High NaCl Diet Research Articles

The salt sensitivity of Drd4-null mice is associated with the upregulations of sodium transporters in kidneys.

To explore the mechanism of the hypertension in dopamine receptor-4 (Drd4) null mice, we determined the salt sensitivity and renal sodium transport proteins in Drd4-/- and Drd4+/+ mice with varied salt diets. On normal NaCl diet (NS), mean arterial pressures (MAP, telemetry) were higher in Drd4-/- than Drd4+/+; Low NaCl diet (LS) tended to decrease MAP in both strains; high NaCl diet (HS) elevated MAP with sodium excretion decreased and pressure-natriuresis curve shifted to right in Drd4-/- relative to Drd4+/+ mice. Drd4-/- mice exhibited increased renal sodium-hydrogen exchanger 3 (NHE3), sodium-potassium-2-chloride cotransporter (NKCC2), sodium-chloride cotransporter (NCC), and outer medullary α-epithelial sodium channel (αENaC) on NS, decreased NKCC2, NCC, αENaC, and αNa+-K+-ATPase on LS, and increased αENaC on HS. NKCC2, NCC, αENaC, and αNa+-K+-ATPase in plasma membrane were greater in Drd4-/- than in Drd4+/+ mice with HS. D4R was expressed in proximal and distal convoluted tubules, thick ascending limbs, and outer medullary collecting ducts and colocalized with NKCC2 and NCC. The phosphorylation of NKCC2 was enhanced but ubiquitination was reduced in the KO mice. There were no differences between the mouse strains in serum aldosterone concentrations and urinary dopamine excretions despite their changes with diets. The mRNA expressions of renal NHE3, NKCC2, NCC, and αENaC on NS were not altered in Drd4-/- mice. Thus, increased protein expressions of NHE3, NKCC2, NCC and αENaC are associated with hypertension in Drd4-/- mice; increased plasma membrane protein expression of NKCC2, NCC, αENaC, and αNa+-K+-ATPase may mediate the salt sensitivity of Drd4-/- mice.

Metabolomic study on the effect of Indonesian propolis in hypertensive rats

Context: Hypertension is a health problem that is a major factor triggering cardiovascular diseases; however, effective treatment has not been found. Aims: To evaluate the effectiveness of Indonesian propolis in hypertension treatment through metabolomic study. Methods: A total of 36 Sprague Dawley rats were divided into 6 groups, including standard diet group, high-NaCl diet group, high-NaCl diet + captopril group (25 mg/kg), high-NaCl diet + propolis from Riau Archipelago group, high-NaCl diet + propolis from Lampung group, high-NaCl diet + propolis from South Sulawesi group. An 8% NaCl diet was used to induce hypertension for 3 weeks, while 200 mg/ kg propolis was administered for 1 week following the induction. The bioactive compounds of ethanol extract of propolis were identified using UHPLC. Moreover, the metabolomic profile was performed using LC-MS/MS. Data was processed using principal component analysis and was used for metabolic pathway analysis. Results: All propolis samples significantly ameliorated the blood pressure of hypertensive rats. The metabolites that were mainly found in rat serum were amino acids, glycerophospholipids, and acylcarnitine. Rats administered with propolis from South Sulawesi had the closest metabolite profile to the standard group, where L-acetylcarnitine could be the potential marker of hypertension. Several metabolic pathways, including the degradation of tryptophan, β-fatty acid oxidation, degradation of threonine, and 2-oxobutanoic acid, were the most impaired pathways in hypertension. Conclusions: Indonesian propolis, especially from South Sulawesi, could be used for controlling blood pressure.

Dissociation of sodium-chloride cotransporter expression and blood pressure during chronic high dietary potassium supplementation.

Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+-free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.

Abstract P312: High Potassium Intake Aggravates Cardiovascular Damage Accompanied By Premature Senescence In Hypertensive Apolipoproteine-ko Mice Under High Salt Diet

