Normal Salt Diet Research Articles

Abstract P045: Endothelin Receptor Blockade Eliminates Sex-differences In Natriuresis In Rats Acclimating To A High Salt Diet

The current understanding of sodium (Na + ) handling, which is critical for blood pressure regulation, is based on studies done primarily in male (M) subjects. Our recent studies showed that female (F) Sprague Dawley (SD) rats reach a steady state in urinary Na + excretion after 1 day of increasing dietary Na + intake while M rats take 3-5 days. RNA sequencing data suggests that renal endothelin-1 (ET-1) signaling plays an important role in this difference, however, the role of ET receptors in sex-differences in acclimation to a high salt (HS) diet is not clear. We hypothesized that ET receptors mediate the advanced natriuretic capacity of F rats, compared to M rats, during acclimation to a HS diet. To test our hypothesis, M and F SD rats were implanted with telemeters and randomly assigned to treatment with A-182086, a dual antagonist of ET receptor A and B, or vehicle. After 2-3 days of treatment, rats were placed into metabolic cages and 24-hour urine samples were collected on a normal salt (NS, 0.3% NaCl) diet and after challenging rats with a HS (4% NaCl) diet for 1 day. Treatment with the ET receptor antagonist increased serum ET-1 and mean arterial pressure (MAP) to a comparable extent in both sexes (ET-1: M; 4.17 ± 0.65 vs 0.32 ± 0.02 pg/ml P=0.0004, F; 4.94 ± 1.01 vs 0.30 ± 0.01 pg/ml P<0.0001; MAP: M; 126 ± 2 vs 116 ± 3 mmHg P<0.0001, F; 114 ± 3 vs 107 ± 3 mmHg P<0.0043). On a NS and HS diet, F rats treated with A-182086 consumed more food than corresponding M rats (NS; P=0.0377, HS; P<0.0001), whereas vehicle-treated rats have no sex-difference in food intake. In vehicle-treated rats, urinary Na + excretion was higher in F compared to M rats on either NS or HS diet. Interestingly, ET receptor antagonism eliminates these sex differences in natriuresis during both dietary phases. In particular, A-182086 promotes urinary Na + excretion in M rats fed a HS diet (65.0 ± 2.3 vs 28.2 ± 4.6 % of Na + intake, P=0.0004). In conclusion, ET receptor antagonism eliminates sex-differences in natriuresis on NS and day 1 of HS. In opposition to our hypothesis, ET receptor antagonism promotes HS-induced natriuresis in M rats rather than attenuating the natriuretic capacity of F rats. A better mechanistic understanding of M-F differences in natriuresis could reveal sex-specific targets for hypertension treatment.

Abstract 026: Sex Specific Regulation Of The Sodium-chloride Cotransporter And The Salt Sensitivity Of Blood Pressure In Age-dependent Hypertension

Objectives: Blood pressure (BP) increases with age in a sex-dependent manner and a high salt diet (HSD) raises BP in salt sensitive individuals. The Na + -Cl - cotransporter (NCC) in renal cortex contributes to the fine-tuning of sodium reabsorption and BP regulation. We hypothesized that 1) increased NCC activity and expression contributes to sex- and age-dependent hypertension (HTN), and 2) age impairs HSD evoked suppression of NCC activity and expression with age to drives the development of salt sensitive hypertension (SSH). Methods: Three different age groups (3, 8, 16 month old) of male and female Sprague-Dawley (SD) rats were fed a normal salt diet (NSD; 0.6% NaCl) or HSD (4% NaCl) for 21 days. On day 21, basal MAP and NCC activity (peak natriuresis to IV hydrochlorothiazide (2mg/kg) infusion) were measured in vivo. The expression of NCC, with-no-lysine [K] kinases (WNK) 1, WNK4, STE20/SPS1-related proline-alanine-rich protein kinase (SPAK), oxidative stress responsive kinase 1 (OxSR1), and their phosphorylation status was assessed via immunoblotting (N=6/gp) Results: Male, but not female, rats develop age-dependent HTN with increased NCC activity and increased expression of NCC, WNK1, SPAK and phosphorylated SPAK/OxSR1 with age during NSD. A HSD evokes SSH with age in male, but not female, rats, and HSD evoked suppression of NCC activity and expression, WNK1, SPAK, OxSR1 and their phosphorylation is impaired in aged male rats. Conclusion: These findings suggest that 1) increased NCC activity and expression contributes to age-dependent HTN in male, but not female, SD rats, and 2) HSD evoked suppression of NCC is impaired with age driving SSH in male but not female SD rats.

Genistein Alleviates Oxidative Stress and Inflammation in the Hypothalamic Paraventricular Nucleus by Activating the Sirt1/Nrf2 Pathway in High Salt-Induced Hypertension.

Hypertension caused by a high-salt (HS) diet is one of the major causes of cardiovascular diseases. Underlining pathology includes oxidative stress and inflammation in the hypothalamic paraventricular nucleus (PVN). This study investigates genistein's (Gen) role in HS-induced hypertension and the underlying molecular mechanism. We placed male Wistar rats on HS (8% NaCl) or normal salt diet (0.3% NaCl). Then, we injected bilateral PVN in rats with Gen, vehicle, or nicotinamide (NAM) for 4weeks. Tail cuff was used weekly to assess the systolic pressure, diastolic pressure, and mean arterial pressure (MAP). Cardiac hypertrophy was analyzed by heart weight/body weight ratio and wheat germ agglutinin staining. ELISA kits, Western blot, or dihydroethidium staining determined the levels of inflammatory cytokines and oxidative stress markers. Western blot measured protein levels of Sirt1, Ac-FOXO1, Nrf2, NQO-1, HO-1, and gp91phox. Our result showed that PVN infusion of Gen significantly reduced the increase of systolic pressure, diastolic pressure, and MAP induced by an HS diet. Additionally, there was a decrease in cardiac hypertrophy and the levels of inflammatory cytokines in PVN and plasma. Meanwhile, PVN infusion of Gen notably inhibited the levels of oxidized glutathione and superoxide dismutase and improved the glutathione level and total antioxidant capacities and superoxide dismutase activities. It also decreased the level of reactive oxygen species and gp91phox expression in PVN. Furthermore, Gen infusion markedly increases the Sirt1, Nrf2, HO-1, and NQO-1 levels and decreases the Ac-FOXO1 level. However, PVN infusion of NAM could significantly block these changes induced by Gen in HS diet rats. Our results demonstrated that PVN infusion of Gen could inhibit the progression of hypertension induced by an HS diet by activating the Sirt1/Nrf2 pathway.

