Model Of Salt-sensitive Hypertension Research Articles

Phillyrin improves myocardial remodeling in salt-sensitive hypertensive mice by reducing endothelin1 signaling.

Prolonged exposure to chronic hypertension places the heart under excessive strain, resulting in myocardial remodeling. Phillyrin, derived from the natural plant Forsythia suspensa, has been found to possess cardioprotective properties. The objective of this study is to investigate the role and mechanism of phillyrin in hypertension-induced myocardial remodeling in mice. We constructed a mouse model of salt-sensitive hypertension. The mice were treated with varying doses of phillyrin, and their blood pressure, cardiac function, cardiac hypertrophy, fibrosis, inflammation, and other conditions were assessed. Our research findings demonstrated that phillyrin has the potential to lower blood pressure, enhance cardiac function, and mitigate cardiac hypertrophy, fibrosis, and inflammatory responses in deoxycorticosterone acetate-salt hypertension mice. In hypertensive mice, there was an elevated expression of endothelin1 (ET-1) in heart tissue, which can be reduced by phillyrin. Additionally, phillyrin effectively reduced the hypertrophy of H9c2 cells induced by ET-1 stimulation. Our research highlights the therapeutic capabilities of phillyrin in the treatment of myocardial remodeling through the reduction of ET-1 signaling. These results contribute to the advancement of novel applications for phillyrin and establish a solid conceptual basis for future investigations in this area.

Sex-specific role for the clock protein PER1 in renal ET-1 regulation in salt-sensitive hypertension

Global knockout (KO) of the clock protein PERIOD1 (PER1) in male, but not female, C57BL/6J mice displayed both blunted blood pressure and urinary endothelin-1 (ET-1) rhythms. More recently, we showed that Dahl salt-sensitive (SS) Per1 KO male rats had exacerbated SS hypertension, alongside increased renal injury, renal ET-1 gene ( Edn1) expression, and ET-1 peptide expression from kidneys collected during the rat’s inactive period (2 PM). Additionally, we see increased long non-coding RNA EDN1-AS, which has been suggested to modulate ET-1 production. However, it is unknown whether PER1 regulates rhythms of ET-1 in SS rats and whether PER1 regulates ET-1 in a sex-dependent way. The study objective was to test the hypothesis that PER1 negatively regulates renal EDN1-AS and Edn1 to modulate ET-1 in a sex- and time-dependent manner. To test this hypothesis, kidneys were collected during rat’s active period (2 AM) from male and female SS Per1 KO and SS control rats on a normal salt (0.4% NaCl) or 3 weeks high salt (4% NaCl) diet ( N=5-6). Renal ET-1 protein levels were measured by ELISA and semi- and quantitative PCR was performed to quantify the levels of EDN1-AS and Edn1. Renal ET-1 levels were significantly higher at 2 AM than our previously reported data at 2 PM in both SS Per1 KO and control male rats ( p<0.0001). Renal ET-1 levels were significantly increased in male SS Per1 KO rats following a high salt diet ( p=0.0362) but not in females ( p=0.9522). This was also reflected in the Edn1 mRNA levels (male, p=0.0005; female, p=0.0814). EDN1-AS levels were also increased in males ( p=0.0123) but not in females. Interestingly, significant sex differences were seen in EDN1-AS levels, where EDN1-AS levels were higher in males than females following both low and high salt diets, suggesting a potential novel sex-dependent mechanism for ET-1 regulation. Together, these data suggest sex-specific action of PER1 in the regulation of ET-1 in a model of salt-sensitive hypertension. Future work aims to investigate blood pressure and renal injury in female SS Per1 KO rats and whether this apparent negative regulation of ET-1 by PER1 contributes to the SS hypertensive and renal injury phenotype seen in male SS Per1 KO rats. This work was supported by the National Institutes of Health grants R35 HL135749 (to A.S.), R01 342 DK109570 (to M.G. and A.S.), and Department of Veteran Affairs grants I01 BX004024 (to A.S.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