Increased sodium intake aggravates cardiovascular diseases. Salt substitute containing potassium chlorideimproves cardiovascular outcome in hypertensive patients with high cardiovascular risk, however it isunknown whether it is attributed to potassium (K+) supplementation or sodium reduction. Here, weinvestigate the role of high K+ intake on the development of hypertensive cardiac damage in the presenceor absence of high salt diet. 8-10 weeks old apolipoproteinE-deficient mice (apoE-KO) were fed a normalK+ (0,55%) or high K+ (5%) diet throughout the experiment. Two weeks after diet start,mice were infusedwith angiotensin (Ang)II (500ng/kg/min) for 28 days. Cardiac function was assessed by MRI. High K+ dietincreased serum K+ levels compared to mice fed a normal K+ diet (5.3±1.2 vs. 3.9±0.4mmol/L, p<0.05).Interestingly, high K+ diet did not affect blood pressure or cardiac function. As expected, aldosteroneexcretion was increased in high K+ diet group compared to normal K+ diet (132±20 vs 8±18 ng/24h,p<0.01). To evaluate the consequence of K+ mediated aldosterone secretion for hypertensivecardiovascular damage, we additionally treated our high K+ group with high salt diet (1%NaCl) in thedrinking water. High K+ induced aldosterone production in the presence of high NaCl intake aggravatedhypertensive cardiac damage characterized by higher left ventricular mass, more cardiac fibrosis andinflammation compared to mice fed a high K+ or a high NaCl diet solely. Furthermore, the simultaneousintake of high K+/NaCl diet, induced higher mitochondrial ROS production in cardiac endothelial cellscompared to high K+ or high NaCl diet alone. As a consequence, cellular senescence markers such as p16and p21 were significantly higher in mice fed a high K+ / NaCl diet. Of note, co-treatment withspironolactone (50mg/kg/day), significantly attenuated cellular senescence and cardiovascular damage inmice fed a high K+/NaCl diet. High K+ diet compared to normal K+ diet does not have a beneficial effecton hypertensive cardiac damage in apoE-KO mice. Moreover, the present study indicates that potassium-induced aldosterone production aggravates the detrimental effect of high salt intake on cardiac health inhypertension by influencing cardiac senescence.

Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4

Prevention of phenotype switching of vascular smooth muscle cells is an important determinant of normal vascular physiology. Hydrogen peroxide (H2O2) promotes osteogenic differentiation of vascular smooth muscle cells through expression of Runt related transcription factor 2 (Runx2). In this study, an increase in dietary NaCl increased endothelial H2O2 generation through NOX4, a NAD(P)H oxidase. The production of H2O2 was sufficient to increase Runx2, osteopontin and osteocalcin in adjacent vascular smooth muscle cells from control littermate mice but was inhibited in mice lacking endothelial Nox4. A vascular smooth muscle cell culture model confirmed the direct involvement of the activation of protein kinase B (Akt) with inactivation of FoxO1 and FoxO3a observed in the control mice on the high NaCl diet. The present study also showed a reduction of catalase activity in aortas during high NaCl intake. The findings demonstrated an interesting cell-cell communication in the vascular wall that was initiated with H2O2 production by endothelium and was regulated by dietary NaCl intake. A better understanding of how dietary salt intake alters vascular biology may improve treatment of vascular disease that involves activation of Runx2.

Dietary sodium and health: How much is too much for those with orthostatic disorders?

High dietary salt (NaCl) increases blood pressure (BP) and can adversely impact multiple target organs including the vasculature, heart, kidneys, brain, autonomic nervous system, skin, eyes, and bone. However, patients with orthostatic disorders are told to increase their NaCl intake to help alleviate symptoms. While there is evidence to support the short-term benefits of increasing NaCl intake in these patients, there are few studies assessing the benefits and side effects of long-term high dietary NaCl. The evidence reviewed suggests that high NaCl can adversely impact multiple target organs, often independent of BP. However, few of these studies have been performed in patients with orthostatic disorders. We conclude that the recommendation to increase dietary NaCl in patients with orthostatic disorders should be done with care, keeping in mind the adverse impact on dietary NaCl in people without orthostatic disorders. Modest, rather than robust, increases in NaCl intake may be sufficient to alleviate symptoms but also minimize any long-term negative effects.

Empagliflozin Reduces Renal Hyperfiltration in Response to Uninephrectomy, but Is Not Nephroprotective in UNx/DOCA/Salt Mouse Models.

