Normal Salt Diet Research Articles

Angiotensin II-stimulated proximal nephron superoxide production and fructose-induced salt-sensitive hypertension.

Angiotensin II (ANG II) increases proximal tubule superoxide (O2-) production more in rats fed a 20% fructose normal-salt diet compared with rats fed a 20% glucose normal-salt diet. A 20% fructose high-salt diet (FHS) increases systolic blood pressure (SBP), whereas a 20% glucose high-salt diet (GHS) does not. However, it is unclear whether FHS enhances ANG II-induced oxidative stress in proximal tubules and whether this contributes to increases in blood pressure in this model. We hypothesized that FHS augments the ability of ANG II to stimulate O2- production by proximal tubules, and this contributes to fructose-induced salt-sensitive hypertension. We measured SBP in male Sprague-Dawley rats fed FHS and GHS and determined the effects of 3 mM tempol and 50 mg/kg losartan for 7 days. We then measured basal and ANG II-stimulated (3.7 × 10-8 M) O2- production by proximal tubule suspensions and the role of protein kinase C. FHS increased SBP by 27 ± 5 mmHg (n = 6, P < 0.006) but GHS did not. Rats fed FHS + tempol and GHS + tempol showed no significant increases in SBP. ANG II increased O2- production by 11 ± 1 relative light units/µg protein/s in proximal tubules from FHS-fed rats (n = 6, P < 0.05) but not in tubules from rats fed GHS. ANG II did not significantly stimulate O2- production by proximal tubules from rats fed FHS + tempol or FHS + losartan. The protein kinase C inhibitor Gö6976 blunted ANG II-stimulated O2- production. In conclusion, FHS enhances the sensitivity of proximal tubule O2- production to ANG II, and this contributes to fructose-induced salt-sensitive hypertension.NEW & NOTEWORTHY A diet containing amounts of fructose consumed by 17 million Americans causes salt-sensitive hypertension. Oxidative stress is an initiating cause of this model of fructose-induced salt-sensitive hypertension increasing blood pressure. This salt-sensitive hypertension is prevented by losartan and thus is angiotensin II (ANG II) dependent. Fructose-induced salt-sensitive hypertension depends on ANG II stimulating oxidative stress in the proximal tubule. A fructose/high-salt diet augments the ability of ANG II to stimulate proximal tubule O2- via protein kinase C.

Abstract 13461: Early Vascular Aging is Associated With Early Cardiac Aging, Upregulation of Left Ventricle Pro-Fibrotic and Inflammatory Genes and Memory Decline in Aged Dahl Salt-Sensitive Rats

Introduction: Blood pressure (BP) begins to increase early in life in Dahl salt-sensitive rats (DSS) that consume a normal salt (NS) diet. Epidemiologic studies have shown that early vascular aging (EVA) is associated with central arterial stiffening (CAS) and hypertension, which develop earlier in life than expected in general population and contribute to cognitive impairment later in life. Hypotheses: (i) DSS rats will develop EVA earlier in life on NS diet, and with advancing age will exhibit early CV aging; (ii) In DSS, CV remodeling and CAS are implicated in memory decline. We tested these hypotheses using a longitudinal repeated measure design in DSS and their parental breed Sprague-Dawley rats (SD) at 3- &amp; 12-mo of age. Methods: Male SD and DSS were kept on NS diet (0.5% NaCl; n=8/group) for entire experiment. Repeated measures of systolic BP (SBP), pulse wave velocity (PWV; an index of CAS), echocardiography, and non-repeated measures of Morris water maze (MWM) to test spatial memory, aortic collagen, elastin (histochemistry), and left ventricle (LV) mRNA expression (qPCR) were assessed at 3- &amp; 12-mo. Data analyses: linear mixed-effect 2-way ANOVA, t-test &amp; linear regression (LR) modeling. A 2-sided p&lt;0.05 was considered significant. Results: SBP, PWV, aortic weight, wall thickness and collagen/elastin ratio were higher in DSS-3 vs. SD-3, and in DSS-12 vs. DSS-3. RWT and LV expression of pro-fibrotic, inflammatory and senescence genes were higher in DSS-12 vs. DSS-3 and vs. SD-12 (Table). DSS-12 spent more time to find a hidden platform in MWM vs. SD-12; their impaired spatial hippocampal memory was positively associated with PWV and LV mass by LR analysis. Conclusions: EVA, indexed as higher BP, PWV and aortic remodeling was associated with early CV aging, manifested by activation of LV senescence, inflammatory and pro-fibrotic genes and by higher rates of age-associated changes in BP, RWT and LV mass in DSS vs. SD. In DSS, early CV aging was associated with memory decline.

