Dahl Salt-resistant Research Articles

Irisin mitigates salt-sensitive hypertension via regulating renal AMPK-Rac1 pathway

ABSTRACT Background Irisin, as a myokine, plays a protective role against cardiovascular disease, including myocardial infarction, atherosclerosis and hypertension. However, whether irisin attenuates salt-sensitive hypertension and the related underlying mechanisms is unknown. Methods Male Dahl salt-resistant (DSR) and Dahl salt-sensitive (DSS) (12 weeks) rats were fed a high salt diet (8% NaCl) with or without irisin treatment by intraperitoneal injection for 8 weeks. Results Compared with DSR rats, DSS rats showed higher systolic blood pressure (SBP), impaired natriuresis and diuresis and renal dysfunction. In addition, it was accompanied by downregulation of renal p-AMPKα and upregulation of renal RAC1 and nuclear mineralocorticoid receptor (MR). Irisin intervention could significantly up-regulated renal p-AMPKα level and down-regulated renal RAC1-MR signal, thereby improving renal sodium excretion and renal function, and ultimately reducing blood pressure in DSS rats. Ex vivo treatment with irisin reduced the expression of RAC1 and nuclear MR in primary renal distal convoluted tubule cells from DSS rats and the effects of irisin were abolished by cotreatment of compound C (AMPK inhibitor), indicating that the regulation of RAC1-MR signals by irisin depended on the activation of AMPK. Conclusions Irisin administration lowered salt-sensitive hypertension through regulating RAC1-MR signaling via activation of AMPK, which may be a promising therapeutic approach for salt-sensitive hypertension.

Sex differences in dietary sodium evoked NCC regulation and blood pressure in male and female Sprague-Dawley, Dahl salt-resistant, and Dahl salt-sensitive rats.

Hypertension affects approximately one in two United States adults and sex plays an important role in the pathogenesis of hypertension. The Na+-Cl- cotransporter (NCC), regulated by a kinase network including with-no-lysine kinase (WNK)1 and WNK4, STE20/SPS1-related proline alanine-rich kinase (SPAK), and oxidative stress response 1 (OxSR1), is critical to Na+ reabsorption and blood pressure regulation. Dietary salt differentially modulates NCC in salt-sensitive and salt-resistant rats, in part by modulation of WNK/SPAK/OxSR1 signaling. In this study, we tested the hypothesis that sex-dependent differences in NCC regulation contribute to the development of the salt sensitivity of blood pressure using male and female Sprague-Dawley (SD), Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats. In normotensive salt-resistant SD and DSR rats, a high-salt diet evoked significant decreases in NCC activity, expression, and phosphorylation. In males, these changes were associated with no change in WNK1 expression, a decrease in WNK4 levels, and suppression of SPAK/OxSR1 expression and phosphorylation. In contrast, in females, there was decreased NCC activity associated with suppression of SPAK/OxSR1 expression and phosphorylation. In hypertensive DSS rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension. Collectively, our findings support the existence of sex differences in male versus female rats with NCC regulation during dietary salt intake involving suppression of WNK4 expression in male rats only and the involvement of SPAK/OxSR1 signaling in both males and females.NEW & NOTEWORTHY NCC regulation is sex dependent. In normotensive male and female Sprague-Dawley and Dahl salt-resistant rats, which exhibit dietary Na+-evoked NCC suppression, male rats exhibit decreased WNK4 expression and decreased SPAK and OxSR1 levels, whereas female rats only suppress SPAK and OxSR1. In hypertensive Dahl salt-sensitive rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension.

Curcumin, an Inhibitor of p300-HAT Activity, Suppresses the Development of Hypertension-Induced Left Ventricular Hypertrophy with Preserved Ejection Fraction in Dahl Rats.

We found that curcumin, a p300 histone acetyltransferase (HAT) inhibitor, prevents cardiac hypertrophy and systolic dysfunction at the stage of chronic heart failure in Dahl salt-sensitive rats (DS). It is unclear whether curcumin suppresses the development of hypertension-induced left ventricular hypertrophy (LVH) with a preserved ejection fraction. Therefore, in this study, we randomized DS (n = 16) and Dahl salt-resistant (DR) rats (n = 10) at 6 weeks of age to either curcumin or vehicle groups. These rats were fed a high-salt diet and orally administrated with 50 mg/kg/d curcumin or its vehicle for 6 weeks. Both curcumin and vehicle treatment groups exhibited similar degrees of high-salt diet-induced hypertension in DS rats. Curcumin significantly decreased hypertension-induced increase in posterior wall thickness and LV mass index, without affecting the systolic function. It also significantly reduced hypertension-induced increases in myocardial cell diameter, perivascular fibrosis and transcriptions of the hypertrophy-response gene. Moreover, it significantly attenuated the acetylation levels of GATA4 in the hearts of DS rats. A p300 HAT inhibitor, curcumin, suppresses the development of hypertension-induced LVH, without affecting blood pressure and systolic function. Therefore, curcumin may be used for the prevention of development of LVH in patients with hypertension.