Large-scale clinical outcome studies demonstrated the efficacy of SGLT2 inhibitors in patients with type II diabetes. Besides their therapeutic efficacy in diabetes, significant renoprotection was observed in non-diabetic patients with chronic kidney disease (CKD), suggesting the existence of glucose-independent beneficial effects of SGLT2 inhibitors. However, the relevant mechanisms by which SGLT2 inhibition delays the progression of renal injury are still largely unknown and speculative. Previous studies showed that SGLT2 inhibitors reduce diabetic hyperfiltration, which is likely a key element in renoprotection. In line with this hypothesis, this study aimed to investigate the nephroprotective effects of the SGLT2 inhibitor empagliflozin (EMPA) in different mouse models with non-diabetic hyperfiltration and progressing CKD to identify the underlying diabetes-independent cellular mechanisms. Non-diabetic hyperfiltration was induced by unilateral nephrectomy (UNx). Since UNx alone does not result in renal damage, renal disease models with varying degrees of glomerular damage and albuminuria were generated by combining UNx with high NaCl diets ± deoxycorticosterone acetate (DOCA) in different mouse strains with and without genetic predisposition for glomerular injury. Renal parameters (GFR, albuminuria, urine volume) were monitored for 4–6 weeks. Application of EMPA via the drinking water resulted in sufficient EMPA plasma concentration and caused glucosuria, diuresis and in some models renal hypertrophy. EMPA had no effect on GFR in untreated wildtype animals, but significantly reduced hyperfiltration after UNx by 36%. In contrast, EMPA did not reduce UNx induced hyperfiltration in any of our kidney disease models, regardless of their degree of glomerular damage caused by DOCA/salt treatment. Consistent with the lack of reduction in glomerular hyperfiltration, EMPA-treated animals developed albuminuria and renal fibrosis to a similar extent as H2O control animals. Taken together, the data clearly indicate that blockade of SGLT2 has the potential to reduce non-diabetic hyperfiltration in otherwise untreated mice. However, no effects on hyperfiltration or progression of renal injury were observed in hypervolemic kidney disease models, suggesting that high salt intake and extracellular volume might attenuate the protective effects of SGLT2 blockers.

Starvation and activity dependent modulation of salt taste behavior in Drosophila

Sodium present in NaCl is a fundamental nutrient required for many physiological processes but high salt consumption in western world is contributing health risk to all age individuals. Although high salt detection pathways have been studied in great detail, the mechanisms that regulate high salt consumption in animals are largely unknown. To understand how pre-exposure to high NaCl diet modulates subsequent feeding behavior, we looked into the neural mechanisms of high NaCl consumption in adult Drosophila. We used Neuro-Genetics, imaging and behavioral assays to determine how flies respond to high NaCl exposure. We studied the neural mechanism by which flies modify their acceptance of high salt as a function of diet, where a long-term high-salt exposure increases taste sensitivities of pharyngeal LSO (Labral sense organ) neurons and enhances high salt intake. We discovered that exposing flies to high NaCl diet(200mM NaCl in fly food) for three days modify their feeding responses to high levels of salt. High NaCl fed flies show decline in high salt aversion under starvation. Genetic suppression of LSO pharyngeal neurons in high NaCl fed flies inhibits excessive salt intake. We found that this modulation requires functional LSO neurons and starvation state, and that multiple independent taste receptor neurons and pathways are involved in this process. Silencing any one of multiple LSO neuronal types inhibits excessive salt intake. Our data support the idea that high dietary salt modulates and reshapes salt and other taste curves to promote over consumption of food in flies. Our study suggest flies can adapt to the amount of salt ingested over several days, indicating the presence of a critical mechanism to reset the salt appetite and related neural circuits. Identification of new molecular sensors for salt and related neural controls such as hormones, neuropeptides, and neurotransmitters may yield insights into the coordination of processes in the nervous system.

Voluntary intake, milk and colostrum production and lamb growth when ewes are fed high-NaCl diets during pre- and post-lambing

Nutritional requirements at the end of gestation in ewes carrying multiple fetuses are rarely fully met in pasture production systems. Thus, concentrate feeds entail an alternative for increasing herd productivity. However, daily offer of complementary feed in extensive livestock production systems is not always possible due to climatic or geographic constraints or to labor shortages. One alternative is to offer large amounts of concentrate feed with the addition of NaCl that limits voluntary consumption, eliminating the need for daily supplement delivery. Our objective was to evaluate the effect of high salt levels added to concentrate feed on pre- and post-lambing twin-gestating ewes and their responses on feed intake, colostrum and milk production and composition, plasma biochemical profile variation and lamb growth. Twenty-four adult Corriedale females were housed in individual stalls and divided into four treatments: S13: ad libitum concentrate feed plus 13 % of salt; C13: the same amount of concentrate feed consumed by S13 ewes the day before, but without salt; S17: ad libitum concentrate feed with 17 % of salt, and C17: the same amount of concentrate feed consumed by S17 ewes the day before, but without salt. Fresh water and forage were offered in ad libitum amounts. Treatments were applied in the period of highest ewe nutrient demand, from day 123 of pregnancy to day 21 of lactation. The four percentage points in salt content resulted in a decrease of organic matter daily intake of concentrate (23 % of reduction in pre-lambing and 35 % of reduction in post-lambing for S17 relative to S13; P = 0.0134 and P = 0.0164, respectively). Lamb birth weights were similar between S13 and C13 (P = 0.1002) and between S17 and C17 (P = 0.8449) treatments. Colostrum and milk production did not differ between S13 and C13 (P = 0.9776 and P = 0.8928, respectively) or between S17 and C17 groups (P = 0.4943 and P = 0.7705, respectively). Plasma insulin, hematocrit and total protein levels were not influenced by the treatments and these parameters remained within normal physiological limits. Thus, the addition of 13 or 17 % of salt to concentrate feed did not affect the reproductive capacity of ewes or offspring growth, showing that ewes are able to withstand high doses of NaCl and supporting the potential of salt as a limiter of voluntary intake in pre- and post-lambing diets.

