Salt-sensitive Rats Research Articles

Impact of bedding on Dahl salt-sensitive hypertension and renal damage.

Salt-sensitive hypertension, increases in blood pressure in response to increased salt intake, is associated with an increased risk of morbidity, mortality, and end-organ damage compared with salt-resistant hypertension. The Dahl salt-sensitive (SS) rat mimics the phenotypic characteristics observed in human hypertension when rats are challenged with a high-salt diet. Our previous work demonstrated that environmental factors, such as dietary protein, alter the severity of salt sensitivity in Dahl SS rats and should be an important consideration in experimental design. The present study investigated how the bedding on which animals were maintained (wood vs. corncob) could impact the SS phenotype in the Dahl SS rat. Animals that were maintained on corncob bedding exhibited a significant attenuation in blood pressure and renal end-organ damage in response to a high-salt diet compared with animals maintained on wood bedding. This attenuation was associated with an improvement in renal function and reduction in immune cell infiltration into the kidneys of Dahl SS rats maintained on corncob bedding. These results indicate that the type of bedding impacts the SS phenotype in the Dahl SS rat and that the bedding used in experiments can be a confounding factor to consider during data interpretation and experimental design.NEW & NOTEWORTHY Results from our present study demonstrate the profound effect of animal bedding on the severity of salt-sensitive hypertension, renal damage, and inflammation in Dahl salt-sensitive rats. This study highlights the important consideration that should be given to environmental factors, namely, the type of bedding in animal facilities, in experimental design and data interpretation.

EDITORIAL: Mitochondrial Function and Blood Pressure Regulation: From Bioenergetics to Pathophysiology

The mitochondrion is the powerhouse of all living cells as it provides the energy needed to maintain obligatory regulatory functions.1 The generation of adenosine triphosphate (ATP) via oxidative phosphorylation underlies the principal role of the mitochondrion in cell survival. Aside this basic contribution to energy generation, the mitochondria has been established to regulate cell death (apoptosis), redox and ion signaling.2 The crosstalk between redox signaling and a myriad of pathological disorders created a nexus between the mitochondrion and the cardiorenal system.3,4 Similarly, the high distribution of mitochondria in organs of the cardiorenal system, meant that these organs such as the kidney, are subject to the effect of mitochondria-induced alterations in redox signaling.5 For instance, mitochondrial dysfunction has been linked to the pathophysiology of kidney disorders.6 Considering the intricate link between the kidneys and blood pressure regulation, mitochondrial dysfunction was suggested to contribute significantly to distortions in renal control of blood pressure. Recently, it was reported that the tricarboxylic acid (TCA) cycle plays a role in the etiology of genetic hypertension.7 This novel iscovery linked the activity of the TCA cycle enzyme, fumarase to a reduction in nitric oxide production and an upregulation in redox signaling in the renal medulla of salt-sensitive rats.7,8 In these animals, an innate mutation in the fumarase enzyme, reduced its activity and increased cellular levels of its substrate, fumarate. Hence, the role of these TCA cycle intermediaries was shifted from being ‘mere’ participants in the generation of energy to endogenous ligands with biochemical targets that alter renal function and by extension, blood pressure. Furthermore, fumarate was shown to reduce blood pressure and modulate the expression of genes that ameliorated hypertension induced renal damage in deoxycorticosterone acetate (DOCA) hypertension, a non-genetic form of hypertension.9 Subsequently, succinate, the upstream product of fumarate was reported to directly stimulate GPR91 receptors to increase blood pressure.10 These actions of fumarate and its intermediaries, exceed the renal system as reports have shown a cardioprotective role via upregulation of nuclear erythroid factor-2 (Nrf2).11 Fumarate is now known to regulate the expression of genes such as hypoxia inducible factor (HIF-1), transforming growth factor (TGF-β), kidney injury molecule (KIM-1) amongst others. What is evident from the foregoing is that the mitochondrion is no longer just an idle energy-generating center. It is now listed as a probable etiology in hypertension, and this has opened new vistas of possibilities as it relates to the pathophysiology of hypertension.8 Is it possible that these intermediaries are involved in the physiological control of blood pressure? Could they also be exerting direct vasoactive effects? Is it likely that they may be modulating the expression of genes that underlie vascular/organ remodeling? And finally, is it possible that mitochondrial dysfunction could partly explain the etiology of idiopathic hypertension? As, far reaching as these insights may be, it is not completely out of place to be optimistic as the foray into these previously uncharted areas of mitochondrial metabolism progress. What is very clear is that there is now a paradigm shift in the function of the mitochondria in blood pressure regulation from that of a bioenergetic center to pathophysiological axis which contributes significantly to the etiology of hypertension.

Abstract P225: Dahl Salt-sensitive And Salt-resistant Rats Exhibit Highly Disparate Blood Pressure Responses To Lifetime High Fat Diet Feeding