Adrenergic regulation of the NCC in the development and maintenance of Dahl Salt‐Sensitive Hypertension occurs via a WNK/SPAK/OxSR1 pathway

BackgroundDietary sodium intake significantly influences blood pressure (BP) regulation, as increased sodium reabsorption is a well‐established precursor of hypertension risk. In a subset of individuals that demonstrate the salt sensitivity of BP, increased dietary sodium intake evokes significant increases in BP and contributes to the development of salt‐sensitive hypertension (SSH). Studies suggest that excessive Sympathetic Nervous System (SNS) release of norepinephrine (NE) stimulates activity of the sodium chloride cotransporter (NCC) via synergistic α1 or β adrenergic receptor pathways to drive SSH.HypothesisExcessive SNS release of NE influences NCC activity via an α1‐adrenoceptor gated pathway that relies upon WNK/SPAK/OxSR1 signaling to drive the development of Dahl SSH.MethodsGroups of naïve Dahl Salt Sensitive (DSS) rats, Dahl Salt Resistant (DSR), DSS rats receiving a continuous subcutaneous (s.c.) terazosin (10 mg/kg/day), or s.c. propranolol (10 mg/kg/day) infusion were fed a normal salt (NS, 0.6% NaCl) or high salt (HS, 4% NaCl) diet for 21 days respectively. On day 21 basal MAP and NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2mg/kg) infusion) were assessed. NCC, phosphoNCC, WNK1, SPAK, and OxSR1 expression were assayed via immunoblotting (N=6/gp). Additional groups of DSS rats were fed a 42 day HS diet. On day 21, rats were given s.c. saline or s.c. terazosin (10 mg/kg/day). On day 42, measurements were taken as described previously (N=6/gp).ResultsDSR rats maintain normal BP when fed a 21 day HS diet and suppress NCC activity, expression, and phosphorylation compared to rats on a NS diet. DSS rats develop SSH when fed a 21 day HS diet and exhibit increases in NCC activity, expression, and phosphorylation compared to rats on a NS diet. Chronic α1‐adrenoceptor antagonism (confirmed pharmacologically) using terazosin attenuates SSH, reduces NCC activity, expression, and phosphorylation in rats on a HS diet. β‐adrenoceptor antagonism using propranolol fails to attenuate SSH or influence NCC activity. Critically, α1‐antagonism reduces BP and NCC activity and expression in DSS rats with established SSH.ConclusionSalt resistance in DSR rats is marked by dietary sodium‐evoked suppression of NCC activity and expression. Salt sensitivity in DSS rats is driven in part by a failure to suppress NCC activity or expression and contributes to the development of SSH. α1‐adreoceptor antagonism attenuates both the development and maintenance of SSH in DSS rats by evoking suppression of NCC activity and expression. Collectively our findings suggest that excessive SNS release of NE influences NCC activity via α1‐adrenoceptor pathway to drive sodium reabsorption and the development of SSH.Support or Funding InformationThis work was supported by NIH R56 AG057687, R01 HL139867, R01 HL141406, R01 HL107330 and K02 HL112718 and AHA 16MM32090001 and 17GRNT33670023 to R.D.W., NIH F31 DK116501 to A.A.F., ASN Foundation for Kidney Research Pre‐Doctoral Fellowship award and APS William Townsend Porter Physiology Development Fellowship award to F.P. Dietary Salt Intake MAP (mmHg) Peak ΔUNaV to HCTZ (μeq/min) NCC Expression (Fold Change) Total pNCCT53 (Fold Change) WNK1 Expression (Fold Change) SPAK Expression (Fold Change) OxSR1 Expression (Fold Change) Naïve DSR rat 21‐day NS 129 ± 3 10.5 ± 1.2 1 ± 0.06 1 ± 0.14 1 ± 0.05 1 ± 0.11 1 ± 0.08 HS 131 ± 2 8.5 ± 1.0 * 0.34 ± 0.04 * 0.23 ± 0.03 * 0.76 ± 0.08 0.83 ± 0.15 0.49 ± 0.05 * Naïve DSS rat 21‐day NS 127 ± 3 9.4 ± 0.6 1 ± 0.14 1 ± 0.17 1 ± 0.08 1 ± 0.11 1 ± 0.05 HS 162 ± 4 * 11.4 ± 0.4 * 1.09 ± 0.14 1.05 ± 0.17 1.04 ± 0.12 0.96 ± 0.15 0.96 ± 0.06 DSS rat 21‐day sc Terazosin HS 143 ± 3 # 5.9 ± 0.6 # 0.25 ± 0.13 # 0.42 ± 0.20 # 0.25 ± 0.08 # 0.30 ± 0.08 # 0.71 ± 0.01 # DSS rat 21‐day sc Propanolol HS 157 ± 4 10.4 ± 0.6 0.88 ± 0.04 0.95 ± 0.06 0.99 ± 0.01 0.37 ± 0.06 # 1.09 ± 0.01 DSS rat 42‐day sc Saline HS 207 ± 5 14.4 ± 0.6 1 ± 0.10 1 ± 0.20 1 ± 0.19 1 ± 0.19 1 ± 0.05 DSS rat 42‐day sc Terazosin HS 191 ± 3 Φ 9.3 ± 2.0 Φ 0.69 ± 0.08 Φ 0.42 ± 0.15 Φ 0.48 ± 0.09 Φ 0.97 ± 0.11 0.88 ± 0.09 *p<0.05 vs. respective naive Normal Salt (NS, 0.6% NaCl) group; #p<0.05 vs. naive DSS 21 Day High Salt (HS, 4% NaCl) group; Φp<0.05 vs. 42 day subcutaneous (sc) saline group.

Role of the afferent renal nerves in sodium homeostasis and blood pressure regulation in rats.

