Regular Salt Diet Research Articles

Chronic High-Salt Diet Activates Tumor-Initiating Stem Cells Leading to Breast Cancer Proliferation.

Several chronic inflammatory diseases have been linked to high-salt (HS) diets. Chronic inflammation is an established causative hallmark of cancer. However, a direct role of HS diets in tumorigenesis is yet to be defined. Previous orthotopic murine breast tumor studies have shown that short-term HS diets caused inhibition of tumor growth through the activation of cytotoxic adaptive immune responses. However, there have been experimental challenges in developing a viable chronic HS-diet-based murine tumor model. To address this, we have developed a novel chronic HS diet tumor model through the sequential passaging of tumor cells in mice under HS dietary conditions. Two orthotopic murine triple-negative breast cancer models, 4T1 tumor cells injected into BALB/c mice and Py230 tumor cells injected into C57Bl/6 mice, were utilized in our study. For the HS diet cohort, prior to orthotopic injection with tumor cells, the mice were kept on a 4% NaCl diet for 2 weeks. For the regular salt (RS) diet cohort, the mice were kept on a 1% NaCl diet. Following syngeneic cancer cell injection, tumors were allowed to grow for 28 days, following which they were collected to isolate immune cell-depleted cancer cells (passage 1, P1). The tumor cells from P1 were reinjected into the next set of non-tumor-bearing mice. This procedure was repeated for three cycles (P2-P4). In P1, compared to the RS diet cohort, we observed reduced tumor kinetics in both murine tumor models on the HS diet. In contrast, by P4, there was significantly higher tumor progression in the HS diet cohort over the RS diet cohort. Flow cytometry analysis demonstrated an 8-fold increase in tumor-initiating stem cells (TISCs) from P1 to P4 of the HS diet cohort, while there were no significant change in TISC frequency with sequential passaging in the RS diet cohort. Molecular studies showed enhanced expression of TGFβR2 and CD80 on TISCs isolated from the P4 HS diet cohort. In vitro studies demonstrated that TGFβ stimulation of these TISCs increased the cellular expression of CD80 molecules. Further, the chronic HS diet selectively induced the glycolytic metabolic phenotype over the mitochondrial oxidative phosphorylation phenotype in TISCs, which is needed for the production of metabolites during tumor cell differentiation and proliferation. The infiltrating CD8 and CD4 T-lymphocytes in P4 tumors demonstrated increased expression of the immune checkpoint inhibitor (ICI) CTLA4, a known binding partner of CD80, to cause immune exhaustion and pro-tumorigenic effects. Interestingly, anti-TGFβ monoclonal antibodies (mAbs) played a synergistic role in further enhancing the anti-tumor effect of anti-CTLA4 mAb. In summary, our findings demonstrated that chronic HS diet increased the frequency of TISCs in tumors leading to blunting of cytotoxic adaptive immune responses causing tumor proliferation. Furthermore, a combination of anti-TGFβ with current ICI-based immunotherapies could exert more favorable anti-cancer clinical outcomes.

Abstract 4850: High salt diet induced tumor initiating stem cells mediate breast cancer progression

Abstract High-salt (sodium chloride) diets have been associated with several chronic inflammatory diseases. While the role of chronic inflammation in cancer is well established, the specific role of high salt diet in carcinogenesis is unknown. Previous studies with syngeneic murine breast cancer models, both in our laboratory and others, have shown that high salt (HS) diet induced tumor regression through inflammatory activation of anti-tumor adaptive immune responses. We tested this counter-intuitive and suspiciously beneficial role of HS diet by performing sequential passage studies in preclinical murine models. Six week old mice were placed on high salt diet for 2 weeks prior to injection with syngeneic 4T1 and Py230 triple negative breast cancer cells (into BALB/c and C57Bl/6J mice, respectively, referred to as passage-1). Tumors were harvested after four weeks of injection. The cancer cells depleted of immune cells were collected from these harvested tumors and reinjected into new non-tumor bearing mice. This cycle was repeated three times. In passage-1 cohort, by day 28, HS diet induced reduced tumor progression in both 4T1-BALB/c (267 ± 59 mm3) and Py230-C57Bl/6J (238 ± 54 mm3) as compared to regular salt (RS) diet cohort (611 ± 94 mm3 and 473 ± 69 mm3, respectively; p<0.05 for both). However, in passage-4 cohort, by day 28, HS diet induced significantly higher tumor progression in both 4T1-BALB/c (806 ± 91 mm3) and Py230-C57Bl/6J (743 ± 81 mm3) models, as compared to regular salt (RS) diet cohort (577 ± 83 mm3 and 462 ± 77 mm3, respectively; p<0.05 for both). Cellular analysis by flow cytometry revealed that there was a 12-19 fold increase in tumor initiating stems cells (TISCs) in passage-4 HS diet cohort compared to RS diet cohort. Mechanistic studies have demonstrated that there was increased TGFβ expression on TISCs obtained from passage-4 HS diet cohort which correlated with enhanced exhaustion phenotype (CTLA4+) of tumor infiltrating adaptive immune cells (CD4 and CD8 T cells) in this cohort. Co-treatment with anti-TGFβ and anti-CTLA4 monoclonal antibodies (mAb) in passage-4 HS diet cohort, significantly reduced tumor progression (p<0.05), as compared to treatment with either mAb alone. Taken together, these data demonstrated that chronic HS diet leads to expansion of TGFβ expressing TISCs leading to host immune exhaustion and tumorigenesis. Importantly, anti-TGFβ mAb exerted a favorable synergistic effect to enhance the anti-tumor efficacy of immune check-point inhibitors. Citation Format: Lisa Tucker, Sonya Reid, Jeffrey C. Rathmell, Venkataswarup Tiriveedhi. High salt diet induced tumor initiating stem cells mediate breast cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4850.