In an effort to understand obesity associated hypertension, we and others are using Dahl salt-sensitive (SS) rats fed a high fat diet as an experimental model. One reason for choosing this rat strain is that it is well-known that SS rats exhibit accelerated hypertension development not just in response to high salt intake but to numerous other stimuli as well, when compared to Dahl salt resistant (SR) rats. However, it is not known whether this general dichotomous sensitivity to hypertension development in SS and SR rats also extends to the impact of high fat feeding. Therefore, in the present study we tested the hypothesis that high fat feeding would cause a smaller increase in blood pressure in SR rats versus SS rats in both females and males (n=8 each). To explore possible strain differences in the mechanisms known to contribute to obesity associated hypertension, we also compared the activity of the renin angiotensin and sympathetic nervous systems in SS and SR rats. Rats were fed a high fat diet (60% kcal fat) from weaning. Blood pressure was measured by telemetry starting at 12 weeks of age. At 12 weeks, mean arterial pressures (MAP, mmHg) were 109±8 and 158±6 in male SR and SS rats, and 108±1 and 150±5 in female SR and SS rats, respectively. By 18 weeks MAP were 109±7 and 175±4 in male SR and SS rats, and 108±1 and 180±4 in female SR and SS rats, respectively. In males, plasma renin was lower, and aldosterone the same, in SS rats than SR rats. However, SS rats showed larger falls in blood pressure during week-long ACE inhibitor treatment and to acute sympathoinhibition with hexamethonium than did SR rats. In females, plasma renin and aldosterone both were higher in SS rats than SR rats. Female SS rats also showed larger falls in blood pressure to chronic ACE inhibition and acute ganglion blockade than did SR rats. These results confirm that both male and female SR rats are highly resistant to obesity associated hypertension, and that less robust activation by high fat diet of the renin angiotensin and sympathetic nervous systems may be a causative factor.

Abstract P224: Perivascular Adipose Tissue May Contribute To Disparate Blood Pressure Responses To Lifetime High Fat Feeding In Dahl Salt-sensitive And Salt-resistant Rats

Dahl salt-sensitive (SS) and salt-resistant (SR) rats show dramatically different blood pressure (BP) responses to being raised on a high fat, but normal salt, diet starting at weaning. Virtually no effect on BP is seen in SR rats, whereas in SS rats mean arterial pressure (MAP) reaches over 160 mmHg by adulthood. The BP difference is similar in female and male rats. The cellular and physiological mechanisms responsible have not been completely elucidated in either sex. Dysfunctional perivascular adipose tissue (PVAT) is proposed to contribute to obesity-associated hypertension through actions on other components of the vascular wall. Here we used female rats to test the hypothesis that differences in adipose tissue amount or function in SS and SR rats play a part in the distinct BP effects of high fat diet in the two strains. Rats (n=8 per strain) were fed a high fat diet (60% kcal fat) from weaning. BP was measured with telemetry. At 18 weeks of age, MAPs (mmHg) were 108±1 and 180±4 in SR and SS rats. Body weights (g) were 274±4 and 323±4. Total and visceral adipose volumes (mm3) measured by μCT were higher in SS rats versus SR rats (visceral: 22372±927 versus 14112±1495; total: 17208±732 versus 10234±984). PVAT volume around the thoracic aorta was larger in SS rats (0.228±0.017 versus 0.167±0.006). Subcutaneous adipose tissue volumes did not differ. Plasma triglycerides and cholesterol were modestly but significantly higher in SS rats. The well-known anticontractile effect of PVAT was compared in thoracic aorta from both strains. Removal of PVAT increased the AUC of the tension/PE contraction in SR rats by 1484±226 units and by a significantly smaller amount (946±162.5 units) in SS rats. Finally, aortic stiffness was estimated from pulse wave velocity measured via Doppler in a subset (n=4 each) of rats from each strain. Pulse wave velocity (M/sec) was markedly higher in SS than SR rats (11.5±1.0 versus 6.4±0.3). We conclude that differences in adipose tissue volume, location or function could account in part for the distinct BP responses of female SS and SR rats to high fat feeding.

Abstract 099: Role Of Nox-induced Oxidative Stress In Mitochondrial Bioenergetic Dysfunction In The Kidney Cortex And Outer Medulla Of Dahl Salt-sensitive Rats During The Development Of Hypertension

Background: Although renal mitochondrial dysfunction has been observed in various models of hypertension, the events leading to this dysfunction remain unclear. The present study focused on the role of salt (NaCl) intake upon renal mitochondrial bioenergetic function in Dahl salt-sensitive (SS) rats hypothesizing that a high salt (HS) diet would impair mitochondrial bioenergetics via stimulation of NOX-induced oxidative stress. Method: The HS induced progressive alteration of oxidative phosphorylation (OxPhos) and ATP synthesis in mitochondria obtained from renal cortex and outer medulla (OM) of male SS rats were compared to those of SS rats with dual knockout of NOX4 and p67 phox subunit of NOX2 (NOX DKO). Mitochondria were isolated from the cortex and OM of groups of age matched rats fed a 0.4% NaCl (LS) diet since weaning and at days 3, 7, 14 & 21 of a 4.0% NaCl (HS) diet feeding. ADP-induced (state 3) O 2 consumption rates (OCR) were measured in mitochondria energized with pyruvate and malate as substrates using an Oroboros Oxygraph-2k Instrument. Results: In SS rats, a biphasic pattern in state 3 OCR was observed with progressive uncoupling of OxPhos occurring in both cortex and OM. In the early phase of hypertension (days 3 & 7), state 3 OCR was increased (155 ± 8.2 to 190 ± 12.2 to 203.4 ± 12.1 nmol/min/mg in cortex and 101.4 ± 5.1 to 121.8 ± 7.3 to 112.8 ± 6.8 nmol/min/mg in OM; n = 5). In the established phase of hypertension (days 14 & 21), this increase was not sustained as state 3 OCR was reduced to 153 ± 7.65 and 132 ± 6.6 nmol/min/mg in cortex and 105 ± 7.3 and 63.3 ± 6.5 nmol/min/mg in OM. In contrast, SS NOX DKO rats fed a HS diet showed no significant differences in state 3 OCR in both cortex and OM, as was also observed in a control group of age matched NOX DKO rats maintained on a LS diet throughout the study. Conclusions: In SS rats, a HS diet causes initial increase in the efficiency of OxPhos for ATP production followed by a progressive decrease in the efficiency of OxPhos for ATP production in the established phase of hypertension. This biphasic pattern with ultimate reduction in the efficiency of OxPhos for energy production was not observed in SS NOX DKO rats demonstrating the important causal role of NOX-induced oxidative stress in the renal mitochondrial bioenergetic dysfunction.