New Findings What is the central question of this study? What are the differential roles of the mechanosensitive and chemosensitive afferent renal nerves in the reno‐renal reflex that promotes natriuresis, sympathoinhibition and normotension during acute and chronic challenges to sodium homeostasis? What is the main finding and its importance? The mechanosensitive afferent renal nerves contribute to an acute natriuretic sympathoinhibitory reno‐renal reflex that may be integrated within the paraventricular nucleus of the hypothalamus. Critically, the afferent renal nerves are required for the maintenance of salt resistance in Sprague–Dawley and Dahl salt‐resistant rats and attenuate the development of Dahl salt‐sensitive hypertension. These studies tested the hypothesis that in normotensive salt‐resistant rat phenotypes the mechanosensitive afferent renal nerve (ARN) reno‐renal reflex promotes natriuresis, sympathoinhibition and normotension during acute and chronic challenges to fluid and electrolyte homeostasis. Selective ARN ablation was conducted prior to (1) an acute isotonic volume expansion (VE) or 1 m NaCl infusion in Sprague–Dawley (SD) rats and (2) chronic high salt intake in SD, Dahl salt‐resistant (DSR), and Dahl salt‐sensitive (DSS) rats. ARN responsiveness following high salt intake was assessed ex vivo in response to noradrenaline and sodium concentration (SD, DSR and DSS) and via in vivo manipulation of renal pelvic pressure and sodium concentration (SD and DSS). ARN ablation attenuated the natriuretic and sympathoinhibitory responses to an acute VE [peak natriuresis (µeq min−1) sham 52 ± 5 vs. ARN ablation 28 ± 3, P < 0.05], but not a hypertonic saline infusion in SD rats. High salt (HS) intake enhanced ARN reno‐renal reflex‐mediated natriuresis in response to direct increases in renal pelvic pressure (mechanoreceptor stimulus) in vivo and ARN responsiveness to noradrenaline ex vivo in SD, but not DSS, rats. In vivo and ex vivo ARN responsiveness to increased renal pelvic sodium concentration (chemoreceptor stimulus) was unaltered during HS intake. ARN ablation evoked sympathetically mediated salt‐sensitive hypertension in SD rats [MAP (mmHg): sham normal salt 102 ± 2 vs. sham HS 104 ± 2 vs. ARN ablation normal salt 103 ± 2 vs. ARN ablation HS 121 ± 2, P < 0.05] and DSR rats and exacerbated DSS hypertension. The mechanosensitive ARNs mediate an acute sympathoinhibitory natriuretic reflex and counter the development of salt‐sensitive hypertension.

Inhibitory Effects of Tofogliflozin on Cardiac Hypertrophy in Dahl Salt-Sensitive and Salt-Resistant Rats Fed a High-Fat Diet.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are drugs for diabetes and might prevent heart failure. In this study, we investigated the effects of tofogliflozin, an SGLT2 inhibitor, on cardiac hypertrophy and metabolism in hypertensive rats fed a high-fat diet. Dahl salt-sensitive (DS) rats, hypertensive model rats, and Dahl salt-resistant (DR) rats, non-hypertensive model rats, were fed a high-salt and high-fat diet containing tofogliflozin (0.005%) for 9 weeks to examine the effects of this drug on cardiac hypertrophy and metabolism. Tofogliflozin tended to suppress a rise of the systolic blood pressure, relative to the control, throughout the treatment period in both DR and DS rats, and significantly suppress a rise of the systolic blood pressure, relative to the control, at the 9th week in DS rats. Tofogliflozin reduced cardiac hypertrophy (heart weight/body weight) not only in DS rats but also in DR rats. Histological analysis showed that tofogliflozin significantly decreased cardiomyocyte hypertrophy and perivascular fibrosis in both DS and DR rats. Tofogliflozin significantly decreased the expression levels of genes related to cardiac hypertrophy (encoding for natriuretic peptides A and B and interleukin-6), and to cardiac fibrosis (encoding for transforming growth factor-β1 and collagen type IV), in DS rats. Recent studies have shown that hypertrophied and failing hearts shift to oxidizing ketone bodies as a significant fuel source. We also performed metabolome analysis for ventricular myocardial tissue. Tofogliflozin reduced 3-hydroxybutyrate, a ketone body, and significantly decreased the expression levels of β-hydroxybutyrate dehydrogenase 1 and 3-oxoacid CoA-transferase, which are related to ketone oxidization. In conclusion, tofogliflozin ameliorated cardiac hypertrophy and fibrosis along with reduction of ketone usage in myocardial tissue.

Contribution of the Renal Lymphatic Circulation to the Development of Salt‐Sensitive Hypertension