544 Interleukin-6 protects renal dysfunction in mouse models of hypertension and salt-sensitive hypertension

OBJECTIVES/GOALS: We are investigating the role of IL-6 in regulating renal function by measuring mean arterial pressure (MAP), renal plasma flow (RPF) and glomerular filtration rate (GFR) in wild type (WT) and IL-6-knockout (KO) mice in established mouse models of angiotensin II (AII)-dependent- hypertension and -salt-sensitive hypertension. METHODS/STUDY POPULATION: Twelve-week-old male WT and KO mice on the C57BL6 background strain were infused with vehicle (V; saline) or angiotensin II (AII; 200 ng/kg/min) for 12-14 days. Half of the AII-treatment groups were maintained on a high salt (HS; 6% NaCl) diet for the duration of the experiment, while the other half of the AII treatment groups and both vehicle groups were fed normal rat chow. MAP was continuously measured by a fluid filled catheter in conscious mice for the duration of the experiment. RPF and GFR were measured on days 12-14 in anesthetized mice by the para-aminohippurate, and fluorescein isothiocynate-Inulin techniques, respectively. All data were analyzed by 2-way ANOVA; *p<0.05 vs. WT, same treatment; #p<0.05 vs.V, same genotype; ^p<0.05, AII vs. AII+HS, same genotype. RESULTS/ANTICIPATED RESULTS: MAP was 31% lower in KO vs WT mice. AII increased MAP (1.2-fold) in WT but not KO mice. HS diet magnified AII-induced increases in MAP in WT and moderately increased MAP in AII-KO mice: [MAP (mmHg): WT+V, 130±7.0; KO+V, 91.0+4.0*; WT+AII, 153±5.0#; KO+AII, 83.0±4.0*; WT+AII+HS, 150±11#; KO+AII+HS, 93.0±4.0#]. AII infusion reduced RPF in the KO but not WT mice. Addition of HS reduced RPF in WT and exacerbated AII-induced reductions in RPF in KO mice [RPF (ml/min/g): WT+V, 1.82±0.23; KO+V, 1.91+0.40; WT+AII, 3.16±0.75#; KO+AII, 1.65±0.42*; WT+AII+HS, 1.10±0.31#^; KO+AII+HS, 1.13±0.XX#^]. The HS diet reduced GFR in AII-infused KO but not WT mice [GFR (µl/min/g): WT+V, 756±XX; KO+V, 788±XX; WT+AII, 1010±63*#; KO+AII, 756±23*; WT+AII+HS, 1100±150#; KO+AII+HS: 540±210*#^]. DISCUSSION/SIGNIFICANCE: The absence of IL-6 in male mice attenuated AII- and/or AII+HS-induced increases in MAP; however, it exacerbated HS-induced reductions in RPF and GFR. These findings suggest inhibiting IL-6 has therapeutic potential as an antihypertensive but not as a renal protective agent in hypertension and salt-sensitive hypertension disease states.

Meningeal interleukin-17-producing T cells mediate cognitive impairment in a mouse model of salt-sensitive hypertension.

Hypertension (HTN), a disease afflicting over one billion individuals worldwide, is a leading cause of cognitive impairment, the mechanisms of which remain poorly understood. In the present study, in a mouse model of HTN, we find that the neurovascular and cognitive dysfunction depends on interleukin (IL)-17, a cytokine elevated in individuals with HTN. However, neither circulating IL-17 nor brain angiotensin signaling can account for the dysfunction. Rather, IL-17 produced by T cells in the dura mater is the mediator released in the cerebrospinal fluid and activating IL-17 receptors on border-associated macrophages (BAMs). Accordingly, depleting BAMs, deleting IL-17 receptor A in brain macrophages or suppressing meningeal T cells rescues cognitive function without attenuating blood pressure elevation, circulating IL-17 or brain angiotensin signaling. Our data unveil a critical role of meningeal T cells and macrophage IL-17 signaling in the neurovascular and cognitive dysfunction in a mouse model of HTN.

Hypoxia-inducible factor-prolyl hydroxylase inhibitor Roxadustat (FG-4592) reduces renal fibrosis in Dahl salt-sensitive rats.