Abstract P082: Exposure To Excess Albumin Causes Inflammatory Activation Of The Proximal Tubule.

In previous studies, we used chronic servo control experiments to show that renal inflammation is a secondary event in salt-sensitive hypertension, with pressure amplifying renal immune cell infiltration in Dahl salt-sensitive (SS) rats. In contrast, albuminuria occurs early in the development of hypertension and facilitates its progression. In this study, we tested the hypothesis that albuminuria causes release of pro-inflammatory cytokines in the proximal tubule, which in turn may initiate renal inflammation.We exposed the apical membrane of human proximal tubule cell monolayers (RPTEC-TERT1) to human serum albumin (HSA, 10 mg/ml, n=8) for 24 and 48hrs. Control cells were kept in standard media without HSA (n=8). The concentrations of 47 cytokines were measured in the basolateral (BM) and apical (AM) media using Luminex assays. After 24hrs we found that 18 cytokines were significantly higher in the BM and 14 in the AM relative to controls. At 48hrs, 17 cytokines were significantly higher in the BM and 15 in the AM. Notably, we found significant increases in basolateral release of several chemokines, including CCL2 (24hrs: 6.1±0.9 vs. 40.6±8.9, 48hrs: 20.1±4.5 vs. 85.8±15.6 pg/mg), CXCL1 (24hrs: 1.3±0.3 vs. 39.5±12.2, 48hrs: 3.4±1.1 vs. 94.2±41.4 pg/mg), and IL-8 (24hrs: 1.3±0.4 vs. 11.6±3.7, 48hrs: 2.1±0.5 vs. 10.1±3 pg/mg). We propose that HSA-induced chemokine secretion facilitates immune cell recruitment and the initiation of renal inflammation. To track the progression of albuminuria and proximal tubule injury in SS rats we performed daily urine collections in rats fed either a high-salt (HS, 4.0% NaCl, n=6) or a control-salt (CS, 0.4% NaCl, n=5) diet for 7-days. In SS rats fed HS, albuminuria preceded proximal tubule injury. Urinary albumin was elevated after 2-days of HS (22.6±6.5 vs. 61.1±18.7 mg/day) whereas Kidney Injury Molecule-1 (KIM-1) was increased after 5-days of HS (15.5±1.5 vs. 31.3±5.6 ng/day).To conclude, in vitro exposure to excess albumin causes significant increases in the levels of multiple cytokines involved in the initiation of inflammation. In SS rats fed HS, albuminuria precedes proximal tubule injury. Together these data suggest that albuminuria may facilitate hypertension through the initiation of renal inflammation.

Fuzzy Dispersion Entropy: A Nonlinear Measure for Signal Analysis

Entropy is a powerful tool for nonlinear analysis of time series. We have recently introduced dispersion entropy (DispEn), which has widely drawn attention from researchers in a variety of settings in data analysis. However, DispEn is sensitive to its parameters, especially number of classes (quantization level). Additionally, in some cases, a small change in the signal amplitude due to noise changes the quantized series and so entropy value. To address these limitations, we develop fuzzy dispersion entropy (FuzzDispEn) on the basis of fuzzy membership functions for the quantization of a signal and Shannon entropy (ShEn). In FuzzDispEn, the concept of fuzzification in ShEn-based methods is introduced for the first time. Several straightforward concepts in signal processing using a set of time series were utilized to show the advantages of FuzzDispEn over DispEn in terms of the detection of the dynamical variability of signals. The results showed that FuzzDispEn has lower sensitivity to noise and signal length compared to DispEn. FuzzDispEn was also applied to five real world applications: mechanical fault detection in gearbox, fault diagnosis of rolling element bearings, damage level detection of bearings, detection of blood pressure in salt-sensitive rats, and detection of seizure and seizure-free electroencephalographics (EEGs). The results suggest that FuzzDispEn outperforms DispEn as well as popular sample entropy and permutation entropy in distinguishing different kinds of dynamical patterns in synthetic and real data.

Abstract P244: Tubular Sites Of Mtorc2 Inhibition Of Sodium Transport In Dahl Salt-sensitive Rats.

Background: There is evidence that the mTOR signaling pathway plays an important role in salt induced hypertension in the Dahl salt-sensitive (SS) rat via regulation of Na + excretion. PP242 inhibition of mTORC1-2 prevented hypertension in SS rats and intrarenal PP242 produced a 4-fold increase of Na + excretion while selective inhibition of mTORC1 with rapamycin had no effect. The goal of the present study was to identify the renal tubular Na + transporters regulated by the mTORC2 pathway. Methods: Natriuretic effectors of mTORC2 were deduced from the differences of Na + excreted with PP242 treatment beyond that observed by the natriuretic actions of benzamil (B; ENaC inhibitor), furosemide (F; NKCC2 inhibitor), hydrochlorothiazide (H; NCC inhibitor) and in combination (B+F+H). Unanesthetized SS rats were first administered B, F, H or B+F+H (i.p.) and 2 days later with co-administration of PP242 with these same reagents. Rats were studied when fed 0.4% NaCl (LS) and then following 3 weeks of a 4.0% NaCl (HS) diet. The effect of PP242 on ENaC activity in freshly isolated cortical collecting ducts was also examined using cell-attached patch clamp electrophysiology. Results: Urine Na + excretion rates determined at 20 min. intervals over 80 minutes increased nearly 2-fold with administration of PP242 + B, compared to B alone [0.10 + 0.02 vs. 0.04 + 0.01 mmol/100gbw (n=8; P<0.05)] and similarly in the F and H comparisons [F: 0.20 + 0.02 vs. 0.10 + 0.02 mmol/100gbw (n=8; P<0.05); H: 0.05 + 0.01 vs. 0.03 + 0.01 mmol/100gbw (n=8; P=0.06)]. PP242 given in the presence of F+B+H did not further enhance the overall natriuretic response. Rats fed HS showed little enhancement of natriuresis when PP242 was administered with any of the drugs. Minimum changes in K + excretion were observed with PP242 in LS or HS studies. Patch clamp studies found that PP242 reduced ENaC activity through reduction of the open channel probability (Po) in both LS and HS fed rats. Western blot data indicated that PP242 reduced pNCC T53 and phosphorylation of SGK1. Conclusion: Together, these studies found evidence that the natriuretic actions of PP242 are a consequence of inhibitory actions on the ENaC and NCC transporters in the distal nephron segments.