Abstract MP22: Dietary Salt Restriction And High Salt Intake Exaggerate Sympathetic Reflexes And Sensitize Central Autonomic Networks: Potential Mechanisms Of The J-shaped Curve

Observational cohort studies suggest that severe salt restriction increases cardiovascular morbidity/mortality, and the relationship between cardiovascular morbidity and dietary salt intake resembles a J-shaped curve. A high salt diet exaggerates sympathetic nerve activity (SNA) and arterial blood pressure (ABP) responses to several cardiovascular reflexes in salt-resistant animals. This study assessed whether salt restriction also exaggerates cardiovascular reflex responses and sensitizes central autonomic networks. To test this hypothesis, male Sprague-Dawley rats were fed low (0.01% NaCl), normal (0.1% NaCl), and high (4.0% NaCl) salt diet for 14-21 days. Baseline mean ABP was not different across groups (low: 104±4, normal: 107±4, high: 107±4mmHg). Activation of sciatic afferents (1ms pulse, 500uA, 5s duration, 2-20Hz) produced significantly greater increases in renal SNA (5Hz; low: 196±12, normal: 136±9, high: 177±8%, n=8, P<0.05) and ABP (5Hz; low: 29±3, normal: 16±1, high: 24±2 mmHg, n=8, P<0.05) of rats fed low and high versus normal NaCl diets. Activation of the aortic depressor nerve (2ms pulse, 500uA, 15s duration, 2-20Hz) produced significantly greater decreases in renal SNA (5Hz; low: -55±9, normal: -34±8, high: -63±13%, n=7-8, P<0.05) and ABP (5Hz; low: -31±3, normal: -15±5, high: -32±5 mmHg, n=7-8, P<0.05) of rats fed low and high versus normal NaCl diets. To test whether dietary salt intake sensitized central sympathetic circuits, microinjection of L-glutamate (0.1-1nmol, 30nL) in the rostral ventrolateral medulla produced significantly greater increases in renal SNA (0.1nmol; low: 212±15, normal: 149±8, high: 183±17%, n=7-8, P<0.05) and ABP (0.1Hz; low: 20±2, normal: 12±2, high: 22±2 mmHg, n=7-8, P<0.05) of rats fed low and high versus normal NaCl diets. Finally, rats fed low or high NaCl versus normal NaCl diets displayed exaggerated cardiovascular responses to cage switch or mild restraint and increased 24-h blood pressure variability. The present findings show that severe salt restriction and excess dietary salt intake exaggerate sympathetic and cardiovascular responses, and may be explained by a parallel change in the sensitivity of central autonomic networks to resemble a J-shaped curve.

Effect of dietary calcium and vitamin D supplementation on blood pressure control following salt loading

Epidemiological and animal studies assert an inverse relationship between calcium intake and risk of clinical hypertension. Nonetheless, there is a paucity of information on the effect of calcium and vitamin D co-administration on sodium chloride (NaCl)-induced blood pressure (BP) elevation in apparently healthy normotensive subjects. Hence, this study evaluated this relationship. For a 5-day study period, a group of 10 subjects (Group A) was placed on placebo while another group of 5 subjects (Group B) had dietary NaCl (200 mmol/day) and a third group of 5 subjects (Group C) had dietary NaCl (200 mmol/day) with calcium/vitamin D supplement (625 mg/200 IU). Before and after the 5-day regimen, blood pressure (BP) was measured, and blood, as well as urine samples, were collected from the subjects for evaluation of electrolyte status. Mean arterial BP (MABP) increased (p<0.01) in group B, but decreased in group C (p<0.01). Plasma potassium level increased in both groups B and C (p<0.01). However, while plasma calcium decreased in the two groups (p<0.01), plasma sodium decreased (p>0.01) only in group C, after the 5-day regimen. Urinary excretion of calcium increased and decreased (p<0.01) in groups B and C, respectively, a converse of the trend in urinary potassium. Whereas that of sodium increased in both groups B and C (p<0.01). These results suggest that oral Ca and Vitamin D supplementation may prevent a rise in BP in subjects on a high NaCl diet by attenuating plasma Na retention, with an augmented urinary excretion.