PS-BPB10-1: INVOLVEMENT OF RENAL TUBULAR NFAT5 IN THE REGULATION OF BLOOD PRESSURE AND BODY FLUID VOLUME

Objective: Nuclear factor of activated T-cells 5 (NFAT5) is an osmoresponsive transcription factor that activates osmoprotective genes to protect cells against hypertonicity. Renal tubular cell-specific NFAT5 knockout (KO) mice have been shown to exhibit polyuria, hypernatremia, low urinary sodium excretion, and increased expression of the epithelial sodium channel (ENaC) in the kidney. In the present study, regulation of blood pressure (BP) as well as that of body fluid volume were examined in the wild type (WT) mice and NFAT5 KO mice. Methods: The BP of KO mice and WT mice was measured using radiotelemetry method. The mice were fed with regular salt diet (RSD, 0.3% NaCl chow) or high salt diet (HSD, 8% NaCl chow) followed by the treatment with amiloride, an ENaC blocker. To assess body fluid volume, the plasma renin activity (PRA) and the plasma aldosterone concentration (PAC) were measured at the condition of RSD and HSD, together with plasma arginine vasopressin (AVP) and hematocrit (Ht) levels. Results: Compared to WT mice, the mean BP was significantly higher in KO mice both at the condition with RSD and that with HSD in dark period, but not in light period. Treatment with amiloride significantly decreased the mean BP in KO mice with HSD to the level of WT mice with HSD in dark period. PRA, PAC, and AVP levels were not significantly different between in WT and KO mice with RSD. HSD significantly decreased PRA and PAC and tended to increase the AVP level both in WT and KO mice. The Ht level was significantly lower in KO mice than in WT mice regardless of RSD or HSD (WT with RSD, 42.6 ± 0.5%; WT with HSD, 42.8 ± 0.6%; KO with RSD, 38.3 ± 0.4%; KO with HSD, 39.0 ± 0.4%, n = 4). Conclusions: The results suggest that the renal tubular cell-specific NFAT5 KO mice exhibit excess body fluid retention induced by sodium reabsorption through ENaC, leading to salt-sensitive hypertension. Such phenotypes seem to be independent of the systemic renin-angiotensin-aldosterone and AVP systems. Renal tubular NFAT5 could integrate urine concentration and sodium reabsorption in the kidney, thus preventing salt-dependent hypertension.

Abstract P113: SOX6 IS NOT A RELEVANT TRANSCRIPTION FACTOR FOR RENIN TRANSCRIPTIONAL REGULATION BY JUXTAGLOMERUALR CELLS

The SRY (sex determining region Y)-box 6, Sox6 , encodes a member of trancription factors (TFs) crucial for organogenesis. It has been suggested that Sox6 is essential for the Renin transcriptional regulation. However, we identified Sox6 expression only in tubular cells. We aim to investigate whether Sox6 contributes to Renin gene expression by the Juxtaglomerular Cells in normal and under stress. Therefore, we generated Ren1 d -Cre;Sox6 -/- mice, where cells expressing Renin lacks the Sox6 gene. We randomized the mice into four groups (n= 6/group): Wild-type (WT) and mutants ( Sox6 cKO) exposed to either regular salt diet or low salt diet and captopril (0.5 mg/mL in water ad libitum for 8 days). The later condition decreases blood pressure and increases Renin transcription and exocytosis. We measured the blood pressure by direct carotid artery catheterization. There were no differences between WT and cKO in mean blood, systolic or diastolic pressures. ELISA assay measured the plasmatic Renin content, without significance in any group (WT: 126775 pg/mL ± 59035; cKO: 200008 pg/mL ± 68556; n=3). In sytu hybridization ( ISH ) for Sox6 has never been visible in the kidney despite all optimizations. Renin ISH had usual distribution in the Juxtaglomerular Apparatus (JGA; n=6). In the captopril treated groups, Renin intensity was higher and no differences were noted. Immunohistochemistry for Renin also reavealed no differences between WT and cKO. Yet, the renin+ cells extended along the afferent arterioles in the captopril treated groups. We calculated the Juxtaglomerular Index, which is the total number of renin+ JGA divided by all glomeruli. WT showed 156 renin+ JGA of 441 glomeruli (n=3), while the cKO exhibited 173 of 449 glomeruli (n=3). In the captopril groups, WT had 302 of 452 glomeruli (n=3), while the ckO exhibited a subtle decrease, 176 of 312 glomeruli (n=4), but without significance. Quantitative RT-PCR from the kidneys showed an increase in the relative R enin gene expression to s14 in cKO group (WT: 1.2 ± 0.8; cKO: 2.1 ± 2.2; n=3), while Sox6 relative gene expression to s14 decreased (WT: 1.02 ± 0.27; cKO: 0.35 ± 0.2; n=3). We conclude that Sox6 is not an important TF for Renin transcriptional regulation by JuxtaGlomerular Cells in neither normal nor stressed conditions.