More than one billion people worldwide suffer from hypertension, which is a leading cause of stroke and cardiovascular disease. According to the National Heart, Lung, and Blood Institute (NHLBI) Working Group, an estimated 40% to 85% of hypertensive patients exhibit salt‐sensitivity, which increases the risk for cardiovascular events and mortality. Salt‐sensitive hypertension is known to be caused by an imbalance between sodium retention and excretion in the kidney. However, the mechanisms that enable the kidney to deliver excessive re‐absorbed salt and volume back to the systemic circulation remain obscure. Interestingly, recent studies indicate that the peripheral lymphatics act as a “salt sink” to store excessive salt and volume, thereby maintaining sodium homeostasis to regulate blood pressure. This finding led us to consider whether renal lymphatics contribute to excessive sodium retention in the kidney during the development of salt‐sensitive hypertension. To test this hypothesis, we initially evaluated lymph‐angiogenesis in the kidneys of rats with or without salt‐sensitive hypertension. Real‐time PCR, immunohistochemistry and Western blots revealed increased expression of the lymphatic‐specific marker VEGFR3 in the kidneys of Dahl salt‐sensitive (DSS) rats fed a high‐salt diet compared to either DSS rats fed regular chow or Dahl salt‐resistant (DSR) rats with or without high‐salt intake. To explore the impact of renal lymph‐angiogenesis on blood pressure, we surgically ligated the renal collecting lymph vessel (LV) in DSS rats. Ligation of the renal LV in DSS rats attenuated the increase in systolic blood pressure caused by a high‐salt diet. Notably, a growing body of evidence has implicated macrophages in lymph‐angiogenesis. Indeed, we also observed that a high‐salt diet promoted renal infiltration of macrophages, which stimulated renal lymph‐angiogenesis via upregulating VEGFR3 and its ligand VEGFC. Collectively, our data suggest that in response to a high‐salt diet, kidney‐infiltrated macrophages stimulate renal lymph‐angiogenesis, which appears to support the development of salt‐sensitive hypertension.Support or Funding InformationSupported by NIH grants T32 GM106999 (BRG) and R21 CA187325‐01A1 (NJR), and a Medical Research Endowment Award (SM) from the Univ. of Arkansas for Medical Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The intestine responds to heart failure by enhanced mitochondrial fusion through glucagon-like peptide-1 signalling.

Glucagon-like peptide-1 (GLP-1) is a neuroendocrine hormone secreted by the intestine. Its receptor (GLP-1R) is expressed in various organs, including the heart. However, the dynamics and function of the GLP-1 signal in heart failure remains unclear. We investigated the impact of the cardio-intestinal association on hypertensive heart failure using miglitol, an α-glucosidase inhibitor known to stimulate intestinal GLP-1 production. Dahl salt-sensitive (DS) rats fed a high-salt diet were assigned to miglitol, exendin (9-39) (GLP-1R blocker) and untreated control groups and treated for 11 weeks. Control DS rats showed marked hypertension and cardiac dysfunction with left ventricular dilatation accompanied by elevated plasma GLP-1 levels and increased cardiac GLP-1R expression as compared with age-matched Dahl salt-resistant (DR) rats. Miglitol further increased plasma GLP-1 levels, suppressed adverse cardiac remodelling, and mitigated cardiac dysfunction. In cardiomyocytes from miglitol-treated DS hearts, mitochondrial size was significantly larger with denser cristae than in cardiomyocytes from control DS hearts. The change in mitochondrial morphology reflected enhanced mitochondrial fusion mediated by protein kinase A activation leading to phosphorylation of dynamin-related protein 1, expression of mitofusin-1 and OPA-1, and increased myocardial adenosine triphosphate (ATP) content. GLP-1R blockade with exendin (9-39) exacerbated cardiac dysfunction and led to fragmented mitochondria with disarrayed cristae in cardiomyocytes and reduction of myocardial ATP content. In cultured cardiomyocytes, GLP-1 increased expression of mitochondrial fusion-related proteins and ATP content. When GLP-1 and exendin (9-39) were administered together, their effects cancelled out. Increased intestinal GLP-1 secretion is an adaptive response to heart failure that is enhanced by miglitol. This could be an effective strategy for treating heart failure through regulation of mitochondrial dynamics.

High salt loading induces urinary storage dysfunction via upregulation of epithelial sodium channel alpha in the bladder epithelium in Dahl salt-sensitive rats

We aimed to investigate whether high salt intake affects bladder function via epithelial sodium channel (ENaC) by using Dahl salt-resistant (DR) and salt-sensitive (DS) rats. Bladder weight of DR + high-salt diet (HS, 8% NaCl) and DS + HS groups were significantly higher than those of DR + normal-salt diet (NS, 0.3% NaCl) and DS + NS groups after one week treatment. We thereafter used only DR + HS and DS + HS group. Systolic and diastolic blood pressures were significantly higher in DS + HS group than in DR + HS group after the treatment period. Cystometrogram showed the intercontraction intervals (ICI) were significantly shorter in DS + HS group than in DR + HS group during infusion of saline. Subsequent infusion of amiloride significantly prolonged ICI in DS + HS group, while no intra-group difference in ICI was observed in DR + HS group. No intra- or inter-group differences in maximum intravesical pressure were observed. Protein expression levels of ENaCα in the bladder were significantly higher in DS + HS group than in DR + HS group. ENaCα protein was localized at bladder epithelium in both groups. In conclusion, high salt intake is considered to cause urinary storage dysfunction via upregulation of ENaC in the bladder epithelium with salt-sensitive hypertension, suggesting that ENaC might be a candidate for therapeutic target for urinary storage dysfunction.