A high salt diet protects interleukin 10-deficient mice against chronic colitis by improving the mucosal barrier function

A high salt diet (HSD) is often associated with a high risk for a variety of diseases, such as obesity and cardiovascular disease. Previous studies have demonstrated that an HSD enhances Th17 responses and increases the severity of autoimmune diseases. In this study, we investigated the effects of HSD (4% NaCl w/w) on colitis in IL-10-/- mice by comparing it with IL-10-/- mice on a normal salt diet (NSD, 1% NaCl w/w). The colonic epithelial barrier integrity in IL-10-/- mice, as well as differentiated Caco-2 cells exposed to high NaCl and proinflammatory cytokines, was also evaluated. Surprisingly, an HSD significantly ameliorated macroscopic colitis, improved the intestinal permeability of FITC-dextran, and decreased multiple proinflammatory cytokines in the colonic mucosa of IL-10-/- mice. While occludin and claudin-1, two major tight-junction proteins, were markedly down-regulated in IL-10-/- mice, HSD effectively restored their expressions. In Caco-2 cells, proinflammatory cytokines (TNF-α and IL-1β) potently decreased the expression of occludin and claudin-1 regardless of salt conditions [0.9% (standard), 1.2%, or 1.5% NaCl]. Under high salt conditions (1.5% NaCl), transepithelial electrical resistance (TEER) was elevated, while the addition of IL-10 further downregulated occludin and claudin-1 expressions by ~50% and lowered TEER. These findings suggest that, in the absence of IL-10, HSD promotes intestinal epithelial integrity and exerts an anti-inflammatory role as demonstrated by alleviated colitis in IL-10-/- mice. Moreover, Caco-2 data indicate that, in an inflammatory environment and under high NaCl conditions, IL-10 may play a proinflammatory role by disrupting colonic epithelial integrity and thus further promoting inflammation.

Abstract P3026: The Effects Of Diet Modification On The Progression Of Heart Failure With Preserved Ejection Fraction

Introduction: Hypertension is a major risk factor for the development of heart failure with preserved ejection fraction (HFpEF). A high-salt diet has shown to induce hypertension and HFpEF in Dahl salt-sensitive rats; however, once developed, it is unclear if HFpEF resolves after a return to low-salt diet. Hypothesis: Dietary modification to reduce salt content will decrease blood pressure and lead to resolution of HFpEF in Dahl salt-sensitive rats. Methods: Dahl salt-sensitive rats (n=12) were fed a high-salt (HS) diet (8% NaCl) from 7 weeks of age to induce HFpEF. Rats fed a normal-salt (NS) diet (0.3% NaCl; n=6) served as controls. The development of HFpEF was verified with an echocardiogram at 14 weeks of age in HS-fed rats. HS diet was then switched to NS diet in a subset of the HFpEF rats (HFpEF s/p NS, n=6) at 16 weeks of age, while the rest of the HFpEF rats continued to consume HS diet (HFpEF s/p HS, n=6). Blood pressure was measured non-invasively using a tail artery cuff. Echocardiography was performed to measure systolic and diastolic function. Programmed electrical stimulation was performed to induce ventricular arrhythmias. Results: In rats with HFpEF, a switch to NS diet improved diastolic function (E/E’ ratio 14.9±3.0 in HFpEF s/p NS vs. 25.3±8.2 in HFpEF s/p HS, p=0.045). However, the diet switch did not reduce systolic blood pressure (204±15 mmHg in HFpEF s/p HS vs. 194±21 mmHg in HFpEF s/p NS, p=0.44). Lung congestion was also significantly reduced after NS diet in rats with HFpEF (lung/body weight 0.39±0.03% in HFpEF s/p NS vs. 0.50±0.06% in HFpEF s/p HS, p=0.0040). Ventricular arrhythmia inducibility did not change after diet switch (100% in both HFpEF s/p HS and HFpEF s/p NS rats), nor did the duration of induced ventricular arrhythmias (75±3 beats in HFpEF s/p HS vs. 67±8 beats in HFpEF s/p NS, p=0.0749). QTc intervals did not decrease after diet changes in rats with HFpEF (226±29 ms in HFpEF s/p HS vs. 225±4 ms in HFpEF s/p NS, p=0.095). Conclusions: Diet modification improves diastolic function and reduces pulmonary congestion in Dahl salt-sensitive rats with HFpEF, but it did not reduce blood pressure or inducibility of ventricular arrhythmias. Further mechanistic study is warranted to further investigate the effects of diet on HFpEF progression.