Pharmacological antagonism of EP2 receptor does not modify basal cardiovascular and respiratory function, blood cell counts, and bone morphology in animal models

The EP2 receptor has emerged as a therapeutic target with exacerbating role in disease pathology for a variety of peripheral and central nervous system disorders. We and others have recently demonstrated beneficial effects of EP2 antagonists in preclinical models of neuroinflammation and peripheral inflammation. However, it was earlier reported that mice with global EP2 knockout (KO) display adverse phenotypes on fertility and blood pressure. Other studies indicated that EP2 activation with an agonist has a beneficial effect of healing fractured bone in animal models. These results impeded the development of EP2 antagonists, and EP2 antagonism as therapeutic strategy. To determine whether treatment with EP2 antagonist mimics the adverse phenotypes of the EP2 global KO mouse, we tested two EP2 antagonists TG11–77. HCl and TG6–10–1 in mice and rats while they are on normal or high-salt diet, and by two different administration protocols (acute and chronic). There were no adverse effects of the antagonists on systolic and diastolic blood pressure, heart rate, respiratory function in mice and rats regardless of rodents being on a regular or high salt diet. Furthermore, chronic exposure to TG11–77. HCl produced no adverse effects on blood cell counts, bone-volume and bone-mineral density in mice. Our findings argue against adverse effects on cardiovascular and respiratory systems, blood counts and bone structure in healthy rodents from the use of small molecule reversible antagonists for EP2, in contrast to the genetic ablation model. This study paves the way for advancing therapeutic applications of EP2 antagonists against diseases involving EP2 dysfunction.

Low-Salt Diet Attenuates B-Cell- and Myeloid-Cell-Driven Experimental Arthritides by Affecting Innate as Well as Adaptive Immune Mechanisms.

A link between high sodium chloride (salt) intake and the development of autoimmune diseases was previously reported. These earlier studies demonstrated exacerbation of experimental autoimmune encephalomyelitis and colitis by excess salt intake associated with Th17- and macrophage-mediated mechanisms. Little is known about the impact of dietary salt intake on experimental arthritides. Here, we investigated if salt restriction can exert beneficial effects on collagen-induced arthritis (CIA) and K/BxN serum transfer-induced arthritis (STIA). CIA depends on both adaptive and innate immunity, while STIA predominantly mimics the innate immune cell-driven effector phase of arthritis. In both models, low salt (LS) diet significantly decreased arthritis severity compared to regular salt (RS) and high salt (HS) diet. We did not observe an aggravation of arthritis with HS diet compared to RS diet. Remarkably, in STIA, LS diet was as effective as IL-1 receptor blocking treatment. Complement-fixing anti-CII IgG2a antibodies are associated with inflammatory cell infiltration and cartilage destruction. LS diet reduced anti-CII IgG2a levels in CIA and decreased the anti-CII IgG2a/IgG1 ratios pointing toward a more Th2-like response. Significantly less inflammatory joint infiltrates and cartilage breakdown associated with reduced protein concentrations of IL-1 beta (CIA and STIA), IL-17 (CIA), and the monocyte chemoattractant protein-1 (MCP-1) (CIA) were detected in mice receiving LS diet compared to HS diet. However, we did not find a reduced IL-17A expression in CD4+ T cells upon salt restriction in CIA. Analysis of mRNA transcripts and immunoblots revealed a link between LS diet and inhibition of the p38 MAPK (mitogen-activated protein kinase)/NFAT5 (nuclear factor of activated T-cells 5) signaling axis in STIA. Further experiments indicated a decreased leukodiapedesis under LS conditions. In conclusion, dietary salt restriction ameliorates CIA and STIA, indicating a beneficial role of LS diet during both the immunization and effector phase of immune-mediated arthritides by predominantly modulating the humoral immunity and the activation status of myeloid lineage cells. Hence, salt restriction might represent a supportive dietary intervention not only to reduce cardiovascular risk, but also to improve human inflammatory joint diseases like rheumatoid arthritis.