Abstract P285: Hydralazine Attenuates the Development of Hypertension in the Female Dahl Salt-sensitive Rat in a T Cell-independent Manner

Introduction: Several studies in Dahl salt-sensitive ( DS ) rats suggest that T cells play a role in salt-sensitive hypertension. To further investigate the role of T cells, we compared T cell profiles in hypertensive DS and normotensive Dahl salt-resistant ( DR ) rats as well as in DS rats treated with hydralazine ( HYD ) to attenuate the development of hypertension. Methods: Mean arterial pressure ( MAP ) was measured by telemetry in DS rats (n=13) from 1 to 4.5 months ( mo ) of age. At 1.5 mo, all of the DR (n=8) and half of the DS rats were treated with vehicle (VEH, n=7). The other half of the DS rats (n=6) received HYD (25 mg/kg/day) in the drinking water. At 4.5 mo, renal T helper ( Th ) and cytotoxic ( Tc ) cells were assessed by multicolor flow cytometry. Results: In the DS kidney, the frequency of CD4 + Th cells [(%): DS-VEH, 76±1.2* vs. DR-VEH, 55±0.7; *p&lt;0.0001; n=7-8/group] was higher while the frequency of CD8 + Tc cells [(%): DS-VEH, 14±1.2* vs. DR-VEH, 35±1; *p&lt;0.0001; n=7-8/group] was lower compared to DR rats. 10 weeks of HYD treatment attenuated the age-associated increase in MAP observed in DS rats [p&lt;0.0001, Two-Way ANOVA (time, treatment); MAP (mmHg): DS-VEH, 157±4 vs. DS-HYD, 133±3; *p&lt;0.0004; n=6-7/group]. HYD had no effect on the frequency of CD4 + [(%):77±1.5] or CD8 + [(%):15.5±0.9] T cells in the kidney of DS rats [(CD4 + ): DS-VEH vs. DS-HYD, p=0.83; (CD8 + ): DS-VEH vs. DS-HYD, p=0.5; n=7-8/group]. In summary, the ratio of Th (CD4 + ) to Tc (CD8 + ) cells is higher in the kidney of DS compared to DR rats and HYD had no effect on the T cell profile in the DS rat kidney under conditions in which the MAP was attenuated by 20 mm Hg. Conclusions: These findings indicate the DS rat has more active Th cells in the kidney compared to the DR rat. Our study also suggests that vasodilators can attenuate the development of hypertension in the DS rat in a Th- and Tc-independent manner.

OS 29-04 SYMPATHETIC NERVOUS SYSTEM REGULATION OF THE RENAL NCC AND BLOOD PRESSURE DURING HIGH DIETARY SALT INTAKE

Objective: These studies tested the hypothesis that the SNS release of norepinephrine modulates NCC activity via a WNK1 mechanism to contribute to the pathophysiology of salt-sensitive hypertension in rats. Design and Method: Male Sprague-Dawley (SD) rats receiving a continuous s.c. saline or NE (600 ng/min) infusion or naïve Dahl Salt-Resistant (DSR) and Dahl Salt-Sensitive (DSS) rats were fed a 0.6% (NS) or 8% NaCl (HS) diet for 14 or 21 days respectively (N = 6/group). On day 14 (SD) or 21 (DSR & DSS) MAP, plasma NE content and NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2 mg/kg) infusion)), was assessed. Kidney cortical tissue was collected for immunoblotting of NCC, STE-20 Alanine/Proline kinase (SPAK), oxidative stress response-1 (OxSR1) and WNK1 (normalized to beta-actin). Results: Salt-resistant SD & DSR rats exhibit HS evoked suppression of plasma NE and NCC expression and activity. In contrast the DSS rat exhibits hypertension, increased plasma NE and a failure to suppress NCC expression and activity during HS-intake. NE infusion in the SD rat resulted in the development of the salt-sensitive hypertension and a failure of HS to suppress NCC activity and expression. In SD & DSR rats HS evoked down regulation of the protein levels of NCC regulatory kinases SPAK, OxSR1 and WNK1. Further, in the DSR we observed significantly reduced endogenous WNK1 levels on NS intake vs SD and DSS phenotypes (P < 0.05). In contrast, in salt sensitive animals (DSS & sc NE infused SD) we observed no change in NCC regulatory kinase protein expression following HS intake. Conclusions: These data provide new mechanistic insight into the impact of the sympathetic nervous system on the kidney in hypertension and support the hypothesis that cross-talk between increased dietary salt intake and elevated SNS release of NE specifically modulates NCC activity to contribute to the pathophysiology of salt-sensitive hypertension.

OS 21-04 UPREGULATION OF CLAUDIN-4 IN THE KIDNEY OF DAHL SALT-SENSITIVE RATS

Objective: Claudins are tight junction proteins that regulate paracellular permeability of renal epithelia, and renal paracellular sodium chloride permeability may be essential to salt homeostasis and blood pressure control. A previous study has shown that the collecting duct-specific claudin-4 knockout animals develop hypotension due to profound renal wasting of chloride. This study was undertaken to investigate whether claudins have a role in the hypertension of Dahl salt-sensitive (DS) rats. Design and Method: Male DS and Dahl salt-resistant (DR) rats, 6 of each, were maintained on an 8% NaCl-containing rodent diet for 4 weeks. Systolic blood pressure (SBP) and plasma and urine electrolytes were weekly measured. At the end of animal experiment, kidneys were harvested for immunoblotting and quantitative PCR analysis of tight junction proteins including claudin-2, claudin-4, claudin-7, claudin-8, occludin, and ZO-1. Results: SBP was significantly higher in DS than in DR rats at the 1st to 4th week of the animal experiment. At the 1st and 2nd week, urine sodium and chloride excretion were significantly reduced in DS as compared with DR rats. At the 2nd week, DS rats had lower concentrations of plasma sodium and chloride than DR rats. The body weight measured at the end of animal experiment was lower in DS than in DR rats. In the kidney, claudin-4 protein and mRNA were significantly increased in DS as compared with DR rats. On the other hand, occludin protein and mRNA were significantly decreased in DS as compared with DR rats. The expressions of claudin-2, claudin-7, and claudin-8 were not significantly different between the two groups. Conclusions: In DS rats, salt-sensitive hypertension was associated with differential changes in renal tight junction protein expression. Upregulation of claudin-4 might increase paracellular sodium chloride transport in the kidney, resulting in impaired pressure natriuresis in DS rats.