Abstract P067: Sex-Specific Protection Of Fermentable Dietary Fiber On Salt-Sensitive Hypertension And Renal Function

Our recent data show that female Dahl Salt-Sensitive (SS) rats are significantly protected from salt-induced hypertension and renal injury and have stark differences in gut microbiota composition compared to males. Gut-derived metabolites are gaining recognition as mechanistic links between the microbiota and disease. One such metabolite is propionate, a protective short-chain fatty acid byproduct of bacterial fiber fermentation. Metabolomics revealed a specific elevation in serum propionate in female versus male SS rats after high salt (HS) (40.3±3.8 vs 24.4±4.2 μM, female vs male, p<0.02, n=6), and propionate administration to male SS rats blunts salt-induced elevations in mean arterial pressure and albuminuria (MAP, HS21: 160.5±6.6 vs 141.8±3.5 mmHg, p<0.05; albuminuria: 437.8±108.6 vs 263.6±50.33 mg/day, p<0.19, control vs propionate, 200mM p.o.; n=3-4). Therefore, the current study hypothesized that substitution of non-fermentable fiber cellulose with the fermentable fiber inulin (AIN-76A, Dyets Inc), a known precursor of short-chain fatty acids, would attenuate salt-sensitivity in males. Males and females were placed on the 0.4% NaCl (LS) inulin diet for one week prior to the switch to a 4.0% NaCl (HS) inulin diet for 4 weeks (inulin-M: n=7, inulin-F: n=6). Controls were maintained on either LS or HS diets containing cellulose (control-M: n=9, control-F: n=11). While there were no significant MAP differences observed in males, the inulin diet significantly reduced albuminuria (370.8±32.4 vs 174.4±30.0 mg/day on HS28, control-M vs inulin-M, p<0.001), reduced CD45+ renal leukocytes (35% reduction, p<0.03), reduced BUN (21.6±2.0 vs 15.4±0.5 mg/dL, p<0.05), and increased creatinine clearance (0.44±0.02 vs 0.55±0.02 ml/min/g KW, p<0.001). In females, inulin significantly lowered MAP (170.2±6.2 vs 154.6±4.5 mmHg on HS28, control-F vs inulin-F, p<0.01), albuminuria (180.3±27.4 vs 68.0±8.3, p<0.001), BUN (21.7±1.2 vs 17.9±0.6 mg/dL, p<0.05), and improved creatinine clearance (0.53±0.04 vs 0.71±0.05 ml/min/g KW, p<0.05). In summary, substitution of the fermentable fiber inulin reduced blood pressure only in females, but all other measures indicate improved renal injury, function, and inflammation in both sexes.

Abstract 028: Toll-Like Receptor 4 Promotes Renal Immune Cell Infiltration And The Development Of Renal Damage During Dahl Salt-Sensitive Hypertension

Previous work in our laboratory has implicated a significant role for the immune system in potentiating the hypertensive and kidney damage phenotypes which occur in the Dahl Salt-Sensitive (SS) rat. Toll-like receptors (TLRs), usually expressed on macrophages, play a key role in the innate immune system. A number of TLRs have been implicated in cardiovascular disease and chronic kidney disease with particular interest in TLR4, which specifically binds gram-negative lipopolysaccharide or endogenous molecules produced as a result of tissue injury. We previously performed a transcriptomic analysis which demonstrated TLR4 upregulation in renal medullary tissue of Dahl SS rats fed a high salt (4.0% NaCl) diet. The present study tests the hypothesis that TLR4 influences high salt diet-induced hypertension and renal damage in the Dahl SS rat. Using CRISPR/Cas9 technology, a mutation was generated in the TLR4 gene on the Dahl SS background, creating a 1-bp frameshift deletion resulting in a premature stop codon. SS TLR4-/- males and parental Dahl SS males (SS TLR4+/+ ), maintained on a 0.4% NaCl chow, underwent carotid artery telemetry implantation at 7 weeks of age. After recovery and baseline mean arterial pressure (MAP) recordings (day 0: 125.6 ± 0.5 vs 124.2 ± 2 mmHg), animals were switched to a 4.0% NaCl chow for 3 weeks. Overnight urine collections occurred once during the low salt period and weekly throughout the high salt challenge. Kidneys were flushed prior to euthanasia. Although SS TLR4-/- MAP was not different from SS TLR4+/+ during the high salt challenge (day 21: 176.8 ± 14 mmHg, n=2 vs 172.3 ± 18 mmHg, n=4), high salt day 21 urinary albumin excretion was significantly reduced in SS TLR4-/- (272.7 ± 65 mg/day, n=5, p<0.01) compared to SS TLR4+/+ (427.5 ± 67 mg/day, n=7), indicating reduced renal damage in TLR4 absence. Furthermore, flow cytometric analysis of isolated renal immune cells indicated a 33% reduction in CD45+ total leukocytes (5.0 ± 1.2 vs 3.4 ± 0.9 x10 6 cells) and a 31% reduction in CD11bc+ monocytes/macrophages (3.9 ± 0.9 vs 2.6 ± 0.7 x10 6 cells) in SS TLR4-/- (n=5) vs SS TLR4+/+ (n=7) kidneys. Together, these data indicate that TLR4 may drive the immune response during the induction and progression of salt-sensitive hypertension and renal disease.