Although hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors have been developed for the treatment of renal anemia, their effects on cardiac and renal dysfunction remain unknown. We previously reported on Dahl salt-sensitive rats, in a rat model of salt-sensitive hypertension, that exhibited anemia and impaired expression of duodenal iron transporters after the development of hypertensive cardiac and renal dysfunction. Therefore, we investigated the effects of Roxadustat (FG-4592), an HIF-PH inhibitor, on anemia, iron regulation, and cardiac and renal dysfunction in Dahl salt-sensitive rats. Six-week-old male Dahl salt-sensitive rats were fed a normal or high-salt diet for 8 weeks. A further subset of Dahl salt-sensitive rats, that were fed a high-salt diet, was administered Roxadustat for 8 weeks. Dahl salt-sensitive rats fed a high-salt diet developed hypertension, cardiac and renal dysfunction, and anemia after 8 weeks of feeding. Roxadustat increased hemoglobin and serum erythropoietin levels in Dahl salt-sensitive rats fed a high-salt diet. With regard to the iron-regulating system, Roxadustat lowered hepatic hepcidin gene expression and increased the gene expression of duodenal iron transporters, such as cytochrome b and divalent metal transporter 1 , in Dahl salt-sensitive rats fed a high-salt diet. Roxadustat did not affect the development of hypertension and cardiac hypertrophy in Dahl salt-sensitive rats with a high-salt diet; however, Roxadustat treatment attenuated renal fibrosis in these rats. Roxadustat ameliorated anemia with affecting the gene expression of the iron-regulating system, and did not affect cardiac hypertrophy but attenuated renal fibrosis in Dahl salt-sensitive rats fed a high-salt diet.

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

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

Abstract 126: Nlrp3 Chromatin Availability And Isolg-adducted Histones In Salt-sensitivity Of Blood Pressure

Salt-sensitivity of blood pressure puts both normotensive and hypertensive individuals at a higher risk for cardiovascular morbidity and mortality. Isolevuglandins (IsoLGs) are increased in multiple oxidative-stress associated diseases, including salt sensitive hypertension. Scavenging of IsoLGs using 2-HOBA in a mouse model of salt-sensitive hypertension blocked NLRP3 inflammasome in dendritic cells (DCs) and abolished salt-induced hypertensive response. IsoLGs adduct to histones, specifically H4. We tested the hypothesis that elevated sodium in antigen-presenting cells leads to IsoLG-adducted H4 histones and increased chromatin availability to NLRP3 inflammasome components contributing to salt-sensitive hypertension. In vivo studies were performed on 19 hypertensive subjects enrolled in an acute inpatient protocol to assess response to salt loading (460 mmoL/24 hrs) and salt depletion (10 mmoL/24 hrs, and furosemide 40 mg x 3). Isolated PBMCs from salt sensitive subject (δ;SBP SD-SL = -12.9) and a salt resistant subject (δ;SBP SD-SL = 0.35) were analyzed by ATACseq. In vitro studies were performed on isolated CD11c+ dendritic cells treated with either normal salt (150mM) or high salt (190mM) and histone protein extraction isolated for western blot and blotted for D11, an ScFv specific to IsoLG-adducted proteins. Additional in vitro studies were performed on isolated monocytes from 11 individuals treated with either normal or high salt and submitted for RNA sequencing. RNAseq data of isolated monocytes demonstrated an overall upregulation in differentially expressed histone modification genes after high salt visualized in a heatmap and PCA plot, specifically, upregulation of gene HIST4H4 encoding the Histone H4 (25.4 ± 4.6 vs 65.5 ± 6.5, [n=11, q=1.5 x 10 -3 ]). Changes in chromatin availability inflammasome component NLRP3 were more salt responsive in DCs of the salt-sensitive subject versus the salt-resistant subject after salt depletion. Additionally, high salt increased IsoLG-histone adduction in CD11c+ DCs. Our results suggest the high sodium environment increase in IsoLG-adducts may epigenetically regulate NLRP3 inflammasome components in SSBP.

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.

κ-Opioid Receptors Improve Vascular Endothelial Dysfunction in Salt-Sensitive Hypertension via PI3K/Akt/eNOS Signaling Pathway.