SO035EFFECT OF DIETARY SALT REDUCTION ON BLOOD PRESSURE IN KIDNEY TRANSPLANT PATIENTS: A RANDOMISED CONTROLLED TRIAL

Abstract Background and Aims Cardiovascular morbidity and mortality are increased in kidney transplant patients. High blood pressure (BP) contributes significantly to this risk and is associated with shortened allograft survival. Dietary salt reduction is widely recommended as a strategy to lower BP in the general population and in chronic kidney disease. Due to a lack of evidence there is currently no consensus on dietary salt restriction in kidney transplant patients. The primary aim of the study was to examine the impact of reduced dietary salt intake on BP in kidney transplant patients. Method Sixty stable kidney transplant patients, ≥ 6-months post-transplantation, with BP ≥120/80 mmHg, and sodium intake ≥80 mmol/24hrs, were randomised in this parallel-designed study to receive either a regular-salt diet (target 150 mmol/24hr) or a low-salt diet (target 80 mmol/24hr) for 8-weeks. The primary outcome measure was systolic and diastolic BP. Secondary outcome measures included 24-hour ambulatory BP (ABP) and proteinuria. Dietary salt intake was assessed by 48-hour urinary sodium excretion. Results At baseline, patients (72% men) were 56±11 years with estimated glomerular filtration rate (eGFR) 53±18 mL/min/1.73m2. Mean urinary sodium was 128±42 mmol/24hr, mean systolic BP was 132±12 mmHg, and mean diastolic BP was 77±10 mmHg. At the end of the intervention period sodium excretion was significantly lower in the low-salt group compared with the regular-salt group (90±37 vs. 132±51 mmol/24hr; adjusted mean difference, -36 [95% CI, -59 to -14] mmol/24hr; P=0.002). We found no difference in systolic BP (adjusted mean difference, -2 [95% CI, -12 to 9] mmHg; P=0.750), diastolic BP (adjusted mean difference, 0 [95% CI, -4 to 4] mmHg; P=0.887), 24-hour systolic ABP (adjusted mean difference, -3 [95% CI, -9 to 2] mmHg; P=0.213) or 24-hour diastolic ABP (adjusted mean difference, -2 [95% CI, -5 to 1] mmHg; P=0.267). There was no significant effect on proteinuria or eGFR. Conclusion In this study baseline urinary sodium was lower than expected and baseline BP was well controlled. Reducing dietary salt by 2g/day did not have a significant effect on office blood pressure readings.

A8009 The effect of capsaicin on glomerular damage and TGF-β1/Smads pathway in rats induced by high salt

Objectives: To investigate the protective effects of dietary capsaicin on progression of glomerular damage in Wistar rats induced by high salt. Methods: Thirty-six male Wistar rats were randomly divided into three groups: normal diet group[n = 12, regular salt diet (0.5%)], high salt diet group[n = 12, high salt diet (4%)], and high salt diet plus capsaicin group[n = 12, high salt diet (4%) with 0.02% capsaicin inside]. Tail artery blood pressure was determined by intelligent noninvasive blood pressure monitor-mouse instrument every 4 weeks. The urinary microalbumin, 24-hour creatinine were measured by biochemical methods. Glomerular sclerosis was observed by HE staining, and renal fibrosis was observed by Masson staining. In addition, the mRNA expressions of TGF-β1, Smad2, Smad3, Smad7 were evaluated by real-time PCR and the protein expressions of Transient Receptor potential vanilloid 1 (TRPV1), TGF-β1, p-Smad2/3, Smad7 were observed by immunochemistry and quantitated by western-blot analyses in renal cortex. Results: Firstly, comparing with the normal diet group, the blood pressure, the fibrosis index of the glomerular, the urinary microalbumin were increased in high salt group, creatinine clearance rate was decreased, the mRNA expression of TGF-β1, Smad2, Smad3, Smad7 and the protein expression of TGF-β1, p-Smad2/3, Smad7 were much higher in high salt group as well. Secondly, after the intervention of capsaicin, the blood pressure, the fibrosis index of the glomerular and the urinary microalbumin were much lower than the high salt group, all of the gene and protein expression were also significantly reduced by TRPV1 agonist capsaicin. Conclusion: Dietary capsaicin may prevent glomerular damage induced by high salt diet via intervening TGF-β1/S-mads signal pathway.

Dietary NaCl affects bleomycin-induced lung fibrosis in mice

ABSTRACT Purpose: High levels of NaCl in the diet are associated with both cardiac and renal fibrosis, but whether salt intake affects pulmonary fibrosis has not been examined. Aim of the Study: To test the hypothesis that salt intake might affect pulmonary fibrosis. Materials and Methods: Mice were fed low, normal, or high salt diets for 2 weeks, and then treated with oropharyngeal bleomycin to induce pulmonary fibrosis, or oropharyngeal saline as a control. Results: As determined by collagen staining of lung sections, and protein levels and cell numbers in the bronchoalveolar lavage (BAL) fluid at 21 days after bleomycin, the high salt diet did not exacerbate bleomycin-induced fibrosis, while the low salt diet attenuated fibrosis. For the bleomycin-treated mice, staining of the post-BAL lung sections indicated that compared to the regular salt diet, high salt increased the number of Ly6c-positive macrophages and decreased the number of CD11c and CD206-positive macrophages and dendritic cells. The low salt diet caused bleomycin-induced leukocyte numbers to be similar to control saline-treated mice, but reduced numbers of CD45/collagen-VI positive fibrocytes. In the saline controls, low dietary salt decreased CD11b and CD11c positive cells in lung sections, and high dietary salt increased fibrocytes. Conclusions: Together, these data suggest the possibility that a low salt diet might attenuate pulmonary fibrosis.