Abstract 002: Reducing Blood Pressure By Decreasing Na/k Pump Activities Using Synchronization Modulation Electric Field

Na + /K + -ATPase is essential in maintenance of electrolyte, acid-base and volume homeostasis in kidneys. Increased renal Na/K pump activity has been reported to be associated with elevated blood pressure. Since blood pressure changes are paralleled by alterations in Na/K pump activity, it is proposed that impaired Na + /K + -ATPase signaling may contribute to imbalanced volume homeostasis and hypertension as well as increased salt sensitivity. Recently, we developed a novel technique called synchronization modulation electric field (SMEF) to electrically modulate the Na/K pump functions. The SMEF first synchronizes the pump molecules so that all the pumps simultaneously extrude Na + ions and pump in K + ions. Then, by accurately and precisely accelerating or decelerating the pumping rate, SMEF can consequently regulate renal tubular reabsorption and excretion capacity. In this study, we tested the effectiveness of SMEF in the controls of the tubular reabsorption and the regulation of blood pressure in health and hypertensive rat models. In normal SD rats (n=5), urinary flow rates were measured for the left and right kidneys of the same rat. When the SMEF was turned on only on the left kidney with backward modulation at the target frequency of 10 Hz to slow Na/K pumping rate, the urinary flow rate of the left kidney was quickly increased to 5.6±0.3 μl/min/g KW from the basal value of 4±0.4 μl/min/g KW. While the right kidney without SMEF application kept a constant basal urinary flow and Na+ excretion rate. In the Ang II induced hypertensive rat models (n=6), the MAP was gradually lowered by 8 mmHg than the control group when the backward modulation of SMEF was applied, accompanied by an increase of about 13 ml/day in water intake. We then applied the SMEF to the Dahl salt sensitive (SS) rats (n=5). The MAP in control group started to increase after 3-5 days of high salt intake. The SMEF was then applied from 7 days after switching to a high salt diet till the end of the experiments. The MAP quickly reduced by >30 mmHg (from 148 ± 7 to 117 ± 8 mmHg). These data demonstrated that backward modulation of SMEF lowered blood pressure possibly by promoting Na + and water excretion. SMEF could be an efficient treatment strategy for salt-sensitive hypertension.

Abstract 064: Hypokalemia Induced By Loss Of Kir5.1 Initiates WNK-SPAK/OSR1 Signaling In Dahl Salt-sensitive Rats

Background: Hypokalemia promotes Na + reabsorption via the Na + -Cl - (NCC) cotransporter of the distal convoluted tubule (DCT), which reduces the K + losses in downstream nephron segments and increases NaCl retention and blood pressure. In dietary K + depletion, activation of NCC is mediated by the with no lysine (K) kinase (WNK)-Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) pathway. Our previous work suggested that deletion of Kcnj16 (the gene that encodes K ir 5.1) in Dahl salt-sensitive (SS) rats (SS Kcnj16-/- ) resulted in hypokalemia with a compensatory increase in expression and activity of NCC. Here, we hypothesized that hypokalemia induced by loss of the K ir 5.1 subunit initiates WNK-SPAK/OSR1 signaling. Methods: 8 to 12-week-old male SS WT and SS Kcnj16-/- rats were separated into 3 groups (N=5 per group) and fed different diets for 3 weeks: SS WT (0.4% NaCl); SS Kcnj16-/- (0.4% NaCl); SS Kcnj16-/- (4% NaCl+2% KCl). At the end of the experiment, kidneys were flushed and processed for Western blot, immunofluorescence, and electron microscopy analysis. Results: Electron micrographs exhibited WNK body clusters in SS Kcnj16-/- compared to SS WT rats. Immunofluorescence staining showed dramatically increased expression of both SPAK and phospho-SPAK (pSPAK) in DCTs of SS Kcnj16-/- rats, where pSPAK colocalized with NCC. Similarly, Western blot analysis revealed significantly elevated expression of total SPAK (3.32± 0.18 vs. 1.00 ± 0.19, P <0.0001) and pSPAK (2.09 ± 0.51 vs. 1.00 ± 0.19, P <0.001) in the kidneys of SS Kcnj16/- compared to SS WT rats. Moreover, supplementing the SS Kcnj16-/- rats with high Na + and high K + (4% NaCl+2% KCl) reverses the expression and activity of SPAK. Conclusion: Our results indicate that hypokalemia resulting from loss of K ir 5.1 subunit stimulates WNK-SPAK/OSR1 signaling. Furthermore, the fact that dietary K + supplementation overturned the altered expression of this pathway implies that plasma K + level is responsible for the activation of the WNK-SPAK/OSR1 pathway.