Abstract P3132: High-salt Diet Increases Blood Cholesterol: Potential Role Of Hepatic Enzyme CYP51

Introduction: The increased consumption of dietary fat has been well established to lead to cardiovascular disease via the elevation of lipids in serum. High blood pressure is also commonly associated with high serum cholesterol levels, but the relationships among blood pressure, dietary sodium intake and serum cholesterol have not been fully elucidated. Hypothesis: An increase in the intake of salt causes an elevation of serum cholesterol through the activation of hepatic enzymes critical to cholesterol synthesis. Methods: Dahl salt-sensitive rats (n=6) were fed a high-salt (HS) diet (8% NaCl) from 7 weeks of age to induce hypertension. Rats fed a normal-salt (NS) diet (0.3% NaCl; n=6) served as controls. Blood pressure was measured non-invasively using a tail artery cuff. Blood was withdrawn from 6-hour fasting HS and NS-fed rats to check serum cholesterol levels. Low-density lipoprotein (LDL), high-density lipoprotein (HDL), and very-low density lipoprotein (VLDL) cholesterols were measured. Gene expression array and western blot were performed in liver protein extracts from HS and NS-fed rats. Results: Systolic blood pressure was elevated in HS-fed rats compared to NS-fed rats at 16 weeks of age (225±23 vs. 156±18 mmHg, p=0.0002). Serum fasting cholesterol was higher in hypertensive rats compared to normotensive rats (194±49 vs. 102±12 mg/dL, p=0.0013). LDL cholesterol was higher in HS-fed rats compared to NS-fed rats (83±9 vs. 60±10 mg/dL, p=0.0024), while HDL and VLDL cholesterol were not. Hepatic cholesterol synthesis gene array revealed significant up-regulation of 3 genes in hypertensive rats in comparison to normotensive rats ( Apoa1 fold change [FC] 2.84, p<0.0001; Tm7sf2 FC 2.45, p=0.0004; Cyp51 FC 2.44, p=0.0002). Western blot of hepatic protein extracts showed a definitive increase in CYP51 in HS-fed rats compared to NS-fed rats (normalized against GAPDH, 2.7-fold increase, p=0.0018). Conclusions: A high-salt diet increased serum cholesterol levels, potentially via activation of liver enzyme CYP51, which is crucial for the synthesis of cholesterol in the liver. Further research is warranted to investigate the mechanisms of how high-salt diets can increase CYP51 in the liver.

Modulation of blood pressure regulatory genes in the Agtrap-Plod1 locus associated with a deletion in Clcn6.

The AGTRAP‐PLOD1 locus is a conserved gene cluster containing several blood pressure regulatory genes, including CLCN6, MTHFR, NPPA, and NPPB. Previous work revealed that knockout of Clcn6 on the Dahl Salt‐Sensitive (SS) rat background (SS‐Clcn6) resulted in lower diastolic blood pressure compared to SS‐WT rats. Additionally, a recent study found sickle cell anemia patients with mutations in CLCN6 had improved survival and reduced stroke risk. We investigated whether loss of Clcn6 would delay the mortality of Dahl SS rats on an 8% NaCl (HS) diet. No significant difference in survival was found. The ability of Clcn6 to affect mRNA expression of nearby Mthfr, Nppa, and Nppb genes was also tested. On normal salt (0.4% NaCl, NS) diets, renal Mthfr mRNA and protein expression were significantly increased in the SS‐Clcn6 rats. MTHFR reduces homocysteine to methionine, but no differences in circulating homocysteine levels were detected. Nppa mRNA levels in cardiac tissue from SS‐Clcn6 rat in both normotensive and hypertensive conditions were significantly reduced compared to SS‐WT. Nppb mRNA expression in SS‐Clcn6 rats on a NS diet was also substantially decreased. Heightened Mthfr expression would be predicted to be protective; however, diminished Nppa and Nppb expression could be deleterious and by preventing or blunting vasodilation, natriuresis, and diuresis that ought to normally occur to offset blood pressure increases. The conserved nature of this genetic locus in humans and rats suggests more studies are warranted to understand how mutations in and around these genes may be influencing the expression of their neighbors.

Reduced salt intake partially restores the circadian rhythm of bladder clock genes in Dahl salt-sensitive rats

AimsTo examine the circadian expression changes in bladder clock genes in Dahl salt-sensitive rats following high salt intake. Main methodsEighteen rats were divided into three groups: the high-salt diet group (HS group), the normal-salt diet group (NS group), and the salt-load interruption group (from a 4 % salt diet to a normal diet; salt-load interruption group [SI group]). Each rat was placed in an individual metabolic cage for 24 h twice weekly. Water intake, urine production, voiding frequency, and voided volume per micturition were recorded. Furthermore, 108 control rats were prepared. Bladders were harvested every 4 h at six time points. Furthermore, the mRNA expression of clock genes and mechanosensors was analyzed. Key findingsIn the HS group, the bladder clock genes showed lower mRNA levels than in the NS group. The amplitude of circadian expression changes in bladder clock genes in the HS group was lower than that in the NS group. However, after changing from a 4 % salt diet to a normal diet, the waveforms of the clock gene expression in the SI group were closer to those of the NS group. The 24-h water intake and urinary volume of the SI group decreased to levels comparable to those of the NS group. SignificanceReduced salt intake partially restored the circadian rhythms of bladder clock genes.

Aromatase inhibition increases blood pressure and markers of renal injury in female rats.

Aromatase is a monooxygenase that catalyzes the rate-limiting step of estrogen biosynthesis from androgens. Aromatase inhibitors are widely used for the treatment of patients with hormone receptor-positive breast cancer. However, the effects of aromatase inhibitors on cardiovascular and renal health in females are understudied. Given that estrogen is protective against cardiovascular and kidney diseases, we hypothesized that aromatase inhibition elevates blood pressure and induces kidney injury in female Sprague-Dawley rats. Twelve-week-old female rats were implanted with radiotelemetry transmitters to continuously monitor blood pressure. After baseline blood pressure recording, rats were randomly assigned to treatment with the aromatase inhibitor anastrozole (ASZ) or vehicle (Veh) in drinking water. Twenty days after treatment initiation, rats were shifted from a normal-salt (NS) diet to a high-salt (HS) diet for an additional 40 days. Rats were euthanized 60 days after treatment initiation. Body weight increased in both groups over the study period, but the increase was greater in the ASZ-treated group than in the Veh-treated group. Mean arterial pressure increased in ASZ-treated rats during the NS and HS diet phases but remained unchanged in Veh-treated rats. In addition, urinary excretion of albumin and kidney injury marker-1 and plasma urea were increased in response to aromatase inhibition. Furthermore, histological assessment revealed that ASZ treatment increased morphological evidence of renal tubular injury and proximal tubular brush border loss. In conclusion, chronic aromatase inhibition in vivo with ASZ increases blood pressure and markers of renal proximal tubular injury in female Sprague-Dawley rats, suggesting an important role for aromatization in the maintenance cardiovascular and renal health in females.NEW & NOTEWORTHY Aromatase enzyme catalyzes the rate-limiting step in estrogen biosynthesis. Aromatase inhibitors are clinically used for the treatment of patients with breast cancer; however, the impact of inhibiting aromatization on blood pressure and renal function is incompletely understood. The present findings demonstrate that systemic anastrozole treatment increases blood pressure and renal tubular injury markers in female rats fed a high-salt diet, suggesting an important role for aromatization in preserving cardiovascular and renal health in females.