Abstract 15250: Increased CD81 is Associated With Enhanced Apoptosis in Acute Kidney Injuries Induced by Candesartan/Low Salt Diet

Introduction: Our previous studies have demonstrated that the upregulation of renal CD81 (cluster of differentiation 81), contributes to the sustained hypertension observed in LPS-preeclampsia rats with kidney injuries. Candesartan (Can) lowers blood pressure by selectively blocking type 1 angiotensin II receptors and results in acute kidney injury (AKI) when used together with dietary sodium restriction. Various cell deaths including apoptosis and necrosis are involved in the pathogenesis of AKI. Hypothesis: Renal CD81 may increased in candesartan/low salt diet(LS+Can) model via activation of apoptosis. Methods: Male SD rats aged 8-10 weeks were fed with LS (0.01% NaCl) or normal salt diets (NS, 0.8% NaCl) (6-10rats/group) respectively for 7 days.All rats were intraperitoneally injected with candesartan (1mg/kg/d) simultaneously.Systolic blood pressure(SBP) was monitored with tail-cuff method.The rats were sacrificed by over-anesthesia after the samples of blood and urine (metabolic cage) were collected.The kidneys were immediately removed for immunoblotting, and TUNEL(TdT-mediated dUTP Nick-End Labeling) measurements.P&lt;0.05 was considered significant for 2 group comparison with t-test. Results: Compared with rats on NS+Can, the serum creatinine (20.3±0.3 vs 50.8±4.2 mmol/L) and BUN(4.05±0.2 vs 18.6±2.5 mmol/L) were more than doubled in LS+Can rats ;Creatinine clearance was decreased (100±4.1 vs 40.8±3.7ml/min); systolic blood pressures was lowered slightly (101.7±1.7 vs 88.9±2.7mmHg, n=7-8). Renal CD81 protein abundance by immunoblotting was increased (258.8±52.4,% of control, n=6/group,p&lt;0.05, same as below).At the same time, there were increases in apoptosis-associated proteins including bax(312.6±71.5),caspase-3(124.6±6) ,cleaved caspase3(133.8±9.6) in Can+LS group.The rate of tunel-positive cells was elevated in the Can+LS group. (100.0±8.5 vs 482.72±59.4,p&lt;0.05,n=10/group).However, necrosis-related inflammatory factors, including Interleukin-6,tumor necrosis factor-α, were not affected in the rat kidneys from LS+Can treated rats. Conclusions: Our findings suggest that Increased CD81 may be associated with enhanced apoptosis in rat kidneys with acute kidney injuries.

Effects of Exercise Training on Mitochondrial Fatty Acid β-Oxidation in the Kidneys of Dahl Salt-Sensitive Rats

Exercise training (Ex) has anti-hypertensive and renal protective effects. In this study, we investigate the effects of Ex on mitochondrial fatty acid metabolism in the kidneys of Dahl salt-sensitive (Dahl-S) rats fed a high-salt (HS) diet. Eight-week-old, male Dahl-S rats were divided into three groups: (1) normal-salt diet, sedentary (NS-Sed), (2) HS diet, sedentary (HS-Sed), and (3) HS-Ex. The NS and HS groups were fed a diet containing 0.6% and 8% NaCl, respectively. The HS-Ex group performed treadmill running for 8 weeks (5 days/week; 60 min/day at 16–20 m/min, 0% gradient). Renal function and the expression of enzymes and regulators of β-oxidation and electron transport chain (ETC) complexes were assessed. HS increased systolic blood pressure and proteinuria, and Ex ameliorated these defects. HS also reduced creatinine clearance, and Ex ameliorated it. HS reduced the renal expression of enzymes of β-oxidation (carnitine palmitoyltransferase type I (CPTI) and acyl-CoA dehydrogenases (CADs)) and the related transcription factors peroxisome proliferator-activated receptor α (PPARα) and PPARγ-coactivator-1α (PGC-1α), and Ex restored this. HS also reduced the renal expression of enzymes in ETC complexes, and Ex restored this expression. Ex ameliorates HS-induced renal damage by upregulating enzymes involved in fatty acid β-oxidation and ETC complexes via increases in PPAR-α and PGC-1α expressions in the kidneys of Dahl-S rats. These results suggest that Ex may have beneficial effects on HS-induced mitochondrial dysfunction in the kidney.

Endothelin mediates sex-differences in acclimation to high salt diet in rats

IntroductionCurrent understanding of sodium (Na+) handling is based on studies done primarily in males. Contrary to the gradual increase in high salt (HS) induced natriuresis over 3–5 days in males, female Sprague Dawley (SD) rats have a robust natriuresis after 1 day of HS. Renal endothelin-1 (ET-1) signaling, through ET receptor A and B, is an important natriuretic pathway and was implicated in our previous dietary salt acclimation studies, however, the contribution of ET receptors to sex-differences in acclimation to dietary Na+ challenges has yet to be clarified. We hypothesized that ET receptors mediate the augmented natriuretic capacity of female rats in response to a HS diet.MethodsTo test our hypothesis, male and female SD rats were implanted with telemeters and randomly assigned to treatment with A-182086, a dual ETA and ETB receptor antagonist, or control. 24-h urine samples were collected and assessed for electrolytes and ET-1. Studies were performed on a normal salt (NS, 0.3% NaCl) diet and after challenging rats with HS (4% NaCl) diet for 1 day.ResultsWe found that A-182086 increased blood pressure in male and female SD rats fed either diet. Importantly, A-182086 eliminated sex-differences in natriuresis on NS and HS. In particular, A-182086 promotes HS-induced natriuresis in male rats rather than attenuating the natriuretic capacity of females. Further, the sex-difference in urinary ET-1 excretion in NS-fed rats was eliminated by A-182086.ConclusionIn conclusion, ET receptors are crucial for mediating sex-difference in the natriuretic capacity primarily through their actions in male rats.