Abstract P319: Genetic Interactions Between Novel Genes Arhgef11 And Cgnl1 Are Involved In Hypertensive Chronic Kidney Disease

Patients with hypertension are at higher risk for developing chronic kidney disease (CKD) and ultimately kidney failure. Previous genetic studies involving the Dahl salt-sensitive (SS) rat, a well-established model of hypertensive CKD, identified multiple regions linked to kidney injury. Extensive work on Chr.2 identified and characterized the role of Arhgef11 in the onset and progression of CKD using SS- Arhgef11 -/- rats. The identification of Arhgef11 and mechanism (regulation of actin cytoskeleton components) has provided an opportunity to expedite gene identification on other chromosomes based on known genetic interactions between loci, such as with Chr. 8. The effect of the Chr. 8 locus on kidney injury was previously validated using a congenic strain [S.SHR(8)]. Bioinformatics analysis of genes residing within the confidence interval, and those mechanistically linked with Arhgef11 , identified cingulin-like 1 ( Cgnl1 ) as a candidate. Cgnl1 plays a role in the regulation of cell-cell contacts via adherens and tight junctions through several interacting proteins [e.g. ZO-1 and PLEKHA7 (linked with hypertension by GWAS and an SS- Plekha7 mutant model)]. Recent studies using a novel model (SS- Cgnl1 -/- ) demonstrates that knockout of Cgnl1 leads to loss of salt-sensitive blood pressure and reduced renal injury compared to wildtype SS, supporting the congenic studies. Using multi-omics approaches incorporating bulk RNAseq, single nuclei RNAseq, spatial transcriptomics, small RNAseq, and mass spectrometry discovery proteomics, a putative mechanism connecting proximal tubule injury to alterations in sodium solute transporters and hypertension was established. The direct physiological interaction between Arhgef11 and Cgnl1 on hypertension and CKD is currently underway using a double knockout model (SS- Arhgef11 -/- Cgnl1 -/- ). In addition, to better elucidate the impact of loss of Arhgef11 , Cgnl1 , or both genes together, gene-edited cell lines using CRISPR technology has been generated and are also being studied. It is anticipated that completion of this work will lead to a better understanding of the role of genetics in hypertensive CKD, the influence of factors that complicate kidney disease, and potential new therapeutic targets.

Abstract P343: Adaptive Immune Mechanisms Exacerbate Cholinergic Activation-mediated Hypertension In Dahl Salt-sensitive Rats

Hypertension affects over one billion people worldwide and significantly contributes to the pathogenesis of cardiovascular and renal disease. Activation of nicotinic acetylcholine receptors (nAChR) in immune cells regulates immune responses. We have shown earlier that the development of hypertension in a genetic model of human essential hypertension, the Spontaneously Hypertensive Rats (SHR), is dependent on nAChR-mediated renal inflammation. Broadening the application of this mechanism, in the present study we hypothesized that nAChR activation plays a role in inflammation and the development of hypertension in a salt-sensitive model of hypertension, the Dahl salt-sensitive (DSS) rat. To test the hypothesis, we infused 4 weeks old DSS (n=12) and Dahl salt resistant (DSR, n=12) rats with either saline (n=6) or nicotine (15mg/kg/day, n=6) for 4 weeks. Animals were fed a 4% NaCl diet for the entire duration and moved into metabolic cages 3 days prior to euthanasia. Systolic blood pressure (SBP) measured by tail cuff method remained unaltered in DSR. However, SBP was significantly elevated in DSS rats that received nicotine as compared to saline (196.33±7.8 vs. 168±5.2 mmHg, p<0.05). SBP changes in nicotine infused DSS were not accompanied by differences in albuminuria or urinary sodium excretion compared to saline controls. RT-PCR revealed significantly increased CD3e mRNA expression (a marker of T-cell, P=0.04) in the immune cells derived from nicotine infused DSS as compared to saline controls. Increased CD3e mRNA expression in the kidney of nicotine infused DSS compared to saline was observed. Nicotine infusion did not change mRNA expression of nAChR subunits in DSR immune cells. In contrast, there was a ≥50% decrease in mRNA expression of α1-α7, α9, α10, β2, and β3 nAChR in immune cells derived from nicotine infused DSS compared to saline controls. There was a small yet significant increase in the mRNA expression of the β1-nAChR subunit in the immune cells of nicotine infused DSS compared to saline controls. We conclude that nAChR activation potentiates hypertension in the DSS independent of sodium retention or renal function. Our preliminary data suggests a possible role for nAChR mediated adaptive immune mechanisms in salt-sensitive hypertension.

Abstract P001: Repurposing A Drug Targeting Inflammatory Bowel Disease For Lowering Hypertension

Both the gut per se and its residents, the gut microbiota are linked to blood pressure (BP) regulation, whereby, the gut can be recognized as a previously neglected target organ for hypertension. Hypertension and gut diseases such as inflammatory bowel disease (IBD) share commonalities of host inflammation in the gut and microbiota dysbiosis. Therefore, we reasoned that current treatments to remedy gut inflammation and dysbiosis in IBD could be repurposed to ameliorate hypertension. IBD is commonly treated with 5-Aminosalicylic acid (5-ASA, Mesalamine), which is a selective PPAR-γ agonist to prevent gut inflammation. Clinical use of 5-ASA is also reported to ameliorate gut dysbiosis in IBD. Based on this premise, in this study we chose to test our hypothesis that 5-ASA is an antihypertensive drug owing to its dual ability to enhance PPAR-γ in the gut and to correct gut dysbiosis. 2 groups of 11-12 weeks old male genetically hypertensive Dahl salt-sensitive (S) rats were radiotelemetrically monitored for BP, and orally gavaged with (n=9) or without (n=7) 5-ASA (150 mg/kg) for 4 weeks. PPAR-γ expression was assessed by RT-qPCR. Fecal microbiota were profiled by 16S gene sequencing. Lastly, for translational significance, F/B ratio of non-IBD (n=611) patients was compared with that of IBD (n=631) patients in the American Gut Project data for their Firmicutes/Bacteroidetes (F/B) ratio, elevation of which is a marker of gut dysbiosis. 5-ASA significantly lowered BP of the S rat (mean BP without 5-ASA, 153±1 mm Hg; mean BP with 5-ASA, 145±1 mm Hg, P <0.0001). PPAR-γ gene expression was significantly higher in proximal colon, but not in the heart or kidney. In addition, treatment with 5-ASA corrected gut dysbiosis as observed by a significant reduction in the F/B ratio compared to control. In accordance, humans without IBD had a similar lower F/B ratio, which was consistent with the data presented here comparing S rats with 5-ASA. Collectively, these data support our hypothesis that 5-ASA, an FDA-approved anti-IBD drug, could be repurposed to treat hypertension by specifically enhancing the gut PPAR-γ mediated anti-inflammatory effect while also correcting microbiota dysbiosis to lower BP.

