlying molecular mechanisms remain unknown. In addition, whether exercise training improves endothelial function in HFpEF is still controversial. The present study therefore aimed to determine the functional and molecular alterations in the endothelium associated with HFpEF, while further assessing the effects of high-intensity interval training (HIT). Female Dahl salt-sensitive rats were randomized for 28 wk into the following groups: 1) control: fed 0.3% NaCl; 2) HFpEF: fed 8% NaCl; and 3) HFpEF + HIT: animals fed 8% NaCl and HIT treadmill exercise. Echocardiography and invasive hemodynamic measurements were used to assess diastolic dysfunction. Endothelial function of the aorta was measured in vitro. Expression of endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate-oxidase [NAD(P)H oxidase], and advanced glycation end product (AGE)-modified proteins were quantified by Western blot, and zymography quantified matrix metalloproteinase (MMP) activity. In this model of HFpEF, endothelium-dependent and -independent vasodilation was impaired. However, this was prevented by HIT. In HFpEF protein expression of eNOS was reduced by 47%, but MMP-2 and MMP-9 activity was elevated by 186 and 68%. The expression of AGE-modified proteins was increased by 106%. All of these changes were prevented by HIT. Endothelial function was impaired in this model of HFpEF, which was associated with reduced expression of eNOS, increased MMP activity, and increased AGE-modified proteins. HIT was able to attenuate both these functional and molecular alterations. These findings therefore suggest HFpEF induces endothelial dysfunction, but this is reversible by HIT.