High Salt Diet Exacerbates Intestinal Radiotoxicity by Promoting Intestinal Epithelial Barrier Dysfunction after RT

Intestinal side effects have a substantial impact on patients' quality of life and constrain radiation therapy (RT) of abdominal and pelvic tumors. We aim at gaining a better understanding of the pathogenesis of intestinal radiotoxicity and identifying contributing factors. A high salt diet is common among countries of the Global North. The effects of an increased sodium-chloride (NaCl) intake on the development of intestinal side effects have not been investigated. C57BL/6 wild-type (WT) mice were fed a high salt diet (HSD) or normal salt diet (NSD) and irradiated with 12 Gy total body irradiation (TBI) or 13 Gy abdominal irradiation (ABI). Following TBI the mice received a syngeneic bone marrow transplant to reconstitute hematopoiesis. Readouts after RT included weight monitoring, histopathological analysis and assessment of intestinal epithelial barrier function. To corroborate our findings in vitro, murine organoids of the small intestine were cultivated, treated with varying NaCl concentrations (110-200 mM) and irradiated with 2 or 4 Gy. Organoid survival and growth were determined using multiple methods. Additionally, colony formation assays (CFA) of murine MC38 colon carcinoma cells were performed after stimulation with NaCl. Mechanistically, we analyzed the apoptosis rates of MC38 cells and intestinal epithelial organoids. Irradiated HSD animals showed increased acute weight loss, reduced weight regeneration, a more severe dysfunction of the intestinal barrier (enhanced FITC-dextran permeability) and increased histopathological damage when compared with NSD animals. Murine intestinal organoids and MC38 cells showed decreased survival after RT in hypernatremic conditions. Interestingly, our preliminary data show that there are no increased rates of apoptotic cells in vitro, suggesting a different mechanism affecting the increased radiosensitivity in hypernatremic conditions. Our data show a NaCl induced intensification of intestinal tissue injury following RT. These findings can potentially pave the way for investigating the effect of high salt diet on intestinal radiotoxicity in clinical trials.

Abstract P401: Caveolin-1 And Flotillin-1 Facilitates The SNX19-mediated D 1 R Endocytosis

Sorting nexin 19 (SNX19) belongs to the SNX-PXA-RGS-PXC subfamily of sorting nexins. It controls blood pressure by regulating the endocytosis of dopamine D1 receptor (D 1 R) in renal proximal tubule cells (RPTCs), allowing it to reside in the lipid rafts (LRs) of the plasma membrane. The exclusion of the D 1 R from LRs in RPTCs is associated with hypertension. However, it is unknown if SNX19 interacts with caveolin-1 and flotillin-1, two of the most important LR components. Thus, we studied if SNX19 associates with these two proteins and whether this interaction impacts D 1 R endocytosis. In RPTCs, SNX19 co-immunoprecipitated with caveolin-1 and flotillin-1 in basal conditions. Fenoldopam (FEN, 25nM, 30 min, n=3), a selective D 1 R/D 5 R agonist, increased the co-immunoprecipitation of SNX19 with both caveolin-1 and flotillin-1. The treatment with a D 1 R/D 5 R antagonist, SCH 39166 (SCH, 1 μM, 30 min), prevented the FEN-mediated increase in the co-immunoprecipitation of SNX19 with caveolin-1 and flotillin-1. In RPTCs, the transfection of SNX19 plasmid with deletion of either caveolin-1 (ΔCav1-SNX19) or flotilin-1(ΔFlot1-SNX19) binding domain impaired the FEN-mediated D 1 R endocytosis compared with the cells that were only transfected with wild-type (WT) SNX19 (WT-SNX19, 100.0±5.0%; ΔCav1-SNX19, 39.1±2.8%; ΔFlot1-SNX19, 59.6±3.4%, n=5). Moreover, the relative mRNA expression of SNX19 did not change in RPTCs transfected with WT-SNX19, ΔCav1-SNX19, or ΔFlot1-SNX19. Thus, SNX19 expression does not depend on its caveolin-1 or flotillin-1 binding motif. In C57Bl/6 mice fed with normal salt diet (0.9% NaCl), the renal subcapsular infusion of Snx19 siRNA (3 μg/day,7 days) significantly increased systolic blood pressure (SBP, mmHg) (mock siRNA: 99.3±1.5, n=8; Snx19 siRNA: 114.1±5.4, n=8). The SBP also increased in mice fed with high salt and aggravated by Snx19 siRNA (4% NaCl; mock siRNA: 119.8 ± 5.6, n=4; Snx19 siRNA: 125.6 ±3.4, n=5). Low salt diet decreased SBP that was not affected by Snx19 siRNA (0.04% NaCl, mock siRNA: 92. 0 ± 3.5, n=3; Snx19 siRNA: 82.7 ±6.7, n=3). Germline deletion of Snx19 in C57Bl/6 mice fed with normal salt diet increased SBP. In conclusion, caveolin-1 and flotillin-1 are important for SNX19-mediated D 1 R endocytosis and blood pressure.

Abstract P383: The Renal Mechanisms Involved In The Response To Salt Intake Are Sex Related In Mice.