High Salt Diet Blunted Baroreflex Sensitivity (BRS) to Bilateral Common Carotid Artery Occlusion (BCCO) in Male Sprague –Dawley Rats – Permissive Effect of Testosterone

Autonomic dysfunction is involved in the onset of salt-sensitive hypertension as baroreflex sensitivity (BRS) is blunted in salt-sensitive hypertension. However, salt sensitivity exhibits sex disparity because in salt-sensitive hypertension, blood pressure (BP) is usually higher in males when compared with females. The mechanism(s) underlying this sexual dimorphism is not clear. We, therefore, designed this study to determine BRS in weanling male Sprague – Dawley rats that were either bilaterally orchidectomized or sham-operated (under ketamine and xylazine anaesthesia (90 mg and 10 mg/kg/body weight i.m) respectively, with or without testosterone replacement (10 mg/kg sustanon 250® i.m once in 3 weeks) and were placed on normal (0.3%) or high (8%) NaCl diet for 6 weeks. Blood pressure (BP) and heart rate (HR) were measured via arterial cannulation under urethane and α-chloralose anesthesia (5 ml/kg body weight i.p). BRS was determined as change in HR per unit change in BP in response to 30 seconds bilateral common carotid artery occlusion (BCCO). Serum concentration of testosterone was measured using enzyme-linked immunosorbent assay (ELISA). High salt diet (HSD) increased both the basal BP and peak BP after BCCO when compared with control. Orchidectomy attenuated but testosterone replacement restored the elevated BP in rats fed HSD. HSD blunted BRS to BCCO. However, this effect of HSD was lost in the absence of testosterone as there was no significant difference in BRS between rat fed a normal or high salt diet. Our results suggest that blunting of the BRS may be one of the mechanisms by which testosterone promotes salt-induced hypertension and serves as one of the bases for sex disparity in salt-sensitive hypertension.

Dietary sodium chloride attenuates increased β-cell mass to cause glucose intolerance in mice under a high-fat diet.

Excessive sodium salt (NaCl) or fat intake is associated with a variety of increased health risks. However, whether excessive NaCl intake accompanied by a high-fat diet (HFD) affects glucose metabolism has not been elucidated. In this study, C57BL/6J male mice were fed a normal chow diet (NCD), a NCD plus high-NaCl diet (NCD plus NaCl), a HFD, or a HFD plus high-NaCl diet (HFD plus NaCl) for 30 weeks. No significant differences in body weight gain, insulin sensitivity, and glucose tolerance were observed between NCD-fed and NCD plus NaCl-fed mice. In contrast, body and liver weights were decreased, but the weight of epididymal white adipose tissue was increased in HFD plus NaCl-fed compared to HFD-fed mice. HFD plus NaCl-fed mice had lower plasma glucose levels in an insulin tolerance test, and showed higher plasma glucose and lower plasma insulin levels in an intraperitoneal glucose tolerance test compared to HFD-fed mice. The β-cell area and number of islets were decreased in HFD plus NaCl-fed compared to HFD-fed mice. Increased Ki67-positive β-cells, and increased expression levels of Ki67, CyclinB1, and CyclinD1 mRNA in islets were observed in HFD-fed but not HFD plus NaCl-fed mice when compared to NCD-fed mice. Our data suggest that excessive NaCl intake accompanied by a HFD exacerbates glucose intolerance, with impairment in insulin secretion caused by the attenuation of expansion of β-cell mass in the pancreas.

Effects of Acute SGLT2 Blockade and Dietary NaCl on Glomerular Hemodynamics in Diabetic Rats