Abstract P077: Targeting Neutrophilia Is A Therapeutic Strategy To Regulate Blood Pressure

Neutrophils are important innate immune cells that are relatively understudied for their contributions to blood pressure (BP) regulation. As neutrophilia is a clinical marker for various pathologies, we hypothesized that neutrophilia is also a feature of hypertension and curtailing neutrophilia is beneficial for lowering BP. Peripheral neutrophil levels were quantitated in Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats consuming either a low salt (0.3% NaCl) or high salt (2% NaCl) diet. Compared to R rats, S rats on a low-salt diet already exhibited neutrophilia (% neutrophils, 33.51±1.6 vs. 39.74±1.2, p<0.05). Introducing a high salt diet further potentiated the neutrophilia in S rats prone to salt-induced hypertension (% neutrophils, 48.21±0.6, p<0.05). These data suggest that neutrophilia is pro-hypertensive. To determine the possible mechanism for the pro-hypertensive effects of neutrophils, we investigated two major functions of neutrophils i.e., generation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs, web-like structures of expelled neutrophil DNA). Peripheral neutrophils stimulated with either PMA or LPS showed a ~ 3-fold induction of ROS and NETs, respectively, in S rats compared to R rats on a low-salt diet. Introducing a high-salt diet increased ROS and NETs (~2-fold), the extent of elevation being higher in S rats. Impressively, treatment of S rats on a high-salt diet with 1,3-butandiol [(β - hydroxybutyrate (βOHB, a ketone body) precursor] in the drinking water (20% v/v, 4 weeks) significantly lowered neutrophilia (vehicle: 1.74±0.5 vs βOHB: 0.73±0.3 x 10 9 cells/l, p<0.05) in tandem with their decreased BP (vehicle: 161.2±9.3 vs βOHB: 138.3±4.5 mmHg systolic BP, p<0.05). Interestingly, βOHB intervention also reduced neutrophil levels in R rats (vehicle: 1.15±0.5 vs βOHB: 0.95±0.5 x 10 9 cells/l), albeit to a lesser degree than S rats. Importantly, βOHB administration to S and R rats also reduced NETs and ROS (~2-fold), predominantly in S rats. Collectively, our data are the first to demonstrate that an exaggerated innate immune response via neutrophilia is pathological in hypertension, whereby curbing the neutrophilic response ( e.g., βOHB) could present therapeutic potential in lowering BP.

Abstract P075: Functional NOX2 In T Cells Amplifies Salt-Sensitive Hypertension And Associated Renal Damage

Infiltrating T cells in the kidney amplify salt-sensitive (SS) hypertension and renal damage, but the mechanisms are not known. Genetic deletion of T cells (SS CD247-/- ) or the p67phox subunit of NOX2 (SS p67phox-/- ) attenuates salt-sensitive hypertension in the Dahl SS rat. We hypothesized that reactive oxygen species (ROS) produced by NOX2 in T cells drive the SS phenotype and renal damage. T cells were reconstituted by adoptively transferring ~10 million splenocytes from the Dahl SS (SS→CD247, n=14), the SS p67phox-/- (p67phox→CD247, n=19), or PBS only (PBS→CD247, n=8), into the peritoneum of SS CD247-/- rats on postnatal day 5. Animals were instrumented with radiotelemeters at 8 weeks of age. After a week of recovery, blood pressure and albumin excretion were measured while rats were fed a low salt (LS, 0.4% NaCl) diet. Rats were then switched to a high salt (HS, 4.0% NaCl) diet for 21 days, with continuous blood pressure measurements and urine collections every 7 days. On day 22, the rats were euthanized, and their kidneys were perfused and collected for histology and renal immune cell characterization by flow cytometry. At baseline there was no detectable difference in mean arterial pressure (MAP) between groups (124±2 vs 123±2 vs 116±2 mmHg; SS→CD247 vs p67phox→CD247 vs PBS→CD247, respectively). After 21 days of HS, MAP was significantly greater in the SS→CD247 compared to the p67phox→CD247 rats (155±3 vs 144±2 mmHg; p<0.001) and the PBS→CD247 rats (155±3 vs 141±3 mmHg; p<0.001). Indices of renal damage identified no difference in albuminuria between SS→CD247 and p67phox→CD247 on LS (25.8±4.7 vs 23.2±3.2 mg/day). After 21 days of HS, albuminuria was significantly greater in the SS→CD247 (242.8±30.3 mg/day) compared to the p67phox→CD247 (175.5 ± 14.3 mg/day, p<0.03) and PBS→CD247 (135.9±27.8 mg/day, p<0.005). There was no difference between p67phox→CD247 and PBS→CD247 in albuminuria or MAP during LS or HS. The lack of CD3+ cells in the PBS→CD247 but presence of CD3+ cells in rats receiving the T cell transfer demonstrated the effectiveness of the adoptive transfer. These results indicate that ROS production by T cells participates in the amplification of salt-sensitive hypertension and renal damage.

Associations of genetic variations in NEDD4L with salt sensitivity, blood pressure changes and hypertension incidence in Chinese adults.