Dopamine receptor D2 (Drd2) plays an important role in maintaining normal blood pressure (BP) by regulating renal Na + transport. Knockdown of Drd2 in renal proximal tubule cells (RPTCs) may affect BP and Na + transport differently between male and female mice. We studied the effects of Drd2 in RPTCs of the mouse kidney by generating Drd2 fl/fl , P SGLT2 ::Cre + mice (D2R PT-/- ) that lack D2R only in RPTC and Drd2 fl/fl , P SGLT2 ::Cre - (D2R PT+/+ ) mice that do not have the deletion. The study was performed in male and female mice on normal (0.8% NaCl, NS) and low (0.025-0.05% NaCl; LS) salt diets. On NS diet mean arterial pressures (MAPs, mm Hg), measured under pentobarbital anesthesia, were similar in male D2R PT+/+ (86.38±2.20, n=13) and D2R PT-/- (91.58±1.63, n=19) and female D2R PT+/+ (82.57±2.33, n=14) and D2R PT-/- (85.17±6.09, n=12) mice . By contrast, in D2R PT-/- mice, LS diet similarly increased MAP in male (108.11±7.04 (n=10) and female (103.00±9.98 (n=5) mice. The renal NHE3 protein expression (%) was higher in male than female mice (D2R PT+/+ : 100±4 (male) vs 30±15 (female); n=8/group; P&lt;0.0001; D2R PT-/- 117±10 (male) vs 44±16 (female); n=8/group; P&lt;0.02 ). However, NKCC expression (%) was higher in female than male D2R PT+/+ (788±10 (female) vs 100±4 (male), n=8/group; P&lt;0.01); there was no difference between D2R PT-/- male and female mice. The expressions of Na + -K + /ATPase, NCC, SGLT2, and ENaC were similar in males and females of the two genotypes. These findings suggest that on LS diet D2R knockdown in the RPTC increases BP in both male and female mice but the changes in NHE3 and NKCC expressions are different between male and female mice. These results suggests that the MAP response to salt intake is not affected by sex but the renal mechanisms involved are sex-related.

Abstract P3157: MicroRNA-16 Exerts Anti-fibrotic Effects Via Suppression Of Tgfbr3 In Rats With Heart Failure And Preserved Ejection Fraction

Background: Cardiosphere-derived cells (CDCs) reverse diastolic dysfunction by attenuating ventricular fibrosis in rats with heart failure and preserved ejection fraction (HFpEF). MicroRNAs packaged in exosomes secreted from CDCs (CDCexo), are discovered to mediate the beneficial effects of CDCs. Hypothesis: The anti-fibrotic effects of CDCs are mediated by microRNAs contained in CDCexo. Methods: Dahl salt-sensitive rats were fed either a high-salt diet to induce HFpEF, or a normal-salt diet to serve as controls. Echocardiogram-verified HFpEF rats were randomized to receive human CDCexo (n=37) vs. vehicle (n=47) via tail vein weekly for 4 weeks. Control rats (n=16) served as the normal phenotype. RNA sequencing of CDCexo was performed to search for candidate microRNAs. Single-cell RNA sequencing was performed in isolated ventricular cells to investigate the mechanisms of CDCexo. Five most abundant microRNAs were selected and injected via tail vein to HFpEF rats. MicroRNA-injected HFpEF rat hearts underwent single-cell RNA sequencing to investigate its mechanisms. Results: HFpEF rats injected with CDCexo demonstrated improved diastolic function compared to HFpEF rats injected with vehicle (E/E’ ratio 12.1±2.6 vs. 18.7±4.6 in vehicle, p&lt;0.0001). Masson’s trichrome staining revealed reduced ventricular fibrosis in CDCexo rats (3.5±1.4 vs. 5.4±1.4 % in vehicle rats, p=0.016). Negative regulation of transforming growth factor beta (TGF-β) receptor signaling pathway (p=0.002) was identified in isolated cardiac fibroblasts after CDCexo injection. MicroRNA-16 was revealed as the most abundant microRNA in CDCexo, and intravenous delivery of microRNA-16 recapitulated the beneficial responses (improvement of diastolic function and attenuation of ventricular fibrosis) of CDCexo in rats with HFpEF. Single-cell RNA sequencing of microRNA-16-injected HFpEF hearts confirmed down-regulation of TGF-β receptor activity (p=0.018) in cardiac fibroblasts via suppression of Tgfbr3. Conclusion: CDCexo improved diastolic function by suppressing ventricular fibrosis in rats with HFpEF. MicroRNA-16, the most abundant microRNA in CDCexo, recapitulated the beneficial responses of CDCexo in HFpEF rats, via suppression of Tgfbr3 in cardiac fibroblasts.

Aortic smooth muscle TRPV4 channels regulate vasoconstriction in high salt-induced hypertension.

Recent studies have focused on the contribution of vascular endothelial transient receptor potential vanilloid 4 (TRPV4) channels to hypertension. However, in hypertension, TRPV4 channels in vascular smooth muscle remain unexplored. In the present study, we performed wire myograph experiments in isolated aortas from endothelial cell specific TRPV4 channel knockout (TRPV4EC-/-) mice to demonstrate that GSK1016790A (a specific TRPV4 channel agonist) triggered aortic smooth muscle-dependent contractions from mice on a normal-salt diet, and the contractions were enhanced in high-salt diet (HSD) mice. Intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ imaging assays showed that TRPV4-induced [Ca2+]i was significantly higher in aortic smooth muscle cells (ASMCs) from HSD-induced hypertensive mice, and application of an inositol trisphosphate receptor (IP3R) inhibitor markedly attenuated TRPV4-induced [Ca2+]i. IP3R2 expression was enhanced in ASMCs from HSD-induced hypertensive mice and the contractile response induced by TRPV4 was inhibited by the IP3R inhibitor. Whole-transcriptome analysis by RNA-seq and western blot assays revealed the involvement of interferon regulatory factor 7 (IRF7) in TRPV4-IRF7-IP3R2 signaling in HSD-induced hypertension. These results suggested that TRPV4 channels regulate smooth muscle-dependent contractions in high salt-induced hypertension, and this contraction involves increased [Ca2+]i, IP3R2, and IRF7 activity. Our study revealed a considerable effect of TRPV4 channels in smooth muscle-dependent contraction in mice during high-salt induced hypertension.