Central and peripheral slow-pressor mechanisms contributing to Angiotensin II-salt hypertension in rats.

High salt intake markedly enhances hypertension induced by angiotensin II (Ang II). We explored central and peripheral slow-pressor mechanisms which may be activated by Ang II and salt. In protocol I, Wistar rats were infused subcutaneously with low-dose Ang II (150 ng/kg/min) and fed regular (0.4%) or high salt (2%) diet for 14 days. In protocol II, Ang II-high salt was combined with intracerebroventricular infusion of mineralocorticoid receptor (MR) blockers (eplerenone, spironolactone), epithelial sodium channel (ENaC) blocker (benzamil), angiotensin II type 1 receptor (AT1R) blocker (losartan) or vehicles. Ang II alone raised mean arterial pressure (MAP) ∼10 mmHg, but Ang II-high salt increased MAP ∼50 mmHg. Ang II-high salt elevated plasma corticosterone, aldosterone and endogenous ouabain but not Ang II alone. Both Ang II alone and Ang II-high salt increased mRNA and protein expression of CYP11B2 (aldosterone synthase gene) in the adrenal cortex but not of CYP11B1 (11-β-hydroxylase gene). In the aorta, Ang II-high salt increased sodium-calcium exchanger-1 (NCX1) protein. The Ang II-high salt induced increase in MAP was largely prevented by central infusion of MR blockers, benzamil or losartan. Central blockades significantly lowered plasma aldosterone and endogenous ouabain and markedly decreased Ang II-high salt induced CYP11B2 mRNA expression in the adrenal cortex and NCX1 protein in the aorta. These results suggest that in Ang II-high salt hypertension, MR-ENaC-AT1R signalling in the brain increases circulating aldosterone and endogenous ouabain, and arterial NCX1. These factors can amplify blood pressure responses to centrally-induced sympatho-excitation and thereby contribute to severe hypertension.

Alterations to the middle cerebral artery of the hypertensive-arthritic rat model potentiates intracerebral hemorrhage.

AimsWe have recently created an age-dependent hypertensive-mono-arthritic animal model from the stroke-resistant spontaneously hypertensive rat to model populations with autoimmune disease who are hypertensive and are prone to stroke. The model exhibits signs of hemorrhagic stroke (HS) subsequent to chronic inflammation and hypertension. HS is also associated with the inability of middle cerebral arteries to undergo pressure dependent constriction (PDC). We investigated alterations in the cerebrovasculature of our hypertensive mono-arthritic animals that develop stroke.Main MethodsAnimals were fed either a high salt diet (HSD) (4% NaCl) or Purina chow (0.58% NaCl) from weaning. Complete Freund’s Adjuvant (CFA) was injected into the left hind paw at 21–28 weeks; controls received saline and histological and functional studies were performed.ResultsBrain damage was more prominent with the high salt, with inflammation exacerbating the damage. High salt alone significantly decreased middle cerebral artery’s (MCA’s) ability to undergo PDC. Inflammation significantly decreased the ability of cerebrovasculature to respond to pressure step in the regular salt diet. The responses to vasoactive peptides were also significantly attenuated in both inflamed groups regardless of diet.ConclusionInduction of chronic systemic inflammation increases brain damage, and affect the MCA’s vasogenic function, decreasing its ability to respond to intraluminal pressure. HSD further exacerbates organ damage associated with chronic inflammation, further compromising cerebrovascular function, and likely increasing the incidence of intracerebral hemorrhage and injury.

Severe Salt-Losing Syndrome and Hyperkalemia Induced by Adult Nephron-Specific Knockout of the Epithelial Sodium Channel α-Subunit.

Systemic pseudohypoaldosteronism type 1 (PHA-1) is a severe salt-losing syndrome caused by loss-of-function mutations of the amiloride-sensitive epithelial sodium channel (ENaC) and characterized by neonatal life-threatening hypovolemia and hyperkalemia. The very high plasma aldosterone levels detected under hypovolemic or hyperkalemic challenge can lead to increased or decreased sodium reabsorption, respectively, through the Na(+)/Cl(-) cotransporter (NCC). However, the role of ENaC deficiency remains incompletely defined, because constitutive inactivation of individual ENaC subunits is neonatally lethal in mice. We generated adult inducible nephron-specific αENaC-knockout mice (Scnn1a(Pax8/LC1)) that exhibit hyperkalemia and body weight loss when kept on a regular-salt diet, thus mimicking PHA-1. Compared with control mice fed a regular-salt diet, knockout mice fed a regular-salt diet exhibited downregulated expression and phosphorylation of NCC protein, despite high plasma aldosterone levels. In knockout mice fed a high-sodium and reduced-potassium diet (rescue diet), although plasma aldosterone levels remained significantly increased, NCC expression returned to control levels, and body weight, plasma and urinary electrolyte concentrations, and excretion normalized. Finally, shift to a regular diet after the rescue diet reinstated the symptoms of severe PHA-1 syndrome and significantly reduced NCC phosphorylation. In conclusion, lack of ENaC-mediated sodium transport along the nephron cannot be compensated for by other sodium channels and/or transporters, only by a high-sodium and reduced-potassium diet. We further conclude that hyperkalemia becomes the determining factor in regulating NCC activity, regardless of sodium loss, in the ENaC-mediated salt-losing PHA-1 phenotype.