Neural precursor cell expressed developmentally downregulated 4‐like (NEDD4L), a member of the E3 ubiquitin‐protein ligases, encoded by NEDD4L gene, was found to be involved in in salt sensitivity by regulating sodium reabsorption in salt‐sensitive rats. The authors aimed to explore the associations of NEDD4L genetic variants with salt sensitivity, blood pressure (BP) changes and hypertension incidence in Chinese adults. Participants from 124 families in Northern China in the Baoji Salt‐Sensitive Study Cohort in 2004, who received the chronic salt intake intervention, including a 7‐day low‐salt diet (3.0 g/day) and a 7‐day high‐salt diet (18 g/day), were analyzed. Besides, the development of hypertension over 14 years was evaluated. NEDD4L single nucleotide polymorphism (SNP) rs74408486 was shown to be significantly associated with systolic BP (SBP), diastolic BP (DBP) and mean arterial pressure (MAP) responses to low‐salt diet, while SNPs rs292449 and rs2288775 were significantly associated with pulse pressure (PP) response to high‐salt diet. In addition, SNP rs4149605, rs73450471, and rs482805 were significantly associated with the longitudinal changes in SBP, DBP, MAP, or PP at 14 years of follow‐up. SNP rs292449 was significantly associated with hypertension incidence over the 14‐year follow‐up. Finally, this gene‐based analysis found that NEDD4L was significantly associated with longitudinal BP changes and the incidence of hypertension over the 14‐year follow‐up. This study indicated that gene polymorphism in NEDD4L serve an important function in salt sensitivity, longitudinal BP change and development of hypertension in the Chinese population.

Association of Antihypertensive Effects of Esaxerenone with the Internal Sodium Balance in Dahl Salt-Sensitive Hypertensive Rats.

Background: The nonsteroidal mineralocorticoid receptor blocker esaxerenone is effective in reducing blood pressure (BP). Objective: In this study, we investigated esaxerenone-driven sodium homeostasis and its association with changes in BP in Dahl salt-sensitive (DSS) hypertensive rats. Methods: In the different experimental setups, we evaluated BP by a radiotelemetry system, and sodium homeostasis was determined by an approach of sodium intake (food intake) and excretion (urinary excretion) in DSS rats with a low-salt diet (0.3% NaCl), high-salt diet (HSD, 8% NaCl), HSD plus 0.001% esaxerenone (w/w), and HSD plus 0.05% furosemide. Results: HSD-fed DSS rats showed a dramatic increase in BP with a non-dipper pattern, while esaxerenone treatment, but not furosemide, significantly reduced BP with a dipper pattern. The cumulative sodium excretion in the active period was significantly elevated in esaxerenone- and furosemide-treated rats compared with their HSD-fed counterparts. Sodium content in the skin, skinned carcass, and total body tended to be lower in esaxerenone-treated rats than in their HSD-fed counterparts, while these values were unchanged in furosemide-treated rats. Consistently, sodium balance tended to be reduced in esaxerenone-treated rats during the active period. Histological evaluation showed that esaxerenone, but not furosemide, treatment attenuated glomerulosclerosis, tubulointerstitial fibrosis, and urinary protein excretion induced by high salt loading. Conclusions: Collectively, these findings suggest that an esaxerenone treatment-induced reduction in BP and renoprotection are associated with body sodium homeostasis in salt-loaded DSS rats.

Abstract P3084: T Helper Cell Polarity Determines Salt-sensitivity And Development Of Hypertension

Background: Sodium chloride drives the stable induction of pathogenic T helper (Th) 17 cells and development of hypertension. However, high-salt intake induces hypertension in salt-sensitive persons but not in salt-resistant persons. Pathogenic Th17 cell plays an important role in the development of hypertension whereas suppressive Treg cell plays an important role in the prevention of hypertension. Objective: We tested the hypothesis that . Methods and Results: Eight-week-old male SS and SR rats were fed chow (NS, 0.4% NaCl) or high-salt (HS, 4% NaCl) diet for 4 weeks. Systolic blood pressure was measured by the tail-cuff method. Cultured splenic T cells from Dahl salt-sensitive (SS) and salt-resistant (SR) rats were exposed to hypertonic salt solution with or without IL-6 or TGFβ. T helper cell (Th17 and Treg) polarity was determined via flow cytometry. The expression of Th17-related genes such as interleukin (IL)-17A, IL-23R and retinoic acid receptor-related orphan receptor (ROR) γt) as well as Treg-related genes such as IL-10, CD25 and forkhead box (Fox) P3 were measured by ELISA or qRT-PCR. Adoptive transfer of Th17 cells isolated from SS or SR donors fed HS were performed intraperitoneally into SS and SR recipients. Upon chow diet, SR has higher Treg cells than SS does. HS intake induced hypertension and increased proinflammatory Th17 cells in SS but not in SR. Hypertonic salt solution induced differentiation of cultured splenic T cells preferentially into proinflammatory Th17 cells and anti-inflammatory Treg cells in SS and SR, respectively. Polarizing cytokine IL-6 also induced differentiation of cultured splenic T cells preferentially into proinflammatory Th17 cells and anti-inflammatory Treg cells in SS and SR, respectively, which were further potentiated by hypertonic salt solution. Adoptive transfer of activated Th17 cells isolated from hypertensive SS or normotensive SR, both of which had been fed HS diet, induced hypertension in SS recipients, but not SR recipients. Conclusions: These results indicate that T helper cell polarity after HS intake determines salt-sensitivity and development of hypertension in SS and SR rats. Th17 cell polarity predicts salt-sensitivity and development of hypertension, whereas Treg cell polarity predicts salt-resistance and maintenance of normotension. SR rats protect against hypertension after HS intake through anti-inflammatory Treg cell polarity.