Anthocyanin attenuates high salt-induced hypertension via inhibiting the hyperactivity of the sympathetic nervous system

ABSTRACT Background Anthocyanin plays a protective role in cardiovascular disease through antioxidant effect. Whether anthocyanin can reduce salt-induced hypertension and the related mechanisms remain unclear. Methods Chronic infusion of vehicle (artificial cerebrospinal fluid, aCSF, 0.4 μL/h) or anthocyanin (10 mg/kg, 0.4 μL/h) into bilateral paraventricular nucleus (PVN) of Sprague-Dawley rats was performed. Then, the rats were fed a high salt diet (8% NaCl, HS) or normal salt diet (0.9%, NaCl, NS) for 4 weeks. Results High salt diet induced an increase in blood pressure and peripheral sympathetic nerve activity (increased LF/HF and decreased SDNN and RMSSD), which was accompanied by increased reactive oxygen species (ROS) production and angiotensin II type-1 receptor (AT1R) expression and function in the PVN. Moreover, the NOD-like receptor protein 3 (NLRP3) and related inflammatory proteins (caspase-1) expression, the pro-inflammatory cytokine levels including IL-1β and TNF-α were higher in PVN of rats with a high salt diet. Bilateral PVN infusion of anthocyanin attenuated NLRP3-dependent inflammation (NLRP3, caspase-1, IL-1β and TNF-α) and ROS production, reduced AT1R expression and function in PVN and lowered peripheral sympathetic nerve activity and blood pressure in rats with salt-induced hypertension. Conclusions Excessive salt intake activates NLRP3-dependent inflammation and oxidative stress and increased AT1R expression and function in the PVN. Bilateral PVN infusion of anthocyanin lowers peripheral sympathetic nerve activity and blood pressure in rats with salt-induced hypertension by improvement of expression and function of AT1R in the PVN through inhibiting NLRP3 related inflammatory and oxidative stress.

Excessive sodium chloride ingestion promotes inflammation and kidney fibrosis in aging mice.

In aging kidneys, a decline of function resulting from extracellular matrix (ECM) deposition and organ fibrosis is regarded as "physiological." Whether a direct link between high salt intake and fibrosis in aging kidney exists autonomously from arterial hypertension is unclear. This study explores kidney intrinsic changes (inflammation, ECM derangement) induced by a high-salt diet (HSD) in a murine model lacking arterial hypertension. The contribution of cold shock Y-box binding protein (YB-1) as a key orchestrator of organ fibrosis to the observed differences is determined by comparison with a knockout strain (Ybx1ΔRosaERT+TX). Comparisons of tissue from mice fed with normal-salt diet (NSD, standard chow) or high-salt diet (HSD, 4% NaCl in chow; 1% NaCl in water) for up to 16 mo revealed that with HSD tubular cell numbers decrease and tubulointerstitial scarring [periodic acid-Schiff (PAS), Masson's trichrome, Sirius red staining] prevails. In Ybx1ΔRosaERT+TX animals tubular cell damage, a loss of cell contacts with profound tubulointerstitial alterations, and tubular cell senescence was seen. A distinct tubulointerstitial distribution of fibrinogen, collagen type VI, and tenascin-C was detected under HSD, transcriptome analyses determined patterns of matrisome regulation. Temporal increase of immune cell infiltration was seen under HSD of wild type, but not Ybx1ΔRosaERT+TX animals. In vitro Ybx1ΔRosaERT+TX bone marrow-derived macrophages exhibited a defect in polarization (IL-4/IL-13) and abrogated response to sodium chloride. Taken together, HSD promotes progressive kidney fibrosis with premature cell aging, ECM deposition, and immune cell recruitment that is exacerbated in Ybx1ΔRosaERT+TX animals.NEW & NOTEWORTHY Short-term experimental studies link excessive sodium ingestion with extracellular matrix accumulation and inflammatory cell recruitment, yet long-term data are scarce. Our findings with a high-salt diet over 16 mo in aging mice pinpoints to a decisive tipping point after 12 mo with tubular stress response, skewed matrisome transcriptome, and immune cell infiltration. Cell senescence was aggravated in knockout animals for cold shock Y-box binding protein (YB-1), suggesting a novel protective protein function.

Intestinal Cckbr-specific knockout mouse as a novel model of salt-sensitive hypertension via sodium over-absorption.