Abstract P250: Central Mechanisms Mediating Angiotensin II-Salt Hypertension in Rats

Circulating Ang II causes persistent activation of brain angiotensinergic pathways through a neuromodulatory aldosterone (aldo)-mineralocorticoid receptors (MR)-epithelial Na + channel (ENaC)-ouabain pathway. The response of BP to circulating Ang II is enhanced by high salt intake. To evaluate the central mechanisms that mediate Ang II-salt induced hypertension, Wistar rats were treated with regular salt diet (0.4% NaCl), high salt diet (2% NaCl), sc Ang II (150 ng/kg / min), or sc Ang II with high salt diet for 14 days. In the 2 nd exp, MR blockers (eplerenone, spironolactone), ENaC blocker (benzamil), AT 1 R blocker (losartan) or vehicles (Veh) were icv infused combined with Ang II-salt. BP was recorded by telemetry. Gene expression was assessed by real-time qPCR. Plasma Ang II and tissue aldosterone were measured by RIA. Ang II alone caused a small increase in MAP (112±1 vs 99±1 mmHg), and BP was markedly increased by Ang II-salt (152±4 mmHg, P<0.05 vs. others). BP increases to Ang II-salt were largely inhibited by central infusion of MR blockers (103±2mmHg), benzamil (100±3mmHg) or losartan (98±4mmHg). Ang II alone or together with salt decreased 11βHSD2 and MR mRNA expression but increased AT 1 R and ENaC γ mRNA expression in the PVN, and increased AT 1 R mRNA level in the RVLM. Sc Ang II or high salt diet had no effect on mRNA levels of CYP11B1, B2, ENaC α or β in the PVN or RVLM. Considering that AT 1 R mRNA expression increases in both PVN and RVLM and central MR-ENaC-AT 1 R blockade prevents the hypertension, these results suggest that activation of Ang II-AT 1 R signaling and MR-ENaC pathway in the brain contribute to both Ang II and Ang II-salt hypertension.

Abstract P142: Regulation of Brain Derived Neurotrophic Fractor (BDNF) Expression by Angiotensin II in the Adrenals and the Brain

Circulating Ang II activates central angiotensinergic pathways, and an amplifying aldosterone (aldo)-MR-ENaC-AT 1 R neuromodulatory pathway which are critical for several forms of hypertension. We hypothesized that in addition Ang II increases BDNF expression in both the CNS and adrenal which plays a balancing role against Ang II excitatory actions. In the 1 st exp, Wistar rats were sc infused with Ang II at the high dose of 500 ng/kg/min for 7days. In the 2 nd exp, rats were treated with regular salt diet (0.4% NaCl), high salt diet (2% NaCl), sc Ang II at the low dose of 150 ng/kg/min, or sc Ang II with high salt diet for 14 days. In the 3 rd exp, MR blockers (eplerenone, spironolactone), ENaC blocker (benzamil), AT 1 R blocker (losartan) or vehicles (Veh) were icv infused combined with Ang II-salt. In the 4 th exp, rats were sc infused with aldo (1μg/hr) with saline as drinking fluid. mRNA levels of BDNF were measured by real-time qPCR. In the adrenals, Ang II dose-related increased BDNF mRNA, and Ang II-salt further enhanced BDNF mRNA. High dose Ang II increased BDNF mRNA in the RVLM but not in the PVN. Central blockades markedly decreased Ang II-salt induced BDNF expression in the adrenal cortex. In contrast, saline alone or aldo-saline decreased BDNF mRNA in the adrenal cortex with no effect in the PVN. Central blockades lower Ang II-salt induced BDNF expression in the adrenal, indicating that a central regulatory mechanism plays a role in adrenal BDNF expression. Activation of BDNF by Ang II -modulated by salt-, may provide an important balancing mechanism both centrally and peripherally for Ang II associated hypertension.

Cardiac sympathetic afferent stimulation induces salt-sensitive sympathoexcitation through hypothalamic epithelial Na+ channel activation.

The cardiac sympathetic afferent (CSA), which plays an important role in heart-brain communication for sympathoexcitation, is stimulated in heart failure. Additionally, high salt intake leads to further sympathoexcitation due to activation of hypothalamic epithelial Na(+) channels (ENaCs) in heart failure. In the present study, we stimulated the CSA in adult male mice by epicardial application of capsaicin and using ethanol as a control to determine whether CSA stimulation led to activation of hypothalamic ENaCs, resulting in salt-induced sympathoexcitation. Three days after capsaicin treatment, an upregulation of hypothalamic α-ENaCs, without activation of mineralocorticoid receptors, was observed. We also examined expression levels of the known ENaC activator TNF-α. Hypothalamic TNF-α increased in capsaicin-treated mice, whereas intracerebroventricular infusion of the TNF-α blocker etanercept prevented capsaicin-induced upregulation of α-ENaCs. To examine brain arterial pressure (AP) sensitivity toward Na(+), we performed an intracerebroventricular infusion of high Na(+)-containing (0.2 M) artificial cerebrospinal fluid. AP and heart rate were significantly increased in capsaicin-treated mice compared with control mice. CSA stimulation also caused excitatory responses with high salt intake. Compared with a regular salt diet, the high-salt diet augmented AP, heart rate, and 24-h urinary norepinephrine excretion, which is an indirect marker of sympathetic activity with mineralocorticoid receptor activation, in capsaicin-treated mice but not in ethanol-treated mice. Treatment with etanercept or the ENaC blocker benzamil prevented these salt-induced excitatory responses. In summary, we show that CSA stimulation leads to an upregulation of hypothalamic α-ENaCs mediated via an increase in TNF-α and results in increased salt sensitivity.