Dietary salt and myeloid NFAT5 (nuclear factor of activated T cells 5) impact on the number of bone-remodelling cells and frequency of root resorption during orthodontic tooth movement

PurposeThe aim of the study was to investigate the impact of dietary salt and the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) in myeloid cells on bone remodelling cells as osteocytes, osteoblasts and osteoclasts and on force-induced dental root resorptions in a mouse model. MethodsControl mice and mice lacking myeloid NFAT5 (nuclear factor of activated T cells 5) were either kept on low, normal or high salt diets. After one week on the specified diet an elastic band was inserted between the first and second molar to induce orthodontic tooth movement. One week later the mice were euthanised and jaws were fixed for histological analysis. Osteocyte, osteoblast and osteoclast numbers as well as extent of root resorptions were assessed histologically. ResultsOsteocyte number was diminished with high salt diet in wildtype mice. Osteoblast numbers increased with low salt diet in control mice and reduced with high salt diet in mice without NFAT5 in myeloid cells. High salt diet tended to increase osteoclast number in control mice. In mice without myeloid NFAT5, numbers of osteoclasts were reduced under high salt diet. Frequency of force-induced root resorptions tended to be dependent on dietary salt content in control mice. ConclusionDuring orthodontic tooth movement dietary salt impacts on the frequency of root resorptions and the number of osteoclasts and osteoblasts in alveolar bone of mice. This can affect bone remodelling during orthodontic treatment. Myeloid NFAT5 impacts on this salt-dependent reaction.

LOSARTAN, ANGIOTENSIN II TYPE 1 RECEPTOR BLOCKER, REDUCED AORTIC STIFFNESS AND IMPROVED COGNITIVE FUNCTION IN DAHL SALT SENSITIVE RAT MODEL OF AGE-ASSOCIATED VASCULAR DEMENTIA

Objective: Cardiovascular diseases are the major contributors to the pathogenesis of vascular dementia. The Dahl salt sensitive (Dahl-S) rat model is characterized by the development of central arterial stiffness (CAS), hypertension and cognitive decline with age on a normal salt diet. We hypothesized that treatment with losartan (LOS), angiotensin II type 1 receptor (AT1R) blocker, via reduction of blood pressure (BP) and CAS, will improve cognitive function in aged Dahl-S rats. Design and method: Male Dahl-S rats (n = 30) were kept on a normal salt diet (0.5% NaCl) for the duration of the study. Baseline measurements were taken at 3- and/or 6-mo of age following by the treatment with LOS in drinking water (30 mg/kg/day, n = 14) or control treatment (n = 16) for 6-mo. Measurements were taken every 3 months and included systolic and diastolic BP (SBP, DBP), pulse wave velocity (PWV), a measure of CAS, left ventricular posterior wall thickness in diastole (LVPWd), fractional shortening (FS) and LV mass (by echocardiography). At 12-mo of age the rats were tested in operant chambers using a simple reaction task to assess attention and impulsivity, and in elevated plus maze (EPM) to assess anxiety-like behavior. Statistical analyses were performed using 2-way ANOVA and t-test. Data are presented as mean ± SEM. Results: Prior to the treatment, LOS and control groups did not display differences in any parameter analyzed. Following the treatment, LOS rats had lower SBP, DBP, PWV, thinner LVPWd, higher FS and smaller LV mass at 9- and 12-mo of age vs. control group (Fig.1A-F). In the reaction task, LOS rats demonstrated more correct responses and fewer premature responses. The LOS rats exhibited lower anxiety level, because they spent more time in the open arm of EPM vs. control rats (Fig.1G,H). Conclusions: Beneficial effect of LOS on cognitive function in aged male Dahl-S rats was associated with the improvement of cardiovascular function and remodeling. The CAS stabilizing and BP reduction by LOS treatment was associated with improved attentional performance, lower impulsivity and lower anxiety. The mechanistic basis of these effects of LOS on cognition via cerebrovascular and brain changes will be further investigated.

SALT SENSITIVITY IS MODULATED BY LANOSTEROL SYNTHASE RS2254524 POLYMORPHISM

Objective: The blood pressure (BP) response to different salt intake is associated with hypertension (HT). Elevated levels of the steroid hormone Endogenous Ouabain (EO) have been also associated with HT and salt sensitivity. We characterized the missense variation rs2254524 (V642: CC variant; L642: AA variant) of the Lanosterol Synthase (LSS), a key enzyme in steroid biosynthesis, because AA patients on a low salt diet showed a greater reduction in BP compared to the LSS CC and only CC patients had an increase in plasma EO after the low salt protocol. Hence, we hypothesized that LSS could affect salt-sensitive HT by regulation of EO biosynthesis. Design and method: We generated a knock-in mouse model carrying the variation ubiquitously. Male mice were fed with Normal Salt (NSD; 0.5% NaCl), High Salt (HSD; 4% NaCl) or Low Salt Diet (LSD; 0.03 % Na) for 15 days. BP was measured by the tail-cuff system, on 5 consequent days in conscious trained mice. Results: LSS AA mice were viable, healthy, and undistinguishable phenotypically from LSS CC. The LSS mRNA and protein levels were reduced in the adrenal gland of LSS AA mice at 3 months. At baseline, the LSS did not affect kidney function, while at 9 and 12 months the Systolic BP (SBP) in LSS CC mice showed a progressive increase with age; this increase was not observed in LSS AA mice. At 3 months, we observed an increase in SBP in both genotypes upon HSD compared to those on an NSD, however only the AA mice showed a significant SBP reduction after an LSD (Fig.1). The same response of BP upon HSD has also been observed in AA e CC mice at 12 months but only AA mice showed statistically significant cardiac hypertrophy (Fig. 2), a common feature of hypertensives. Conclusions: The new LSS mouse model resembles what was observed in hypertensive patients and provides a good model to prove our hypothesis. In the perspective of clinical practice, these findings allow the identification of patients whose SBP can seriously benefit from a low salt diet as a lifestyle modification.