To investigate the value of CCKBRfl/fl villin-Cre mice as a mouse model of salt-sensitive hypertension (SSH). In the first part, 2-month-old CCKBRfl/fl villin-Cre mice (CKO) and control CCKBRfl/fl mice (WT) were fed with normal diet (0.4% NaCl) or high salt diet (4% NaCl), separately for 6 weeks. In the rescue study, one week of hydrochlorothiazide or saline injection were treated with the CKO mice fed high salt diet. The blood pressure, biochemical indexes, and the expression of small intestinal sodium transporters (NHE3, NKCC1, eNaC) was detected. The organ injury markers (MMP2/MMP9) and the histopathological changes of kidneys were observed, whereas the changes of duodenal sodium absorption were detected by small intestinal perfusion in vivo. The CCKBRfl/fl villin-Cre mice with high salt intake exhibited high blood pressure, increased duodenal sodium absorption and urinary sodium excretion, and with renal injury. The protein expression of NHE3, NKCC1 and eNaC were also significant increase in the intestine of CKO-HS mice. Treatment with hydrochlorothiazide remarkably attenuated the elevated blood pressure by high salt absorption in the CCKBRfl/fl villin-Cre mice, but no significant histopathological changes were observed. These results support a crucial role of intestinal Cckbr deficiency on SSH development and the diuretic antihypertension effect in CCKBRfl/fl villin-Cre mice. The CCKBRfl/fl villin-Cre mice with the high salt intake may serve as a stable model of salt-sensitive hypertensive induced by sodium overloading.

Kir7.1 knockdown and inhibition alter renal electrolyte handling but not the development of hypertension in Dahl salt-sensitive rats.

High K+ supplementation is correlated with a lower risk of the composite of death, major cardiovascular events, and ameliorated blood pressure, but the exact mechanisms have not been established. Inwardly rectifying K+ (Kir) channels expressed in the basolateral membrane of the distal nephron play an essential role in maintaining electrolyte homeostasis. Mutations in this channel family have been shown to result in strong disturbances in electrolyte homeostasis, among other symptoms. Kir7.1 is a member of the ATP-regulated subfamily of Kir channels. However, its role in renal ion transport and its effect on blood pressure have yet to be established. Our results indicate the localization of Kir7.1 to the basolateral membrane of aldosterone-sensitive distal nephron cells. To examine the physiological implications of Kir7.1, we generated a knockout of Kir7.1 (Kcnj13) in Dahl salt-sensitive (SS) rats and deployed chronic infusion of a specific Kir7.1 inhibitor, ML418, in the wild-type Dahl SS strain. Knockout of Kcnj13 (Kcnj13-/-) resulted in embryonic lethality. Heterozygous Kcnj13+/- rats revealed an increase in K+ excretion on a normal-salt diet but did not exhibit a difference in blood pressure development or plasma electrolytes after 3 wk of a high-salt diet. Wild-type Dahl SS rats exhibited increased renal Kir7.1 expression when dietary K+ was increased. K+ supplementation also demonstrated that Kcnj13+/- rats excreted more K+ on normal salt. The development of hypertension was not different when rats were challenged with high salt for 3 wk, although Kcnj13+/- rats excrete less Na+. Interestingly, chronic infusion of ML418 significantly increased Na+ and Cl- excretion after 14 days of high salt but did not alter salt-induced hypertension development. Here, we found that reduction of Kir7.1 function, either through genetic ablation or pharmacological inhibition, can influence renal electrolyte excretion but not to a sufficient degree to impact the development of SS hypertension.NEW & NOTEWORTHY To investigate the role of the Kir7.1 channel in salt-sensitive hypertension, its function was examined using complementary genetic and pharmacological approaches. The results revealed that although reducing Kir7.1 expression had some impact on maintaining K+ and Na+ balance, it did not lead to a significant change in the development or magnitude of salt-induced hypertension. Hence, it is probable that Kir7.1 works in conjunction with other basolateral K+ channels to fine-tune membrane potential.

#4375 LANOSTEROL SYNTHASE RS2254524 POLYMORPHISM MODULATES SALT SENSITIVITY AND RENAL EXPRESSION IN A NOVEL MICE MODEL, LSSV643L/V643L

Abstract Background and Aims The blood pressure (BP) response to different salt intakes (salt sensitivity-SS), shows variability among individuals, with more frequency in hypertensives compared to the general population. Elevated levels of the Endogenous Ouabain (EO) have been associated with hypertension (HT) and SS. We characterized the missense variation rs2254524 (Val642Leu; C&amp;gt;A) of the Lanosterol Synthase gene (LSS) coding for an enzyme in steroid biosynthesis, since AA patients on a low-salt diet showed a greater reduction in BP compared to the LSS CC, with only CC having an increase in plasma EO. Moreover, AKI incidence following cardiovascular surgery was greater among those with the LSS rs2254524 AA genotype than in those with the CC genotype. We aim at dissecting the functional correlation between LSS polymorphism, EO, SS-HT, and renal function. Method We generated by CRISPR-Cas9 a knock-in mouse model carrying the Lss V643L (LssV643L/V643L), homologous to V642L to dissect the functional correlation between LSS polymorphism, EO and salt-sensitive hypertension, and kidney expression. Male mice were fed with a normal-salt diet (0.5% NaCl), high-salt diet (4% NaCl), or low-salt diet (&amp;lt;0.03% Na) and BP was measured every two days by the tail-cuff system. Results LssV643L/V643L mice were viable, healthy, and undistinguishable phenotypically from WT. At baseline, the Lss AA affected kidney weight that was significantly enlarged at 3 (p = 0.02) and 12 months of age, compared to WT (kidney p = 0.04; liver p = 0.003). The Lss V643L mutation did not affect EO and SBP at 3 and 12 months, per se, but affects SBP responsiveness to salt intake. Indeed, we observed an increased SBP upon a high-salt diet only in LssV643L/V643L mice 12 months old compared to the control diet (p = 0.01). Moreover, the 12-month-old LssV643L/V643L mice in high-salt diet showed cardiac hypertrophy and showed a higher incidence of heart fibrosis. At 12 months, in the adrenal gland of LssV643L/V643L mice, Lss mRNA level was reduced upon high-salt diet (p = 0.03), while RNA-seq analysis of renal differentially expressed genes revealed a different regulation of multiple Slc genes, both in control and high-salt diet, but also of Cbr2, and Arhgap26. Conclusion The new LssV643L/V643L mouse model resembles the SS-HT phenotype together with EO non-responsiveness observed in HT patients, thus providing a good model of SS-HT. Our results reveal a role of Lss gene in the regulation of BP upon salt stimulus, and its influence at renal level at 3 and 12 months of age.