Renal Urotensin II System Plays Roles in the Regulation of Blood Pressure in Dahl Salt-Resistant Rat.

Introduction. Dahl salt-resistant (SR) animal models are similar to peritoneal dialysis patients with fluid volumes overload with normal blood pressure in hemodynamic profiles. We will verify the roles of UII in the regulation of blood pressure in these animal models. Methodology. The Dahl salt-sensitive (SS) and SR rats and UII receptor gene knocked out (KO) mice were placed on a high-salt diet. Renal tissues were performed for the expression of UII in Dahl groups. Results. After high-salt diet for 6 weeks, the systolic blood pressure (SBP) in SR group was significantly lower, accompanied with higher urinary UII levels, higher 24-hour urinary sodium excretion, and higher urinary creatinine clearance in the SR rats in comparison to SS group. The expressions of UII and UT were both upregulated in the kidney tissues of SR group in comparison to SS group (P < 0.05). After high-salt diet for 8 weeks, the SBP of the KO group is significantly higher than that of the wild type group. Conclusion. We first demonstrate that renal UII system can play important roles in the regulation of blood pressure in Dahl SR rats which can be highly correlated to its effect on renal tubular sodium absorption.

Underlying mechanisms of urine storage dysfunction in rats with salt-loading hypertension.

Spontaneous hypertensive rats provide a genetic model for exploring the pathogenesis of urine storage dysfunction related to hypertension (HT). In humans, however, HT develops by both genetic and environmental factors including lifestyle factors such as a high-calorie diet, excessive salt intake and stress. We investigated the influence of salt-loading on bladder function and the underlying mechanisms of storage dysfunction related to HT. Six-week-old male Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were fed with a normal or high-salt diet for 12weeks. Micturition parameters were obtained from a metabolic cage. Whole bladders were excised from 18-week-old rats and distended in an organ bath. The releases of adenosine triphosphoric acid (ATP) and prostaglandin E2 (PGE2) from the distended bladder epithelia were measured. Changes in bladder blood flow (BBF) were determined with a laser-speckle-blood-flow imaging system. An increase in mean blood pressure (BP) was noted only in DS rats after salt-loading. During the inactive (sleeping) period, voided volume per micturition gradually increased in DR rats fed a normal or high-salt diet and normal-diet DS rats, while it did not change in the DS rats fed a high-salt diet. Bladder distension significantly increased ATP and PGE2 release from the urothelium in DS rats fed a high-salt diet. BBF was significantly decreased in high-salt-diet DS rats. One mechanism behind the relationship between salt-sensitive HT and urine storage dysfunction may be an increase in ATP and PGE2 release from the urothelium via suppression of BBF.

Abstract P027: GCH1 and GULP1 Play a Role in Hypertension Associated With Ovarian Hormone Loss Independently of Body Weight Gain

Background and Objective: Ovarian hormone loss is associated with an increased incidence of hypertension, body weight (BW) gain and inflammation. Since BW gain is also associated with an increased incidence of hypertension, it has been difficult to separate the role of the immune system in hypertension from BW gain. Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats gain BW after ovariectomy; however, only the DS rat becomes hypertensive. In this study, we took advantage of the DS/ DR model to determine which T cell genes are associated with hypertension induced by ovarian hormone loss-independently of body weight gain. Methods and Analysis: DS and DR rats (1 month) were either ovariectomized (OVX) or subjected to sham surgery (Sham). At 4 months of age, a microarray analysis was conducted on isolated splenic T-cells from these 4 groups and differential expression was confirmed by real-time PCR. Results: In contrast to DR rats which remain normotensive, the mean arterial pressure (MAP) significantly increased by 14 mm Hg in DS rats after ovariectomy [MAP (mmHg): DS-OVX, 143 ± 3.4 vs DS-Sham, 129 ± 9.4; n=10-11/group; p=0.0002; DR-OVX, 123 ± 15 vs DR-Sham, 126 ± 4.8; n=11-12/group; ns]. Both rat strains, however, exhibited a similar 17-20% increase in BW after ovariectomy compared to sham treatment [BW (g): DS-OVX, 372 ± 34 vs. DS-Sham, 308 ± 13; n=6-7/group; p&lt;0.05; DR-OVX, 323 ± 22 vs, DR-Sham, 276 ± 49; n=7/group; p&lt;0.05]. Microarray analysis suggested that among many others, the genes GTP Cyclohydrolase 1 (GCH1) and Engulfment Adaptor PTB Domain Containing 1 (GULP1) were specifically associated with resistance or susceptibility to hypertension induced by ovarian hormone loss. Real-time PCR confirmed that GCH1 and GULP1 were selectively elevated by ovariectomy in DS but not DR splenic T cells. Conclusion: GCH1 and GULP1 play a role in hypertension associated with ovarian hormone loss independently of BW gain, which has implications for women with ovarian hormone deficiency due to premature ovarian failure or elective oophorectomy.