Inhibitors of the main proximal tubular Na‐glucose cotransporter (SGLT2) mitigate diabetic glomerular hyperfiltration and are approved by FDA for slowing the progression of diabetic kidney disease. It has been proposed that SGLT2 inhibitors improve hard renal outcomes by reducing the glomerular capillary pressure gradient (ΔP) through TGF signaling, which begins with a decrease in fluid reabsorption by the proximal tubule (Jprox). However, the effect of SGLT2 inhibition on ΔP has not been measured up to now.We studied effects of acute SGLT2 blockade (ertugliflozin) on Jprox and glomerular hemodynamics by 2‐period micropuncture in STZ‐diabetic rats fed high or low NaCl diets (HS/LS). ΔP was measured by direct capillary puncture in Wistar Froemter rats (MWF), which have surface glomeruli suitable for this purpose. ΔP was computed from tubular stop‐flow pressure in Wistar rats (Wis), which lack surface glomeruli. Hence, TGF was allowed to operate normally while measuring ΔP in MWF but rendered inoperative while measuring ΔP in Wis. SGLT2 blockade was confirmed to reduce Jprox by collecting fluid from late proximal tubules for volume and inulin clearance.Summary and Conclusions about the diabetic kidney under conditions of moderate hyperglycemia: SGLT2 blockade acutely reduces Jprox, which leads to a decrease in glomerular capillary pressure, but only when TGF is permitted to operate normally. Baseline glomerular capillary pressure varies inversely with dietary NaCl and the impact of SGLT2 blockade applies across a wide range of dietary NaCl.Support or Funding InformationNIDDK RO1 DK112042 Merck Group (n) Glucose (mg/dl) GFR(ml/min) ΔP (mmHg) Jprox' (nl/min) control SGLT2 blocked control SGLT2 blocked control SGLT2 blocked MWF LS (6) 344±41 4.2±0.5 3.5±0.5 * 41.2±1.5 33.0±1.9 * MWF HS (8) 408±35 4.1±0.3 3.3±0.4 * 36.6±1.3 ** 32.4±1.6 * Wis LS (6) 355±41 4.8±0.4 3.8±0.5 * 43.6±1.9 43.7±2.4 18.9±1.3 10.2±1.1 * Wis HS (9) 400±52 4.5±0.5 3.8±0.6 * 32.6±2.3 ** 35.7±3.0 18.1±1.3 12.6±1.1 * *P&lt;0.01 vs control period. **P&lt;0.01 vs LS. Jprox’ is Jprox adjusted for single nephron GFR by ANCOVA to obtain the "primary" effect of SGLT2 blockade on the tubule.

Effects of Potassium Supplementation and the Accompanying Anion on Cardiovascular Parameters

In general, a high NaCl intake is associated with increased blood pressure (BP), whereas dietary potassium intake has a negative correlation with BP, cardiovascular (CV) outcomes and reduces the ‘salt sensitivity’ of BP. High K+ intake reduces activity of the sodium‐chloride cotransporter NCC, promoting natiuresis, which is a proposed major mechanism for mediating the effects of K+ on BP. However, the role of the accompanying anion during K+ supplementation is a matter of debate, especially considering that low plasma chloride levels are associated with better CV outcomes. This study sought to establish an independent role of the anion during chronic potassium supplementation on cardiovascular parameters. Male mice implanted with telemetric BP monitors were initially normalized to a control diet of NaCl (0.3% Na+) and KCl (1.05% K+), and subsequently maintained on this control diet or a high NaCl (1.57% Na+) diet for 7 weeks. Subsequently, mice were stratified to receive either a high KCl (5.25% K+) or a high K‐citrate (KCit, 5.25% K+) diet during control or high NaCl intake for a subsequent 3 weeks. Finally, a subset of mice were switched to a zero KCl diet with either control or high NaCl level. Throughout the experiment BP, heart rate, activity, urinary ion excretion and plasma Na+/K+/Cllevels were monitored.Fractional excretion (FE) of K+ was significantly increased in both high K+ diets after 3 weeks and was independent of NaCl load. KCl and KCit feeding significantly increased FE of Na+ similarly but did not augment an already increased FE Na+ in mice fed high NaCl. High NaCl intake alone had no significant effect on BP, and chronic high K+ diets did not significantly reduce BP. However, overall plasma K+ levels inversely correlated with BP. Interestingly, plasma Cl− levels also inversely correlated with BP. KCl fed mice exhibited lower heart rate relative to KCit fed animals. Regression analysis points to KCit fed mice having lower BP, of about 5–10mmHg, if at the same heart rate as KCl fed animals. In contrast, 2 weeks of low K+ diet increased BP, more notably in high NaCl fed animals (BP increase of 7mmHg). NaCl‐KCit fed mice exhibited a significant 14% reduction in heart weight tibia length ratio (HW:TL) compared to NaCl‐control fed animals. No significant reduction in HW:TL was evident from NaCl‐KCl fed mice.In summary, in the timeframe of this study, potassium supplementation had limited effects on BP in normotensive mice during normal or high NaCl intake despite increasing the FE Na+. However, chronic KCit feeding may have some benefits to cardiovascular health above those promoted by KCl. The molecular mechanisms underpinning these effects are under investigation.Support or Funding InformationThis work was funded by grants from Novo Nordisk Foundation, Danish Independent Research Council and the Leducq Foundation.