κ-Opioid receptors (κ-OR) are widely used to regulate the activity of the cardiovascular system. To explore the effect and mechanism of κ-OR on salt-sensitive hypertensive endothelial dysfunction, we used Dah1 rats to construct a rat model of salt-sensitive hypertension on a high-salt (HS) diet. Then, the rats were treated with κ-OR activators U50,488H (1.25 mg/kg) and inhibitor nor-BNI (2.0 mg/kg) for 4 weeks, respectively. The rat aortas were collected to detect the contents of NO, ET-1, AngII, NOS, T-AOC, SO, and NT. Protein expression was determined for NOS, Akt, and Caveolin-1. In addition, the vascular endothelial cells were extracted, and the levels of NO, TNF-α, IL-1, IL-6, IL-8, IL-10, p-Akt, and p-eNOS in cell supernatants were detected. In vivo results showed that compared with the HS group, treated with U50,488H promoted rats' vasodilation by increasing the NO content and decreasing ET-1 and AngII contents. U50,488H reduced endothelial cell apoptosis and attenuated vascular, smooth muscle cell and endothelial cell injury. U50,488H also enhanced the rats' response to oxidative stress by increasing the NOS and T-AOC contents. Moreover, U50,488H increased the eNOS, p-eNOS, Akt, and p-AKT expression and decreased the iNOS and Caveolin-1 expression. In vitro results showed that U50,488H promoted NO, IL-10, p-Akt, and p-eNOS levels in endothelial cell supernatants versus the HS group. And U50,488H reduced the adhesion of peripheral blood mononuclear cells and polymorphonuclear neutrophils to endothelial cells and the migration function of polymorphonuclear neutrophils. Our study suggested that κ-OR activation may improve vascular endothelial dysfunction in salt-sensitive hypertensive rats through the PI3K/Akt/eNOS signaling pathway. This may be a potential therapeutic approach in the treatment of hypertension.

S-18-3: SGLT2 INHIBITOR CANAGLIFLOZIN ANTAGONIZES SALT-SENSITIVE HYPERTENSION THROUGH MODIFYING TRPC3 MEDIATED VASCULAR CALCIUM HANDLING

Objectiv: Salt-sensitive hypertension is highly prevalent and associated with cardiorenal damage. Large clinical trials have demonstrated that Sodium-glucose cotransporter 2 (SGLT2) inhibitors exert hypotensive effect and cardiorenal protective benefits in hypertensive patients with and without diabetes. However, the underlying mechanism remains elusive. Methods: Dahl salt-sensitive (SS) rats and salt-insensitive controls were fed with 8% high salt diet to establish salt-sensitive hypertensive model, some of them were treated with canagliflozin. The blood pressure, urinary sodium excretion, vascular function and remodeling were detected. Rat aortic proteomics and TRPC3 knockout mice were used to explain the mechanism of the effect of canagliflozin on salt-sensitive hypertension. Results: Canagliflozin treatment significantly reduced high salt induced hypertension and this effect was not totally dependent on urinary sodium excretion in SS hypertensive rats. Assay of vascular function and proteomics showed that canagliflozin significantly inhibited vascular cytoplasmic calcium increase and vasoconstriction in response to high salt diet. High salt intake increased vascular expression of TRPC3 in salt-sensitive rats, which could be alleviated by canagliflozin treatment. Overexpression of TRPC3 mimicked salt-induced vascular cytosolic calcium increase in vitro and knockout of TRPC3 erased the anti-hypertensive effect of canagliflozin. Mechanistically, high-salt-induced activation of sodium/calcium exchanger 1 (NCX1) reverse mode increased cytoplasmic calcium level and vasoconstriction, which required TRPC3, and this process could be blocked by canagliflozin. Conclusions: We define a previously unrecognized role of TRPC3/NCX1 mediated vascular calcium dysfunction in the development of high salt induced hypertension, which can be improved by canagliflozin treatment. This pathway is potentially a novel therapeutic target to antagonize salt sensitive hypertension.