#5907 RENAL INJURY IN RELATION TO OBESITY AND THE ADDITIVE EFFECT OF HYPERTENSION IN FEMALE AND MALE OBESE AND LEAN ZSF1 RATS

Abstract Background and Aims Chronic kidney disease co-exists with heart failure with preserved ejection fraction (HFpEF), which is characterized by the presence of obesity, hypertension, and diabetes mellitus type 2. Obese ZSF1 rats, a model of HFpEF, exhibit multiple of these comorbidities that can disturb cardiac function. Little attention has been paid to how these comorbidities affect renal disease in the ZSF1 rats. HFpEF is found predominantly in women in whom obesity and hypertension are particularly prevalent. Therefore, we aimed to characterize the renal phenotype in female and male, lean and obese, ZSF1 rats and investigated additional effects of worsened hypertension on disease severity. Method Systolic blood pressure and renal function were assessed biweekly in female and male, lean and obese, ZSF1 rats from 12 to 26 weeks of age by tail-cuff plethysmography, urine collection via metabolic cages and plasma collection, respectively. From 19 weeks of age, rats were implanted with either a deoxycorticosterone acetate pellet (DOCA) and fed high salt diet (6% NaCl) or with a placebo pellet and fed a normal salt diet. At 26 weeks of age, terminal GFR was assessed via inulin clearance under isoflurane. Renal sections were processed for histological analysis. Results Lean and obese placebo ZSF1 rats, both female and male, were mildly hypertensive (systolic blood pressure 140-150 mmHg). All obese rats showed HFpEF, evidenced by elevated E/e’ ratio. In female normoglycemic ZSF1 rats, obesity associates with mild proteinuria, decreased GFR and glomerular hypertrophy. The addition of DOCA-salt worsened hypertension in female obese ZSF1 rats, but not in their lean counterparts. DOCA-salt-worsened hypertension enhanced proteinuria and triggered glomerulosclerosis in female obese rats. Male obese placebo ZSF1 rats were hyperglycemic and showed proteinuria, glomerular hypertrophy and sclerosis, and tubulo-interstitial damage. DOCA-salt worsened hypertension in both male lean and obese rats and further aggravated proteinuria, tubulo-interstitial damage, glomerular hypertrophy and sclerosis in obese male rats. No effect of DOCA-salt was observed on GFR in both male and female ZSF1 rats. Conclusion Female obese ZSF1 rats developed mild renal disease and DOCA-salt-worsened hypertension deteriorated renal function and structure in normoglycemic female obese ZSF1 rats similar to hyperglycemic male obese ZSF1 rats.

SALT SENSITIVITY IS MODULATED BY LANOSTEROL SYNTHASE RS2254524 POLYMORPHISM

Objective: The blood pressure (BP) response to different salt intakes (salt sensitivity-SS), shows variability among individuals, with more frequency in hypertensives compared to the general population. Elevated levels of the Endogenous Ouabain (EO) have been associated with hypertension (HT) and SS. We characterized the missense variation rs2254524 (Val642Leu; C&gt;A) of the Lanosterol Synthase gene (LSS) coding for an enzyme in steroid biosynthesis, since AA patients on a low-salt diet showed a greater reduction in BP compared to the LSS CC, with only CC having an increase in plasma EO. We aim at dissecting the functional correlation between LSS polymorphism, EO and SS-HT. Design and method: We generated by CRISPR-Cas9 a knock-in mouse model carrying the Lss V643L (LssV643L/V643L), homologous to V642L. Male mice were fed with a normal-salt diet (0.5% NaCl), high-salt diet (4% NaCl), or low-salt diet (&lt;0.03% Na) and BP was measured every two days by the tail-cuff system. Results: LssV643L/V643L mice were viable, healthy, and undistinguishable phenotypically from WT. At baseline, the Lss AA affected kidney weight that was significantly enlarged at 3 and 12 months of age, compared to WT. The Lss V643L mutation did not affect EO and SBP at 3 and 12 months, per se, but affects SBP responsiveness to salt stimulus. We observed a decreased SBP after 10 days of a low-salt diet and increased SBP upon a high-salt diet only in LssV643L/V643L mice compared to the control diet at 3 and 12 months, respectively. Moreover, 12-month-old mice in high-salt diet showed cardiac hypertrophy, and LssV643L/V643L showed a higher incidence of heart fibrosis. In the adrenal gland of LssV643L/V643L mice, Lss mRNA level was reduced upon high-salt diet. At 12 months, RNASeq analysis of renal differentially expressed genes revealed a different regulation of multiple Slc genes, both in control and high-salt diet, but also of Cbr2, and Arhgap26. Conclusions: The new LssV643L/V643L mouse model resembles the SS-HT phenotype together with EO non-responsiveness observed in HT patients, thus providing a good model of SS-HT. Our results reveal a role of Lss gene in the regulation of BP upon salt stimulus.