Hypertensive Dahl Salt‐Sensitive Rats Demonstrate a Reduction in Central Blood Pressure Following the Short‐Term Treatment with Ivabradine and Spironolactone

IntroductionA proper management of central blood pressure (cBP) has been recently recognized as a novel therapeutic target to reduce cardiovascular risks among hypertensive (HT) patients. Considering that the hypotensive action of most antihypertensive drugs does not persist after treatment interruption, the search for compounds which would permanently reduce the level of cBP remains vital. It has been previously reported that a mineralocorticoid receptor antagonist spironolactone (SL) as well as a “pure” heart rate lowering drug ivabradine (IVA) can both affect the level of cBP and cardiac structure in hypertension.HypothesisIt was hypothesized that combined treatment with SL and IVA could permanently reduce cBP in chronically HT animals via alterations in myocardial properties.MethodsSelf‐sustaining hypertension was induced in 20 male (seven‐week‐old) Dahl salt‐sensitive rats by feeding them a high‐salt (HS) diet (8% NaCl) for 7 weeks. Then all HT rats were switched back to a normal‐salt (NS) diet (0.3% NaCl) and randomized in two experimental groups to receive ether a combined treatment (HT‐T) with IVA (10 mg/kg/day) and SL (20 mg/kg/day) or the vehicle alone (HT‐V) for 8 weeks via intraperitoneal ALZET osmotic pumps. The ten age‐matched male rats fed a NS diet only were used as normotensive controls (NT‐C). Heart rate and peripheral blood pressure were evaluated every week in conscious rats using a CODA tail‐cuff plethysmography system. One week following treatment termination, the central (aortic) and cardiac hemodynamic parameters were assessed in anesthetized rats using an intravascular/intracardiac Millar micro‐tip pressure catheter and a PowerLab data acquisition system. Subsequently, rats were euthanized, and their hearts were excised, weighed and processed to paraffin for histology and quantitative morphology.ResultsAfter 7 weeks on a HS diet, all experimental rats had markedly elevated mean arterial pressure (MAP) and heart rate. Subsequent replacement of a HS diet with a NS diet did reduce the level of MAP by ~12%, but in both HT groups it remained significantly higher than in NT‐C rats throughout the rest of the study on average by ~27% (P≤0.001). On the other hand, during eight weeks of treatment, heart rate was persistently reduced only in HT‐T rats on average by ~38% and ~27% (P≤0.001) compared to HT‐V and NT‐C groups, respectively. At the end of the study, the invasive hemodynamic measurements showed that although cBP remained markedly elevated in all HT rats compared to a NT‐C group, the level of cBP in HT‐T rats was significantly lower than that in HT‐V group (149.4±4.3 vs. 167.5±3.3 mmHg in MAP, P≤0.01). Moreover, a reduction in cBP among HT‐T rats was associated with decreased left ventricular (LV) contractility and delayed active relaxation as compared to HT‐V rats. At the same time, histological examination of the hearts has confirmed that sustained hypertension caused significant LV hypertrophy (P≤0.001) and myocardial fibrosis (P≤0.01) in both HT groups. Most important, compared to a HT‐V group, the HT‐T rats had thicker LV free wall (3.20±0.06 vs. 2.94±0.11 mm, P≤0.05) and a markedly higher myocardial density of monocytes/macrophages (228.8±12.5 vs 62.4±7.1 CD68‐positive cells/mm2, P≤0.0001).ConclusionOur findings demonstrated that concurrent treatment of chronically HT rats with IVA and SL led to a sustained reduction in cBP and that such hypotensive effect was associated with ongoing myocardial remodeling in treated animals.

A High Salt Diet Does Not Alter the Kinetics or the Magnitude of Renal Blood Flow Autoregulation in Conscious Salt‐Resistant and Salt‐Sensitive Rats