Stroke-prone salt-sensitive spontaneously hypertensive rats show higher susceptibility to spreading depolarization (SD) and altered hemodynamic responses to SD

Spreading depolarization (SD) occurs in a plethora of clinical conditions including migraine aura, delayed ischemia after subarachnoid hemorrhage and malignant hemispheric stroke. It describes waves of near-breakdown of ion homeostasis, particularly Na+ homeostasis in brain gray matter. SD induces tone alterations in resistance vessels, causing either hyperperfusion in healthy tissue; or hypoperfusion (inverse hemodynamic response = spreading ischemia) in tissue at risk. Observations from mice with genetic dysfunction of the ATP1A2-encoded α2-isoform of Na+/K+-ATPase (α2NaKA) suggest a mechanistic link between (1) SD, (2) vascular dysfunction, and (3) salt-sensitive hypertension via α2NaKA. Thus, α2NaKA-dysfunctional mice are more susceptible to SD and show a shift toward more inverse hemodynamic responses. α2NaKA-dysfunctional patients suffer from familial hemiplegic migraine type 2, a Mendelian model disease of SD. α2NaKA-dysfunctional mice are also a genetic model of salt-sensitive hypertension. To determine whether SD thresholds and hemodynamic responses are also altered in other genetic models of salt-sensitive hypertension, we examined these variables in stroke-prone spontaneously hypertensive rats (SHRsp). Compared with Wistar Kyoto control rats, we found in SHRsp that electrical SD threshold was significantly reduced, propagation speed was increased, and inverse hemodynamic responses were prolonged. These results may have relevance to both migraine with aura and stroke.

Genetic Engineering of Lymphangiogenesis in Skin Does Not Affect Blood Pressure in Mouse Models of Salt-Sensitive Hypertension.

Recent studies have indicated that sodium storage is influenced by macrophages that secrete VEGF-C (vascular endothelial growth factor) during salt stress thus stimulating lymphangiogenesis, thereby acting as a buffer against increased blood pressure (BP). We aimed to explore the role of dermal lymphatics in BP and sodium homeostasis. Our hypothesis was that mice with reduced dermal lymphatic vessels were more prone to develop salt-sensitive hypertension, and that mice with hyperplastic vessels were protected. Mice with either hypoplastic (Chy), absent (K14-VEGFR3 [vascular endothelial growth factor receptor 3]-Ig), or hyperplastic (K14-VEGF-C) dermal lymphatic vessels and littermate controls were given high-salt diet (4% NaCl in the chow), deoxycorticosterone acetate (DOCA)-salt diet and 1% saline to drink or nitric oxide blocker diet L-NG-nitro arginine methyl ester (followed by high salt diet). BP was measured by telemetric recording, and tissue sodium content by ion chromatography. In contrast to previous studies, high salt diet did not induce an increase in BP or sodium storage in any of the mouse strains investigated. DOCA-salt, on the other hand, gave an increase in BP in Chy and K14-VEGFR3-Ig not different from their corresponding WT controls. DOCA induced salt storage in skin and muscle, but to the same extent in mice with dysfunctional lymphatic vessels and WT controls. Lymph flow as assessed by tracer washout was not affected by the diet in any of the mouse strains. Our results suggest that dermal lymphatic vessels are not involved in salt storage or blood pressure regulation in these mouse models of salt-sensitive hypertension.

ARRB2 (β-Arrestin-2) Deficiency Alters Fluid Homeostasis and Blood Pressure Regulation

GPCRs (G protein-coupled receptors) are implicated in blood pressure (BP) and fluid intake regulation. There is a developing concept that these effects are mediated by both canonical G protein signaling and noncanonical β-arrestin mediated signaling, but the contributions of each remain largely unexplored. Here, we hypothesized that β-arrestin contributes to fluid homeostasis and blood pressure (BP) regulation in deoxycorticosterone acetate (DOCA) salt hypertension, a prototypical model of salt-sensitive hypertension. Global β-arrestin1 (Arrb1) and β-arrestin2 (Arrb2) knockout mice were employed to evaluate drinking behavior, and BP was evaluated in Arrb2-knockout mice. Age- and sex-matched C57BL/6 mice served as controls. We measured intake of water and different sodium chloride solutions and BP employing a 2-bottle choice paradigm with and without DOCA. Without DOCA (baseline), Arrb2-knockout mice exhibited a significant elevation in saline intake with no change in water intake. With DOCA treatment, Arrb2-knockout mice exhibited a significant increase in both saline and water intake. Although Arrb2-knockout mice exhibited hypernatremia at baseline conditions, we did not find significant changes in total body sodium stores or sodium palatability. In a separate cohort, BP was measured via telemetry in Arrb2-knockout and C57BL/6 mice with and without DOCA. Arrb2-knockout did not exhibit significant differences in BP before DOCA treatment when provided water alone, or when provided a choice of water and saline. However, Arrb2-knockout exhibited an increased pressor response to DOCA-salt. These findings suggest that in salt-sensitive hypertension, ARRB2, but not ARRB1 (β-arrestin 1), might counterbalance the canonical signaling of GPCRs.