Abstract 329: Blood Pressure Variability during High Salt Intake Is Associated with Cardiovascular Events in Patients with Essential Hypertension

Introduction: Accumulating evidence suggests that blood pressure variability (BPV) is a strong predictor of cardiovascular events in patients with hypertension. However, it is unknown whether high salt intake may affect BPV and its predictive value. Method: We analyzed the prevalence of cardiovascular events in 47 patients with essential hypertension in whom systolic BPV was determined during three different 7 day salt intake diets: regular (10g/day NaCl diet), low (3g), and high (20g). Supine blood pressures were measured every hour from 6:00 through 23:00. Mean and standard deviation (SD) in the supine systolic blood pressure (SBP) was used to calculate BPV. Results: SDs in SBP during the regular salt (12.5 ± 4.8 mm Hg) and high salt (12.8 ± 5.4 mm Hg) diets were significantly increased compared with the low salt (10.2 ± 4.0 mm Hg). Coefficients of variation (CV) in SBP for the regular, low, and high salt diets were 8 ± 3%, 7 ± 2% and 8 ± 3%, respectively. Salt loading significantly increased both SD and CV (p < 0.05). During a 22 ± 6 year follow-up period, 25 cardiovascular events were observed including 9 fatal events. ROC analyses revealed SD of SBP during regular salt diet had an AUC = 0.66 (95% CI, 0.53 - 0.74) and high salt diet of AUC = 0.72 (0.58 - 0.79), but the low salt diet was not associated with adverse events. CV of SBP during high salt diet, AUC = 0.72 (0.60 - 0.79), was also significantly associated with events. Kaplan Meier analysis also showed that patients with CV of SBP larger than 8% had significantly greater number of cardiovascular events (p < 0.05). Conclusion: These results suggest that high salt diet increased BPV, thereby leading to increased incidence of cardiovascular diseases.

Abstract MP05: Mineralocorticoid And Angiotensin II Type 1 Receptors In The Subfornical Organ Mediate Angiotensin II Hypertension In Rats

Circulating Ang II increases BP through both peripheral and central mechanisms. Activation of both AT 1 R and MR in the CNS plays a critical role in Ang II-induced hypertension. The subfornical organ (SFO) contains both MR and AT 1 R and can relay the signals of circulating Ang II to downstream nuclei such as the PVN. In this study, we evaluated the effect of intra-SFO infusion of AAV-MR-siRNA or AAV-AT 1a R-siRNA on MR or AT 1 R mRNA expression in the SFO and PVN and Ang II induced hypertension as assessed by telemetry. Two week sc infusion of Ang II at 500 ng/kg/min in Wistar rats on regular salt diet increased BP by 60~65 mmHg. This pressor effect of Ang II was largely prevented by AAV-MR-siRNA or AAV-AT 1 R-siRNA in the SFO (Figure). Ang II increased AT 1 R mRNA expression (1.7± 0.4 vs 0.8 ± 0.04 х10 -1 , p<0.05) in the SFO. Both MR- and AT 1 R-siRNA in the SFO prevented this increase (1.1 ± 0.2х10 -1 and 1.0± 0.3х10 -1 ). In contrast, Ang II decreased MR mRNA expression (3.2 ± 0.2 vs. 4.1 ± 0.2х10 -2 , p<0.05) in the SFO. Both MR- and AT 1 R-siRNA further decreased MR mRNA expression (2.7 ± 0.2 х10 -2 and 2.3 ± 0.3 х10 -2 ). In the PVN, Ang II increased AT 1 R mRNA by ~100% and MR mRNA by ~20%. Only AT 1 R-siRNA in the SFO prevented these increases in the PVN. AAV mediated eGFP fluorescence expression was observed in the SFO but not the PVN. These results suggest that circulating Ang II differentially regulates MR and AT 1 R expression in the SFO. Prevention of Ang II induced AT 1 R up-regulation by knockdown of either AT 1 R or MR suggests a MR dependent regulation of AT 1 R in the SFO. MR-AT 1 R signaling in the SFO appears to play a major role in Ang II-induced hypertension in rats. * p<0.05, vs. baseline values; a: p<0.05, vs. AAV-SCM-siRNA

Abstract 154: Knockdown of Mineralocorticoid or AT 1 Receptor Gene in the Paraventricular Nucleus Prevents Ang II Induced Hypertension in Rats