Blood pressure (BP) salt‐sensitivity is a major public health problem that augments the risk of kidney disease. Both BP dependent and independent mechanisms are thought to contribute to the increased risk of kidney injury in salt‐sensitive (SS) populations. Previous studies have reported that high dietary salt intake impairs renal blood flow (RBF) autoregulation, which increases the susceptibility to hypertensive renal injury. However, in these studies, RBF autoregulation was assessed in anesthetized rats or in isolated renal resistance vessels, which do not fully characterize the dynamic nature of RBF autoregulation in the conscious state. Our group has recently developed novel analytical methods to characterize both the magnitude and kinetics of the RBF autoregulatory response to spontaneous BP fluctuations in conscious, chronically instrumented rats. The goal of this study was to assess the effects of a high salt diet on the dynamics of RBF autoregulation in conscious, salt‐resistant and SS rat strains. At 8‐13 weeks of age, male Sprague‐Dawley (SD), Dahl SS, Consomic SS.BN1, Spontaneously Hypertensive (SHR), and Stroke‐prone SHR (SHRsp) rats were instrumented with a BP radiotelemeter and an ultrasonic RBF probe was positioned on the left renal artery. Following 1 week of recovery, BP and RBF were assessed for 2‐3 hours/day over 2‐3 consecutive days while rats were maintained on normal salt diet (0.4‐1% NaCl). All rats were then administered a high salt diet (4‐8% NaCl) and 2‐3 days later, BP and RBF were assessed for 2‐3 hours/day over 3‐5 consecutive days. All recordings were divided into segments of varying length (0.5‐20 seconds), and adjacent segments which exhibited a BP difference of 5 mmHg or greater were used to calculate RBF autoregulatory indices (i.e., % change in RBF / % change in BP). Different segment lengths were used to infer the time required for the preglomerular vasculature to restore RBF to levels observed prior to the change in BP. As shown in Figure 1, BP was significantly different among strains during normal salt diet with SD < SS.BN1 < SS = SHR < SHRsp. A high salt diet significantly increased BP in SS and SHR rats. No significant differences in RBF were found among all strains during normal or high salt intake, and a high salt diet did not significantly alter the RBF autoregulatory response in any strain (Figure 2). As also shown in Figure 2, all strains exhibited > 60% RBF autoregulatory compensation by 2.5 seconds following a change in BP, and the response was significantly faster in SHRsp vs. SD, SS, and SS.BN1 strains. All strains except SS and SS.BN1 rats exhibited complete RBF compensation by 10‐20 seconds following a change in BP. We conclude that a high salt diet does not significantly alter RBF autoregulatory responses in the conscious state in the salt‐resistant or salt‐sensitive strains examined in this study. There are strain differences in the initial magnitude of the RBF autoregulatory response with those strains exhibiting higher ambient BP’s having a stronger RBF autoregulatory response. The implications of the weaker RBF autoregulatory responses at later time points (i.e., 10‐20 seconds) following a change in BP in SS and SS.BN1 rats requires further investigation.

Loss of Clcn6 Alters Expression of Nearby Regulatory Blood Pressure Genes but Does Not Affect High Salt Induced Mortality in Dahl Salt‐Sensitive Rats

Genome‐wide association studies in both humans and Dahl salt‐sensitive (SS) rats have identified the AGTRAP‐PLOD1 locus gene cluster as an area of interest that contains several genes with the ability to regulate blood pressure, including CLCN6, MTHFR, NPPA,and NPPB. We previously demonstrated that a functional knock‐out of Clcn6 on the Dahl SS rat background (SS‐Clcn6) had significantly reduced diastolic blood pressure (BP) compared to WT rats when challenged with a high 4% NaCl (HS) diet to induce hypertension. Increased stroke risk from high blood pressure is well established, and genetic variants, such as those in CLCN6 that reduce BP may also be expected to lower the risk of stroke within this population. Furthermore, a recent paper reported that loss of function mutations in CLCN6 were associated with long‐term survival in a small cohort of sickle cell anemia patients with lower stroke rates. We hypothesized that loss of Clcn6 would delay HS‐induced mortality of Dahl SS rats on a 8% NaCl diet by slowing or preventing lethal cerebrovascular events. However, we observed no significant differences in longevity or neuronal cell death between the SS‐Clcn6 knock‐out and SS‐WT groups. Further assessment led us to examine whether a mutation in Clcn6 could alter mRNA expression of nearby Mthfr, Nppa, and Nppb genes, thereby complicating the genetic phenotype of our knockout animal. We found that on normal salt (NS) diets, renal Mthfr mRNA and protein expression were significantly increased in the SS‐Clcn6 rats, while no differences were observed between WT and knockout rats on a HS diet. MTHFR encodes Methylene tetrahydrofolate reductase (MTHFR), which is involved in reducing homocysteine to methionine, and elevated levels of circulating homocysteine (tHcy) are associated with higher cardiovascular and thromboembolic risk. No significant differences in circulating tHCy between SS‐WT and SS‐Clcn6 (10.63 ± 0.47 vs 10.05 ± 1.12 nM/mL, respectively), were observed on a NS diet; however, there was a trend toward decreased tHcy in SS‐Clcn6 rats on a HS diet (11.85 ± 2.55 vs 6.90 ± 0.40 nM/mL, p=0.08). Genetic ablation of Clcn6 resulted in significant differences in NppamRNA levels in cardiac tissue from SS‐WT and SS‐Clcn6 rat in both normotensive (153.0 ± 23.3 vs 17.61 ±10.67 a.u., p<0.01) and hypertensive (614.9 ± 115.2 vs 153.0 ± 23.3 a.u., p<0.01) conditions. A substantial reduction in NppbmRNA expression in SS‐Clcn6 rats on a NS diet (66.04 ± 4.50 vs 24.01 ± 8.80 a.u., p<0.01) was also found. The observed genetc variances are controversial; while increased Mthfrexpression would be predicted to be protective, reduced Nppaand Nppbexpression could potentially be deleterious and prevent increased vasodilation, natriuresis, and diuresis that should normally occur to compensate for BP increases. Due to the similarity of this genetic locus in humans and rats, more studies are warranted to fully understand how genetic mutations in this cluster may be altering the expression of their neighbors as well as contributing to BP homeostasis.

SK Channel Dysfunction in the Hypothalamic Paraventricular Nucleus Contributes to Sympathoexcitation in Dahl Salt‐Sensitive Rats