Renal histaminergic system and acute effects of histamine receptor 2 blockade on renal damage in the Dahl salt-sensitive rat.

Histamine is involved in the regulation of immune response, vasodilation, neurotransmission, and gastric acid secretion. Although elevated histamine levels and increased expression of histamine metabolizing enzymes have been reported in renal disease, there is a gap in knowledge regarding the mechanisms of histamine-related pathways in the kidney. We report here that all four histamine receptors as well as enzymes responsible for the metabolism of histamine are expressed in human and rat kidney tissues. In this study, we hypothesized that the histaminergic system plays a role in salt-induced kidney damage in the Dahl salt-sensitive (DSS) rat, a model characterized with inflammation-driven renal lesions. To induce renal damage related to salt sensitivity, DSS rats were challenged with 21 days of a high-salt diet (4% NaCl); normal-salt diet (0.4% NaCl)-fed rats were used as a control. We observed lower histamine decarboxylase and higher histamine N-methyltransferase levels in high-salt diet-fed rats, indicative of a shift in histaminergic tone; metabolomics showed higher histamine and histidine levels in the kidneys of high-salt diet-fed rats, whereas plasma levels for both compounds were lower. Acute systemic inhibition of histamine receptor 2 in the DSS rat revealed that it lowered vasopressin receptor 2 in the kidney. In summary, we established here the existence of the local histaminergic system, revealed a shift in the renal histamine balance during salt-induced kidney damage, and provided evidence that blockage of histamine receptor 2 in the DSS rat affects water balance and urine concentrating mechanisms.NEW & NOTEWORTHY Histamine is a nitrogenous compound crucial for the inflammatory response. The knowledge regarding the renal effects of histamine is very limited. We showed that renal epithelia exhibit expression of the components of the histaminergic system. Furthermore, we revealed that there was a shift in the histaminergic tone in salt-sensitive rats when they were challenged with a high-salt diet. These data support the notion that histamine plays a role in renal epithelial physiological and pathophysiological functions.

Renal injury in relation to obesity and the additive effect of hypertension in female and male obese and lean ZSF1 rats.

Heart failure with preserved ejection fraction (HFpEF) is characterized by obesity, hypertension, diabetes mellitus and chronic kidney disease. Obese ZSF1 rats, a model of HFpEF, exhibit multiple such comorbidities that can disturb cardiac function. Little attention has been paid to how these comorbidities affect renal disease in ZSF1 rats. HFpEF is found predominantly in women in whom obesity and hypertension are particularly prevalent. Therefore, we characterized renal phenotype in female and male, lean and obese, ZSF1 rats and investigated additional effects of worsened hypertension on disease severity. Systolic blood pressure and renal function were assessed biweekly from 12 to 26 weeks. From 19 weeks, rats were implanted with either a deoxycorticosterone acetate pellet and fed high salt diet (DS) or a placebo pellet and fed normal salt diet. At 26 weeks, terminal GFR was assessed via inulin clearance under isoflurane. Renal sections were processed for histological analysis. Lean and obese ZSF1 rats, both female and male, were mildly hypertensive (systolic blood pressure 140-150 mmHg). All obese ZSF1 rats showed HFpEF. In female normoglycemic ZSF1 rats, obesity associates with mild proteinuria, decreased glomerular filtration rate and glomerular hypertrophy. DS-worsened hypertension enhanced proteinuria and triggered glomerulosclerosis. Male obese ZSF1 rats were hyperglycemic and showed proteinuria, glomerular hypertrophy and sclerosis, and tubulo-interstitial damage. DS-worsened hypertension aggravated this phenotype in male ZSF1 rats. In conclusion, female obese ZSF1 rats develop mild renal dysfunction and DS-worsened hypertension compromises renal function and structure in normoglycemic female obese ZSF1 rats as in hyperglycemic male obese ZSF1 rats.

Reduced urine volume and changed renal sphingolipid metabolism in P2ry14-deficient mice.

The UDP-glucose receptor P2RY14, a rhodopsin-like G protein-coupled receptor (GPCR), was previously described as receptor expressed in A-intercalated cells of the mouse kidney. Additionally, we found P2RY14 is abundantly expressed in mouse renal collecting duct principal cells of the papilla and epithelial cells lining the renal papilla. To better understand its physiological function in kidney, we took advantage of a P2ry14 reporter and gene-deficient (KO) mouse strain. Morphometric studies showed that the receptor function contributes to kidney morphology. KO mice had a broader cortex relative to the total kidney area than wild-type (WT) mice. In contrast, the area of the outer stripe of the outer medulla was larger in WT compared to KO mice. Transcriptome comparison of the papilla region of WT and KO mice revealed differences in the gene expression of extracellular matrix proteins (e.g., decorin, fibulin-1, fibulin-7) and proteins involved in sphingolipid metabolism (e.g., small subunit b of the serine palmitoyltransferase) and other related GPCRs (e.g., GPR171). Using mass spectrometry, changes in the sphingolipid composition (e.g., chain length) were detected in the renal papilla of KO mice. At the functional level, we found that KO mice had a reduced urine volume but an unchanged glomerular filtration rate under normal chow and salt diets. Our study revealed P2ry14 as a functionally important GPCR in collecting duct principal cells and cells lining the renal papilla and the possible involvement of P2ry14 in nephroprotection by regulation of decorin.