Both Dietary Salt Restriction and Excess Dietary Salt Intake Exaggerate Cardiovascular Reflex Responses to Activation of the Sciatic Afferents, Aortic Depressor Nerve, and Vagal Afferents

Excess dietary salt intake exaggerates sympathetic nerve activity (SNA) and arterial blood pressure (ABP) responses to several cardiovascular reflexes in salt‐resistant animals. Recently, observational cohort studies suggest that salt restriction increases cardiovascular morbidity/mortality in several cardiovascular diseases, and the relationship between cardiovascular morbidity and dietary salt intake resembles a J‐shaped curve. However, little evidence exists to document the impact of salt restriction on cardiovascular reflex responses and whether this relationship resembles a J‐shaped curve. Thus, the aim of the present study was to determine whether salt restriction affects SNA and ABP responses to activation of sciatic afferents, the aortic depressor nerve, and cervical vagal afferents. To address this hypothesis, male Sprague‐Dawley rats were fed low (0.01% NaCl, D17010, Research diets), normal (0.1% NaCl, D17020, Research Diets), and high (4.0% NaCl, D17013, Research Diets) salt diet for 14–21 days. Then, reflex responses were measured in Inactin‐anesthetized animals. Baseline mean ABP was not different across groups (low: 112±4, normal: 101±5, high: 109±3mmHg; P&gt;0.1 overall ANOVA). Electrical stimulation of sciatic afferents (1ms pulse, 500uA, 5s duration, 2–20Hz) produced frequency‐dependent increases in ABP. Interestingly, the pressor response to sciatic nerve stimulation was significantly greater in rats fed low and high versus normal NaCl (5Hz; low: 22±4, normal: 12±2, high: 27±2 mmHg, n=5–10, P&lt;0.05). Activation of the aortic depressor nerve (2ms pulse, 500uA, 15s duration, 2–20Hz) produced frequency‐dependent decreases in ABP. The depressor response to aortic nerve stimulation was significantly greater in rats fed low and high versus normal NaCl (5Hz; low: −29±6, normal: −11±3, high: −29±4 mmHg; n=3–7, P&lt;0.05). Finally, electrical stimulation of the cervical vagal afferents (1ms pulse, 500uA, 5s duration, 2–20Hz) produced frequency‐dependent decreases in ABP. Again, the depressor response to vagal nerve stimulation was significantly greater in rats fed low and high versus normal NaCl (5Hz; low: −25±3, normal: −15±2, high: −29±2 mmHg; n=5–10, P&lt;0.05). Simultaneous recordings of lumbar, renal, and splanchnic SNA followed the changes in ABP across low, normal, and high NaCl diets. The present findings showed that both dietary salt restriction and excess dietary salt intake exaggerates sympathetic and cardiovascular responses. These cardiovascular responses to activation of sciatic afferents, the aortic depressor nerve, and vagal afferents as a function of dietary salt resemble a J‐shaped curve.Support or Funding InformationNIH R01 HL128388 (WBF, SDS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Salt-sensitive transcriptome of isolated kidney distal tubule cells.

In the distal kidney tubule, the steroid hormone aldosterone regulates sodium reabsorption via the epithelial sodium channel (ENaC). Most studies seeking to identify ENaC-regulating aldosterone-induced proteins have used transcriptional profiling of cultured cells. To identify salt-sensitive transcripts in an in vivo model, we used low-NaCl or high-NaCl diet to stimulate or suppress endogenous aldosterone, in combination with magnetic- and fluorescence-activated cell sorting to isolate distal tubule cells from mouse kidney for transcriptional profiling. Of the differentially expressed transcripts, 162 were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, and 161 were more abundant in distal tubule cells isolated from mice fed high-NaCl diet. Enrichment analysis of Gene Ontology biological process terms identified multiple statistically overrepresented pathways among the differentially expressed transcripts that were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, including ion transmembrane transport, regulation of growth, and negative regulation of apoptosis. Analysis of Gene Ontology molecular function terms identified differentially expressed transcription factors, transmembrane transporters, kinases, and G protein-coupled receptors. Finally, comparison with a recently published study of gene expression changes in distal tubule cells in response to administration of aldosterone identified 18 differentially expressed genes in common between the two experiments. When expression of these genes was measured in cortical collecting ducts microdissected from mice fed low-NaCl or high-NaCl diet, eight were differentially expressed. These genes are likely to be regulated directly by aldosterone and may provide insight into aldosterone signaling to ENaC in the distal tubule.