Cardioprotective Role for Paraoxonase-1 in Chronic Kidney Disease.

Paraoxonase-1 (PON-1) is a hydrolytic enzyme associated with HDL, contributing to its anti-inflammatory, antioxidant, and anti-atherogenic properties. Deficiencies in PON-1 activity result in oxidative stress and detrimental clinical outcomes in the context of chronic kidney disease (CKD). However, it is unclear if a decrease in PON-1 activity is mechanistically linked to adverse cardiovascular events in CKD. We investigated the hypothesis that PON-1 is cardioprotective in a Dahl salt-sensitive model of hypertensive renal disease. Experiments were performed on control Dahl salt-sensitive rats (SSMcwi, hereafter designated SS-WT rats) and mutant PON-1 rats (SS-Pon1em1Mcwi, hereafter designated SS-PON-1 KO rats) generated using CRISPR gene editing technology. Age-matched 10-week-old SS and SS-PON-1 KO male rats were maintained on high-salt diets (8% NaCl) for five weeks to induce hypertensive renal disease. Echocardiography showed that SS-PON-1 KO rats but not SS-WT rats developed compensated left ventricular hypertrophy after only 4 weeks on the high-salt diet. RT-PCR analysis demonstrated a significant increase in the expression of genes linked to cardiac hypertrophy, inflammation, and fibrosis, as well as a significant decrease in genes essential to left ventricular function in SS-PON-1 KO rats compared to SS-WT rats. A histological examination also revealed a significant increase in cardiac fibrosis and immune cell infiltration in SS-PON-1 KO rats, consistent with their cardiac hypertrophy phenotype. Our data suggest that a loss of PON-1 in the salt-sensitive hypertensive model of CKD leads to increased cardiac inflammation and fibrosis as well as a molecular and functional cardiac phenotype consistent with compensated left ventricular hypertrophy.

Abstract 053: Increased Arterial Pressure Precedes Renal Dysfunction And Elevated Renal Sympathetic And Afferent Nerve Activity In Deoxycorticosterone-Salt Rat

Hypertension (HTN) is a primary risk factor for cardiovascular disease - the leading cause of death worldwide. HTN is multifaceted but a key factor is aberrant autonomic signaling, including renal sympathetic (RSNA) and afferent nerve activity (ARNA). ARNA is elevated in several models of HTN and renal afferent nerve ablation can mitigate the HTN. It is unknown if ARNA rises before changes in blood pressure (BP) or renal function in HTN. This study aimed to determine when renal nerve activity changes vis a vis BP and renal function in a model of salt sensitive HTN. We hypothesized increased ARNA and RSNA would coincide with a rise in BP and decline in renal function. To test this hypothesis, 24 uninephrectomized male Sprague Dawley rats (300g) received a deoxycorticosterone acetate (DOCA; 100mg, SC) implant and 0.9% NaCl drinking water. Renal function (serum creatinine; Cr) and systolic BP (SBP; tail cuff) were measured weekly. Animals were randomly allocated to acute renal nerve recording on protocol day (D) 0, 7, 14, or 21. Multiunit RSNA and ARNA were recorded serially, as ARNA was isolated after sectioning proximal side. RSNA is expressed as bursts/heartbeat, ARNA and expressed as %ARNA max (response to intrapelvic capsaicin). Data were analyzed by one-way ANOVA with Bonferroni post hoc. Data presented as mean±SEM; *p<.05 vs D0. SBP (mmHg) progressively increased on D7 (*152 ± 3), D14 (*171 ± 3), and D21 (*197 ± 7) vs D0 (138 ± 3). ARNA (%) increased at D14 (*23.0 ± 4.7) and D21 (*34.2 ± 4.4) vs D0 (10.1 ± 0.8), but no changes were detected at D7 (13.4 ± 2.1). No changes in RSNA from D0 were detected. Cr (mg/dL) increased at D14 (*1.2 ± 0.1) and D21 (*1.2 ± 0.1) vs D0 (0.9 ± 0.0), indicating a decline in kidney function. These data do not support the hypothesis that ARNA and RSNA increase in parallel to increasing BP. Rather, BP appears to initially increase independent of detectable changes in RSNA, ARNA or renal dysfunction. Interestingly, renal dysfunction and elevated ARNA were detected on D14, which coincides with our previous report that renal inflammation is first detected at D14. These data do not support a causal role of ARNA in this model. Rather, ARNA may subsequently drive disease progression. Further studies on the cause of increased ARNA and inflammation interaction are underway.