Salt‐sensitive hypertension is characterized by marked activation of the sympathetic nervous system. Previous studies have consistently demonstrated that increased excitability or activity of pre‐sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) contribute to the increased blood pressure and sympathetic nerve activity (SNA) in salt‐sensitive hypertension; however, the underlying mechanisms have not been fully elucidated. Small conductance calcium‐activated potassium (SK) channels act as negative feedback regulators of neuronal excitability in PVN neurons, and inhibition of SK channel function in the PVN is known to augment SNA and arterial blood pressure. In the present study, we tested the hypothesis that reduced SK channel function in the PVN contributes to sympathoexcitation in Dahl salt‐sensitive rats fed a high salt (HS, 4%) diet compared to a normal salt (NS, 0.4%) diet. Sympathoexcitatory responses to bilateral microinjection of the SK channel blocker apamin into the PVN were assessed in anesthetized 12–14‐week‐old male Dahl salt‐sensitive rats fed a high salt (n=7) or a normal salt (n=6) diet for 4 weeks. Baseline mean arterial pressure (MAP) was significantly elevated in rats fed a HS diet compared to NS (HS 129±4 vs. NS 114±4 mmHg, p<0.05), however baseline heart rate (HR) was not different between groups (HS 411±10 vs. NS 385±13 beats/min, p=0.07). Bilateral microinjection of apamin (12.5pmol, 50nL) into the PVN significantly increased splanchnic SNA in both HS and NS rats compared to baseline, however responses were significantly attenuated in HS rats compared to NS (HS 97±22% vs. NS 249±57%, p<0.01). In contrast, increases in MAP in response to PVN microinjection of apamin were similar between HS and NS rats (HS 24±4 vs. NS 29±4 mmHg, p=0.25), whereas HR responses were variable but not significantly different between groups (HS ‐8.5±9 vs. NS 7.5±7 beats/min, p=0.11). Next, we utilized qPCR to assess mRNA expression of SK1, SK2, and SK3 channels in PVN tissue from HS (n=5) and NS (n=6) rats. Interestingly, there was no significant difference in mRNA levels of either SK1, SK2, or SK3 channels between HS and NS fed rats. In summary, increases in splanchnic SNA due to PVN SK channel blockade with apamin were significantly attenuated in Dahl salt‐sensitive rats fed a HS diet, whereas there was no difference in SK channel expression between rats fed a HS vs. NS diet. These results suggest that dysfunction of SK channels in the PVN, but not decreased expression, contributes to sympathoexcitation Dahl salt‐sensitive rats fed a HS diet.

Gut microbiota depletion with antibiotics enhances fructose induced salt‐sensitive hypertension in normal rats

Introduction‐We reported that 20% fructose in drinking water induced salt‐sensitive hypertension in normal rats. High fructose (HF) or high salt (HS) intake reportedly alter the gut microbiota, which influences a variety of microbial‐regulated bioactive metabolites that can affect blood pressure and renal function. The contribution of the gut microbiota to fructose induced salt‐sensitive hypertension or renal function has not been studied. Aim‐ We hypothesized that gut microbiota depletion with antibiotics (ABX) would change fructose induced salt‐sensitive hypertension, renal excretory function and plasma levels of bacterial metabolites (lactate, β‐hydroxy‐butyrate).Methods‐Sprague Dawley rats were treated with a cocktail of antibiotics (ABX group, in g/L 1.0 ampicillin, 1.0 neomycin sulphate, 1.0 metronidazole, and 0.5 vancomycin in drinking water) for 4 weeks followed by a 0.33 fractional maintenance dose for up to 12 weeks) or control diet (no‐ABX). On week 5, all rats were given 20% fructose (HF) in drinking water and fed a diet with normal salt (NS, 0.4% Na) or high salt content (HS, 4% Na). Rats were divided as: Group 1 (HF + NS, no‐ABX, n= 6), Group 2 (HF + NS + ABX, n=6), Group 3 (HF + HS, no‐ABX, n= 8), Group 4 (HF + HS + ABX, n= 8). We trained rats and measured systolic blood pressure (SBP) for 12 weeks by tail‐cuff and collected feces before and after ABX, to measure gut c‐DNA content. On the 12th week, we collected 24‐ hour urine and plasma before the end of the protocol.Results‐ Antibiotics induced microbiome depletion (ABX) decreased fecal DNA content by 83%. Baseline SBP (in mmHg) or urine albumin excretion was not affected by ABX. Fructose with normal salt diet did not change SBP with (125±4) or without ABX (124±3). HS significantly increased SBP in rats fed fructose without ABX (from 123±3 to 141±4, p<.05) but the increase was greater in ABX rats (from 124±4 to 155±3, p<.05). Average increase on SBP was 26±2 in HF+HS with ABX, but only 15±2 without ABX (p<.001). In HF + HS groups, ABX decreased plasma lactate from 1657±259 to 905±144 µM (p<.01), and β‐hydroxy‐butyrate from 861±144 vs 220±53 µM (p<.001). Non‐fasted plasma insulin was also decreased by ABX in HF+HS rats (0.77±0.13 to 0.36±0.06 ng/mL, p<.03). The increase in body weight was similar in HF+HS rats with or without ABX.Conclusion‐ Chronic antibiotic treatment induced gut microbiota depletion and enhanced fructose induced salt‐sensitive hypertension in normal rats, an effect that was associated with decreased plasma beta‐hydroxy‐butyrate, lactate and insulin. Additional studies should focus on direct renal effects of ABX on salt handling.

Maternal angiotensin II‐induced hypertension impairs salt sensitivity and estrogen regulation of salt/water intakes in female adult offspring

Blood pressure (BP) is associated with body fluid homeostasis and the intake and excretion of water and salt. Female sex hormones play an important role in regulating salt and water intake. Recent evidence indicates that prenatal insults can have a strong influence on BP salt sensitivity in offspring. In this study, we investigated if maternal angiotensin II‐induced hypertension (MHT) alters BP salt sensitivity in female offspring exposed post‐weaning to a high salt diet (HSD, 4% salt, 12 weeks), and if estrogen modulates the HSD‐induced changes in BP and salt/water intake. Post‐weaning HSD feeding elicited a significant increase in BP in intact female offspring of normotensive (NT) dams when compared with normal salt diet (NSD) fed female offspring (MAP, 114.5±0.7 to 124.1±1.6 mmHg). However, HSD feeding did not alter BP in intact female offspring of MHT dams (MAP, 114.7±1.7 to 110.6±1.2 mmHg), indicating that MHT blunted salt sensitivity in the offspring. Ovariectomy (OVX) diminished the HSD‐induced BP increase in offspring of NT dams (111.2±1.6 mmHg) but surprisingly significantly increased BP in HSD fed offspring of MHT dams (118.5±1.2 mmHg). There were no differences in HSD and water intakes and body fluid composition (measured by nuclear magnetic resonance spectroscopy) between intact offspring of NT and MHT dams. OVX in offspring of NT dams resulted in significant increases in the intake of HSD and water, and in body fluid composition. The offspring of MHT dams had only a slight increase in HSD intake (i.e., less than that of offspring of NT dams), and they had no changes in water intake or body fluid composition. This suggests that estrogen effects on salt and water intakes were compromised by MHT. The results indicate that MHT induced by angiotensin II during pregnancy may downregulate salt sensitivity and compromise the regulatory action of estrogen to salt and water intakes, thereby altering the BP response to HSD. It is notable that these results seem to be contrary to current dogma that prenatal insults sensitize the development of hypertension and that estrogen plays protective role. These observations need further investigation.