Abstract 139: Renal Afferent Nerve Sensitivity and the Pathophysiology of Salt-sensitive Hypertension

Aim: Altered renal afferent nerve responsiveness contributes to hypertension in the Spontaneously Hypertensive rat. We hypothesized that increased salt-intake differentially impacts the renal afferent nerve sensitivity in salt-resistant vs salt-sensitive rats. Methods: Groups of naïve Sprague-Dawley (SD), Dahl Salt-Resistant (DSR), Dahl Salt-Sensitive (DSS) or SD rats receiving a s.c. norepinephrine (NE:600ng/min) infusion were fed a 0.4% (NS) or 8% NaCl (HS) diet for 14 (SD) or 21 days (DSR &amp; DSS). Following HS intake MAP and plasma NE content were determined. Via a renal pelvis preparation afferent nerve activity was assessed as NE-evoked (6250 pmol) substance P (SP) release (N=5/gp). Results: Salt-resistant phenotypes (SD &amp; DSR) remain normotensive and exhibit HS-evoked suppression of plasma NE and increased renal afferent nerve release of substance P. In contrast in salt-sensitive phenotypes (DSS and NE infused SD) a high salt diet evoked hypertension, increased plasma NE and abolished sodium evoked increased responsiveness of the renal afferent release of substance P. Significance symbols: *p&lt;0.05 vs. resp. NS gp; τ p&lt;0.05 vs. resp. SD Naïve gp. value. Conclusion: These data support a role of the afferent renal nerves in blood pressure regulation during high salt-intake. Increased responsiveness of the renal afferent nerves contributes to the maintenance of normotension in a salt-resistant rat phenotype. Conversely, our data suggest that in salt-sensitive phenotypes excess release of NE triggers impaired activation of the renal afferent nerves, a factor we postulate contributes to the development of salt-sensitive hypertension.

ROS Production Is Increased in the Kidney but Not in the Brain of Dahl Rats With Salt Hypertension Elicited in Adulthood

Enhanced production of superoxide radicals by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase in the brain and/or kidney of salt hypertensive Dahl rats has been proposed to participate in the pathogenesis of this form of experimental hypertension. Most information was obtained in young Dahl salt-sensitive (DS) rats subjected to high salt intake prior to sexual maturation. Therefore, the aim of our study was to investigate whether salt hypertension induced in adult DS rats is also accompanied with a more pronounced oxidative stress in the brain or kidney as compared to Dahl salt-resistant (DR) controls. NADPH oxidase activity as well as the content of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (oxidative index), which indicate a degree of lipid peroxidation, were evaluated in two brain regions (containing either hypothalamic paraventricular nucleus or rostral ventrolateral medulla) as well as in renal medulla and cortex. High salt intake induced hypertension in DS rats but did not modify blood pressure in DR rats. DS and DR rats did not differ in NADPH oxidase-dependent production of ROS, TBARS content or oxidative index in either part of the brain. In addition, high-salt diet did not change significantly any of these brain parameters. In contrast, the enhanced NADPH oxidase-mediated ROS production (without significant signs of increased lipid peroxidation) was detected in the renal medulla of salt hypertensive DS rats. Our findings suggest that there are no signs of enhanced oxidative stress in the brain of adult Dahl rats with salt hypertension induced in adulthood.

Impaired Regulation of the Renal Sodium Chloride Cotransporter (NCC) in Animal Models of Salt‐Sensitive Hypertension

Aim These studies test the hypothesis that excess norepinephrine (NE) and a high salt intake impairs renal NCC regulation evoking salt-sensitive hypertension. Methods: Sprague-Dawley (SD) rats receiving a s.c. saline or NE (600ng/min) infusion were fed a 0.4% (NS) or 8% NaCl (HS) diet for 14 days. Naïve Dahl Salt-Resistant (DSR) and Dahl Salt-Sensitive (DSS) rats were placed on a NS or HS diet for 21 days. Immunoblotting for NCC, STE-20 Alanine/Proline kinase (SPAK), and oxidative stress response-1 (OxSR1) was performed on kidney cortex tissue and normalized to β-actin (N=5/6). Results: Salt-resistant phenotypes (saline-infusion & DSR) show dietary sodium evoked suppression of NCC. Salt-sensitive phenotypes (NE-infusion & DSS) fail to suppress NCC expression on a high salt diet. Dietary sodium evoked suppression of SPAK was observed in salt-resistant phenotypes and this was prevented in salt-sensitive phenotypes. A high salt diet did not alter OxSR1 expression but, salt-resistant phenotypes have lower endogenous levels of OxSR1 than salt-sensitive phenotypes. s.c Saline s.c Saline s.c. NE s.c. NE Type of Diet NS HS NS HS MAP (mmHg) 124±2 124±1 151±3 τ 171±4 *τ Plasma NE (nmol/L n.d. n.d. 99±3 125±20 NCC (ODU/mm2) 1.7±0.4 1.1±0.3 1.9±0.3 2.9±0.9 SPAK (ODU/mm2) 5.9±0.1 0.8±0.3 * 6.4±1.5 ≠ 7.34±1.5 τ OxSR1 (ODU/mm2) 6.7±1.8 3.9±1.6 12.7±2.7 11.6±2.3 DSR DSR DSS DSS Type of Diet NS HS NS HS MAP (mmHg) 129±3 131±2 138±6 166±6 * Plasma NE (nmol/L) 48±6 29±4 47±6 76±4 τ NCC (ODU/mm2) 1.2±0.3 0.5±0.01 2.4±0.8 2.9±0.4 τ SPAK (ODU/mm2) 2.42±1 0.7±0.2 * 4.2±1.6 4.17±0.6 OxSR1 (ODU/mm2) 2.2±0.6 1±0.2 11.2±4.5 11.4±2.7 Significance symbols:*p<0.05 vs. resp. NS gp; τp<0.05 vs. resp. control gp; ≠p<0.05 vs. resp. control + HS gp Conclusion These data support an interaction of increased dietary salt intake and excess NE in the dysregulation of the NCC to drive the development of salt-sensitive hypertension. Our findings suggest that NCC dysregulation may depend on impaired upstream dietary sodium evoked suppression of SPAK and an underlying phenotypic difference in endogenous OxSR1 levels.