Circulating Ang II activates an aldosterone-MR-AT 1 R pathway in the hypothalamus. To obtain insights into the actual aldosterone-MR- AT 1 R neuronal pathway, the present study examined whether intra-PVN infusion of AAV-AT 1a R-siRNA or AAV-MR-siRNA prevents the Ang II induced increases in AT 1 R and MR expression and BP. Two weeks sc infusion of Ang II at 500ng/kg/min in Wistar rats on regular salt diet increased BP by telemetry by 66 ± 3 mm Hg, and AT 1 R and MR protein in the SFO, SON and PVN. Intra-PVN AT 1a R-siRNA decreased AT 1 R but not MR expression in the PVN by sc Ang II, and MR-siRNA decreased MR but not AT 1 R expression in the PVN. AT 1 R and MR expression in both SFO and SON were not significantly changed by the two AAV-siRNA. Specific knockdown of AT 1 R or MR in the PVN by AAV-siRNA prevented ~80% of the increase in BP induced by sc Ang II. These results suggest that circulating Ang II up-regulates brain Ang II and aldosterone and increases MR and AT 1 R in the SFO, SON and PVN. Prevention of Ang II induced increases in MR but not AT 1 R by knockdown of MR and vice versa suggests an independent regulation of MR and AT 1 R in the PVN. Both AT 1 R and MR in the PVN play a critical role in Ang II-induced hypertension in rats. * p<0.05 versus AAV-SCM alone. a: p<0.05 versus AAV-SCM + Ang II.

Mineralocorticoid receptors/epithelial Na+ channels in the choroid plexus are involved in hypertensive mechanisms in stroke-prone spontaneously hypertensive rats

Increase in cerebrospinal fluid (CSF) Na(+) concentration ([Na(+)]) precedes hypertension and is a key step in the development of salt-induced hypertension. In the choroid plexus (CP), epithelial Na(+) channels (ENaCs) have an important role in Na(+) transport from the blood into the CSF. However, it remains unknown whether the mineralocorticoid receptors (MR)/ENaCs pathway in the CP of stroke-prone spontaneously hypertensive rats (SHRSP) is involved in neural mechanisms of hypertension. Therefore, we examined the role of the MR/ENaCs pathway in the CP in the development of hypertension in SHRSP associated with an increase in CSF [Na(+)]. As a marker of MR activation, serum/glucocorticoid-inducible kinase 1 (Sgk1) expression levels in the CP were measured and found to be greater in SHRSP than in Wistar-Kyoto (WKY) rats. CSF [Na(+)] levels were also higher in SHRSP than in WKY rats. In SHRSP, high-salt intake (8%) increased blood pressure and urinary norepinephrine excretion compared with those in animals fed a regular salt diet (0.5%) for 2 weeks. Furthermore, the expression levels of MR, Sgk1 and ENaCs in the CP and the increase in CSF [Na(+)] were greater in SHRSP fed a high-salt diet than in those fed a regular salt diet. These alterations were attenuated by intracerebroventricular infusion of eplerenone (10 μg kg(-1) per day), except for α-ENaC and β-ENaC. We conclude that activation of the MR/ENaCs pathway in the CP contributes to hypertension via an increase in CSF [Na(+)], thereby exaggerating salt-induced hypertension with sympathetic hyperactivation in SHRSP.

Mineralocorticoid Receptors Activation in Choroid Plexus and PVN Exaggerates Salt‐Induced Development of Hypertension in Stroke‐Prone Spontaneously Hypertensive Rats

BackgroundIn salt sensitive models, the increases in cerebrospinal fluid Na+ concentration (CSF [Na]) derived from choroid plexus (CP) play a pivotal role in high salt (HS) induced hypertension. Mineralocorticoid receptors (MR) contribute to Na+ handling. Therefore, we investigated whether MR in CP and paraventricular nucleus of hypothalamus (PVN) exaggerates salt‐induced development of hypertension in stroke‐prone spontaneously hypertensive rats (SHRSP).Methods and ResultsSerum glucocorticoid inducible kinase 1 (sgk1) expression levels in CP and PVN were greater in SHRSP than those in Wistar‐Kyoto (WKY) rats (N=6, P<0.05) associated with increases in CSF[Na] (155.6 ± 0.81 vs. 152.8 ± 0.49 mM, P<0.01). The depressor response to intracerebroventricular (ICV) infusion of MR blocker, eplerenone (10 ng) was greater in SHRSP than in WKY (Δarterial pressure (AP) −25.2 ± 1.4 vs. −8.2 ± 1.4 mmHg, N=5, P<0.001). In SHRSP, HS intake (8%) increased AP and urinary norepinephrine excretion (uNE) compared with those by regular salt diet (2 weeks after ΔAP 41.7 ± 3.2 mmHg, ΔuNE 0.24 ± 0.07 μg/day, N=5, P<0.05) associated with increases in MR and sgk1 expression in CP and PVN. These alterations were attenuated by ICV infusion of eplerenone (10 μg/kg/day).ConclusionMR activation in CP might contribute to increases in CSF[Na], thereby exaggerates salt‐induced development of hypertension via MR activation in PVN in SHRSP.