Abstract P320: Transgenic Cre Drivers For Harvesting Podocytes And Endothelial Cells From Dahl Salt-sensitive Rats

Complex phenotypes such as blood pressure regulation involve multiple organ systems, tissues, and specific cell types. Transgenic rodent models with the expression of fluorescent proteins under the control of tissue- and cell-type-specific promoters are powerful tools for identifying and isolating specific cells but have been primarily limited to mice. Our long-term goal is to establish a Cre-driver rat resource for conditional and physiologically predictive rat models of human diseases. We developed two Cre-driver transgenic strains of rats (SS/PodocinCreERT2 and SS/VECadCre) capable of activating a second Cre-sensitive tdTomato reporter transgene integrated into the rat Rosa26 locus in the Dahl salt-sensitive hypertension model background. Cells with reporter activation were identified under fluorescence microscopy or harvested using fluorescence-activated cell sorting (FACS). A tamoxifen-inducible Cre transgene under the control of the Nphs2 (podocin) gene promoter was used to harvest fluorescently labeled podocytes after tamoxifen injection. A constitutive Cre under the control of the Cdh5 (VE-Cadherin) gene promoter was used for the isolation of endothelial cells. Correspondingly, tens to hundreds of thousands of podocytes or endothelial cells were isolated from a single kidney or mesenteric artery. Isolated cells were enriched for specific marker genes and could be used in downstream assays including single-cell transcriptional profiling. These new Cre/reporter dual transgenic rat models provide a much-needed resource for the biomedical research community to study the role of these cell types in a broad range of health and disease modeling studies.

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

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

SGLT2 inhibition effect on salt-induced hypertension, RAAS, and Na+ transport in Dahl SS rats.

Na+-glucose cotransporter-2 (SGLT2) inhibitors are the new mainstay of treatment for diabetes mellitus and cardiovascular diseases. Despite the remarkable benefits, the molecular mechanisms mediating the effects of SGLT2 inhibitors on water and electrolyte balance are incompletely understood. The goal of this study was to determine whether SGLT2 inhibition alters blood pressure and kidney function via affecting the renin-angiotensin-aldosterone system (RAAS) and Na+ channels/transporters along the nephron in Dahl salt-sensitive rats, a model of salt-induced hypertension. Administration of dapagliflozin (Dapa) at 2 mg/kg/day via drinking water for 3 wk blunted the development of salt-induced hypertension as evidenced by lower blood pressure and a left shift of the pressure natriuresis curve. Urinary flow rate, glucose excretion, and Na+- and Cl--to-creatinine ratios increased in Dapa-treated compared with vehicle-treated rats. To define the contribution of the RAAS, we measured various hormones. Despite apparent effects on Na+- and Cl--to-creatinine ratios, Dapa treatment did not affect RAAS metabolites. Subsequently, we assessed the effects of Dapa on renal Na+ channels and transporters using RT-PCR, Western blot analysis, and patch clamp. Neither mRNA nor protein expression levels of renal transporters (SGLT2, Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter 2, Na+-Cl- cotransporter, and α-, β-, and γ-epithelial Na+ channel subunits) changed significantly between groups. Furthermore, electrophysiological experiments did not reveal any difference in Dapa treatment on the conductance and activity of epithelial Na+ channels. Our data suggest that SGLT2 inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in the RAAS or the expression or activity of the main Na+ channels and transporters.NEW & NOTEWORTHY The present study indicates that Na+-glucose cotransporter-2 (SGLT2) inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development and magnitude of salt-induced hypertension. Chronic inhibition of SGLT2 increases glucose and Na+ excretion without secondary effects on the expression and function of other Na+ transporters and channels along the nephron and hormone levels in the renin-angiotensin-aldosterone system. These data provide novel insights into the effects of SGLT2 inhibitors and their potential use in hypertension.