Abstract 26: PVN GaI2 Subunit Proteins - The Key to a Salt-resistant Phenotype?

Aim: Endogenous up-regulation of PVN Gαi 2 proteins facilitates sympathoinhibition and normotension during chronic Na + challenge in Sprague-Dawley rats via a renal-nerve dependent mechanism. Therefore, we examined the role of PVN Gαi 2 proteins in MAP regulation during high salt-intake in intact and renal denervated (RDNX) Dahl salt-resistant (DSR) &amp; salt-sensitive (DSS) rats and naive Brown Norway (BN) and DSSBN8 congenic rats. Methods: Sham (S) or RDNX DSR &amp; DSS rats receiving a continuous i.c.v. infusion of a scrambled (SCR) or Gαi 2 oligodeoxynucleotide (25 μg/day), and naïve BN and DSSBN8 rats, were fed a 21-day normal 0.4% (NS) or high 8% NaCl (HS) diet. MAP was recorded continuously by radiotelemetry and Na + homeostasis was assessed via metabolic balance studies. On day-21 plasma norepinephrine (NE), autonomic function and PVN Gαi 2 protein levels were determined (N=6/gp). Results: HS-intake did not alter MAP, suppressed plasma NE (P&lt;0.05), and evoked a site-specific increase in PVN Gαi 2 protein levels in BN &amp; DSR (4.8 and 4.5-fold respectively, P&lt;0.05), but not DSS rats. In DSR rats Gαi 2 down-regulation evoked rapid renal nerve-dependent hypertension (MAP [mmHg] Day 4 HS S 121±2* vs RDNX 101±2, Day 21 HS S 127±2* vs RDNX 101±1), Na + retention and sympathoexcitation (plasma NE [nmol/L] S 87±7* vs RDNX 37±3, peak ΔMAP to chlorisondamine [mmHg] S -68±4* vs RDNX -37±3). In DSS rats, Gαi 2 down-regulation exacerbated salt-induced hypertension (MAP [mmHg] Day 4 HS S 148±5* vs RDNX 133±4, Day 21 HS S 175±4* vs RDNX 145±3), Na + retention, and sympathoexcitation (plasma NE [nmol/L] S 118±9* vs RDNX 88±6) in a renal nerve dependent manner. DSSBN8 rats exhibited PVN specific Gαi 2 protein up-regulation, attenuated hypertension (MAP [mmHg] Day 21 DSSBN8 HS 142±2 τ , DSS HS 155±4), Na + retention and global sympathoexcitation (plasma NE [nmol/L] DSSBN8 HS 52±4 τ , DSS HS 87±6). *p&lt;0.05 vs RDNX + HS, τ p&lt;0.05 vs DSS. Conclusion: PVN Gαi 2 protein-gated pathways represent a conserved central molecular pathway mediating sympathoinhibitory renal-nerve dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Owing to SNP’s in the human GNAI2 gene (which encodes Gαi 2 ) correlating with hypertension our findings have translational implications.

Abstract 474: High Salt Diet Modulates Ca 2 +-Sensing Receptor and Cytochrome P450 Isoform Expression in Mesenteric Arteries in Dahl Salt-Sensitive Rats

The Ca 2+ -sensing receptor (CaSR) mediates relaxation of isolated, phenylephrine-contracted mesenteric arteries, which express the receptor in the sensory network. We tested the hypothesis that high salt alters the expression of CaSR and its CYP enzymes downstream of the receptor in salt-sensitive hypertension. Dahl salt−sensitive (DS) and Dahl salt-resistant (DR) rats were placed on an 8% NaCl diet (HS) for 1, 2, and 4 weeks. Using tail cuff plethysmyography, blood pressures were measured before and after HS diet treatment. Our results show a significant increase in blood pressure in DS animals on HS diet (Control : 116 ± 4.8 mmHg; 1 week HS: 164.4 ± 8.7 mmHg; 2 weeks HS: 175.1 ± 7.2 mmHg; p &lt; 0.05 , ANOVA). Western blot analysis of proteins extracted from mesenteric arteries showed an increase in CaSR, CYP1A1, and CYP2J2 expression in DS rats on HS diet, but not in arteries from DR rats on the same diet. The data show that HS increased mean arterial blood pressure, as expected, and increased the expression of the mature form of the CaSR while decreasing expression of the immature form. Increased CYP isoform expression was observed as well, however, these changes did not reduce blood pressure. These new findings suggest an abnormality in CaSR activity in salt −sensitive hypertension, which may provide a molecular target for anti-hypertensive therapies.