Beneficial effects of AT1 antagonists treatment, losartan, on cardiovascular and cognitive function on aged male Dahl salt sensitive rats

Introduction and objectiveCardiovascular diseases and hypertension are the major contributors to the pathogenesis of vascular dementia. The Dahl salt‐sensitive (DSS) rat model is characterized by age‐associated cardiovascular remodeling and the development of central arterial stiffness (CAS) in the presence of renin‐angiotensin system disbalance, accompanied by cognitive decline. We hypothesized, that the anti‐hypertensive treatment, antagonist for angiotensin II receptors losartan (LOS), via reducing CAS, will improve cardiovascular parameters and cognitive function in aged DSS male rats.MethodsMale DSS rats (n=30) were kept on a normal salt diet (0.5% NaCl) for the duration of the study. At 6 months of age, rats were given either LOS in drinking water (30mg/kg/day, n=14) or control treatment (n=16) for 6 months. Measurements, including systolic and diastolic BP (SBP, DBP) and pulse wave velocity (PWV), a measure of CAS (by echocardiology), were taken after 6 months of LOS treatment. Rats were tested in an operant chambers (OCh) attention challenge to assess attention and impulsivity; elevated plus maze (EPM) was performed to estimate anxiety‐like behavior. Statistical analyses were performed using 1‐way ANOVA, t‐test and Pearson correlation. Data are presented as mean ± SEM.Resultsfollowing 6 months of treatment, at 12‐mo of age, LOS treated rats had lower BP and PWV vs. control (Table 1). Performance on the OCh indicated that LOS rats had more correct responses vs. control rats (Fig. 1). The LOS group exhibited lower anxiety levels, because they spent less time on the closed shoulder in EPM (Table 1) and demonstrate 1.5‐fold increase of time in the open shoulder (p < 0.01). PWV and SBP was negatively correlated with time on open shoulder, total distance traveled and total correct answers at 12‐mo of age (Fig.2).ConclusionAged male DSS rats show beneficial effects of LOS on cardiovascular and cognitive function. The CAS and BP improvement by LOS was associated with improved attention and lower anxiety levels. The mechanistic basis of LOS effects on cognition via cerebrovascular and brain changes will be further investigated.

Protective Role of Angiotensin II type 2 receptor (AT2R) in High Salt Diet‐Induced Low Molecular Weight Proteinuria and Loss of Megalin in the Kidney

Proteinuria is a cause and a risk factor for tubulointerstitial injury with the progression of end‐stage kidney disease. Proteinuria is a function of the glomerular filtration of protein and the reabsorption of protein from the post‐glomerular filtrate by the endocytic receptors, megalin and cubilin, localized in the renal proximal tubules. We have shown that treatment with the angiotensin‐II type 2 receptor (AT2R) agonist compound 21 (C21) for 2 weeks reduces proteinuria in obese Zucker rats (OZR) fed with high salt diet (HSD). OZR is a salt‐sensitive model but has no effect on blood pressure in the first 3 days of HSD treatment. However, the early cellular and molecular mechanism of proteinuria in response to HSD and the protective effect of AT2R activation is unknown. Therefore, the present study is designed to determine the molecular/cellular mechanism of AT2R‐mediated protection against HSD‐induced proteinuria, independent of glomerular injury in obesity. We hypothesize that AT2R activation prevents the loss of megalin in proximal tubule epithelial cells and thereby decreases the urinary excretion of low molecular weight (LMW) proteins in HSD fed obese rats. Male OZR (11–14 weeks of age) were fed on normal salt diet (NSD) (0.4%) or HSD (4%) for 2 days with and without treatment with C21. The animals were placed in metabolic cages for collecting urine at various time intervals namely 6, 12, 24 and 48 hours. We observed that HSD caused proteinuria at 24 hrs. and was prevented by C21 treatment. Further analysis by gel electrophoresis revealed that proteinuria was based on LMW protein (<60 kDa) excretion in the urine. This observation was confirmed by no change in albuminuria by HSD and C21. Dot blot analysis and confocal immunofluorescence microscopy revealed that megalin and not cubilin, was significantly decreased in HSD treatment group and the remaining megalin was mostly present in the cytosolic compartment with negligible expression on plasma membrane, suggesting megalin recycling impairment. The C21 treatment restored megalin back to the plasma membrane. FcRn is an exclusive receptor for albumin transport, which remained unaltered by HSD and C21. Western blot of nephrin and podocin, indices of glomerular filtration apparatus, remained unchanged suggesting structural integrity of the glomerular filtration assembly. Consistent to this notion, GFR measured by creatinine clearance method remained same in all the three groups. HSD caused a significant increase in the urinary sodium excretion, which was not affected by C21 treatment and the plasma sodium concentration in all the groups remained the same. Overall, the data suggest that acute feeding of HSD causes LMW proteinuria due to reduction as well as cytosolic expression of megalin in proximal tubule and not due to the change in the glomerular filtration. AT2R activation restores megalin expression and prevents proteinuria with a potential of long‐term protection against tubule‐interstitial injury and kidney disease.