Kidney-specific KO of the circadian clock protein PER1 alters renal Na+ handling, aldosterone levels, and kidney/adrenal gene expression.

PERIOD 1 (PER1) is a circadian clock transcription factor that is regulated by aldosterone, a hormone that increases blood volume and Na+ retention to increase blood pressure. Male global Per1 knockout (KO) mice develop reduced night/day differences in Na+ excretion in response to a high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP), a model of salt-sensitive hypertension. In addition, global Per1 KO mice exhibit higher aldosterone levels on a normal-salt diet. To determine the role of Per1 in the kidney, male kidney-specific Per1 KO (KS-Per1 KO) mice were generated using Ksp-cadherin Cre recombinase to remove exons 2-8 of Per1 in the distal nephron and collecting duct. Male KS-Per1 KO mice have increased Na+ retention but have normal diurnal differences in Na+ excretion in response to HS + DOCP. The increased Na+ retention is associated with altered expression of glucocorticoid and mineralocorticoid receptors, increased serum aldosterone, and increased medullary endothelin-1 compared with control mice. Adrenal gland gene expression analysis revealed that circadian clock and aldosterone synthesis genes have altered expression in KS-Per1 KO mice compared with control mice. These results emphasize the importance of the circadian clock not only in maintaining rhythms of physiological functions but also for adaptability in response to environmental cues, such as HS + DOCP, to maintain overall homeostasis. Given the prevalence of salt-sensitive hypertension in the general population, these findings have important implications for our understanding of how circadian clock proteins regulate homeostasis.NEW & NOTEWORTHY For the first time, we show that knockout of the circadian clock transcription factor PERIOD 1 using kidney-specific cadherin Cre results in increased renal Na+ reabsorption, increased aldosterone levels, and changes in gene expression in both the kidney and adrenal gland. Diurnal changes in renal Na+ excretion were not observed, demonstrating that the clock protein PER1 in the kidney is important for maintaining homeostasis and that this effect may be independent of time of day.

Abstract P203: Time-restricted Feeding Attenuates Hypertension In Mice

Hypertension is associated with inflammation and decreased kidney function. Studies of cardiovascular function have observed that time-restricted feeding (TRF), a form of intermittent fasting, is associated with decreased blood pressure, decreased inflammation, and improved kidney function. We hypothesized that implementation of a time-restricted feeding protocol in hypertensive mice would decrease systolic blood pressure and increase kidney function. C57BL6/J mice were randomly assigned to either an L-arginine methyl ester hydrochloride (LNAME)-induced hypertension (LHTN) model, where they received LNAME in their drinking water, or a salt-sensitive hypertension (SSHTN) model, where they received a 4% high salt diet following LNAME priming and a washout period. Two days following introduction of LNAME or the high salt diet, mice were either provided food ad libitum or placed on a 12-hour TRF protocol, where they were only allowed to eat from 8PM to 8AM. Hypertensive mice receiving TRF treatment displayed a significantly decreased systolic blood pressure (SBP) after 4 weeks when compared to the control hypertensive groups (LHTN SBP: 164±1 vs. 149±2 mmHg, p<0.001; SSHTN SBP: 139±1 vs. 130±1 mmHg, p<0.001). When kidney function was examined in the LHTN group, TRF mice had decreased serum creatinine (Sc) levels along with decreased fractional excretion of sodium (FENa) when compared to their respective control mice (Sc: 0.19±0.01 vs. 0.13±0.01 mg/dL, p=0.007; FENa: 0.59±0.05 vs. 0.32±0.05 %, p=0.009). Glomerular filtration rate was significantly increased in TRF treated LHTN mice. Overall, these data indicate that TRF treatment reduces blood pressure in hypertensive mice, which is associated with an improvement in renal function. These findings could establish TRF as a potential therapeutic option for hypertensive patients.