Nitric oxide mediates anomalous tubuloglomerular feedback in rats fed high-NaCl diet after subtotal nephrectomy.

Tubuloglomerular feedback (TGF) responses become anomalous in rats fed high-NaCl diet after subtotal nephrectomy (STN), such that stimulating TGF causes single nephron GFR (SNGFR) to increase rather than decrease. Micropuncture experiments were performed to determine whether this anomaly results from heightened nitric oxide response to distal delivery, which is a known mechanism for resetting TGF, or from connecting tubule TGF (cTGF), which is a novel amiloride-inhibitable system for offsetting TGF responses. Micropuncture was done in Wistar Froemter rats fed high-NaCl diet (HS) for 8-10 days after STN or sham nephrectomy. TGF was manipulated by orthograde microperfusion of Henle's loop with artificial tubular fluid with or without NOS inhibitor, LNMMA, or the cell-impermeant amiloride analog, benzamil. SNGFR was measured by inulin clearance in tubular fluid collections from the late proximal tubule. TGF responses were quantified as the increase in SNGFR that occurred when the perfusion rate was reduced from 50 to 8 nl/min in STN or 40 to 8 nl/min in sham animals. The baseline TGF response was anomalous in STN HS (-4 ± 3 vs 14 ± 3 nl/min, P < 0.001). TGF response was normalized by perfusing STN nephron with LNMMA (14 ± 3 nl/min, P < 0.005 for ANOVA cross term) but not with benzamil (-3 ± 4 nl/min, P = 0.4 for ANOVA cross term). Anomalous TGF occurs in STN HS due to heightened effect of tubular flow on nitric oxide signaling, which increases to the point of overriding the normal TGF response. There is no role for cTGF in this phenomenon.

Abstract 087: NPFF Receptors NPFF-R1 and NPFF-R2 in the Kidney: A Target for the Negative Control of Sodium Transport and Blood Pressure

The kidney is critical in the overall regulation of fluid, electrolyte balance and blood pressure (BP). The renal dopaminergic system inhibits sodium transport in almost all nephron segments and is responsible for ≥50% of renal sodium excretion under moderate sodium excess conditions and is considered anti-hypertensive. Neuropeptide FF (NPFF), a morphine-modulating peptide, regulates food consumption and cardiovascular function through its interaction with two receptors, NPFFR1 and NPFFR2. NPFFR2 is anti-hypertensive under “normal” salt intake but becomes pro-hypertensive when salt intake is “high”. The ultimate phenotype depends upon protein/protein interaction. Hence, we tested NPFFR2 and dopamine receptor interaction in vitro using renal proximal tubule cells (RPTCs) and blood pressure in vivo in C57Bl/6J mice. NPFF is synthesized in RPT where NPFFR2 is expressed and co-localize with D 1 R. In RPTCs (n= 3-4/group), the D 1 R/D 5 R agonist fenoldopam (1 μM/30 min), increased cAMP production (4.23±0.56 pmol/mg/min vs . 2.54±0.19, vehicle; P&lt;0.05) but was abrogated by co-treatment with NPFF (1 μM/30 (2.36±0.29 pmol/mg/min). Low NPFF (10 -10 and 10 -9 M) concentrations increased while higher concentrations had no effect (10 -8 and 10 -7 M), and slightly decreased (10 -6 M) cAMP production in RPTCs. Fenoldopam (1 μM/30 min) added to the basolateral side of polarized RPTCs grown in Transwells, increased intracellular Na + (117±4% versus the vehicle-treated control), indicating inhibition of basolateral Na + transport. This effect was prevented by co-treatment with NPFF (98±5%). Basolateral NPFF treatment alone decreased the intracellular Na + (-86.10±4.6%), indicating increased basolateral Na + transport and that renal NPFF may be a negative regulator of dopamine. In C57Bl/6J mice on 0.9% NaCl diet , renal-selective silencing of Npffr2 increased BP (115±3 vs 102±1 mmHg; P&lt;0.05; n=3-5/group). By contrast, on 4% NaCl diet, renal-selective silencing of Npffr2 decreased BP (98±4 vs 113±3 mmHg; P&lt;0.05; n=3-5/group), indicating that Npffr2 is antihypertensive on normal NaCl diet but pro-hypertensive on high NaCl diet. These contrasting effects may be due to D 1 R/D 5 R negative regulation of Npffr2 on normal salt diet which is lost when NaCl intake is increased.

Abstract 062: Renal Hydrogen Peroxide Prevents Salt-Sensitive Hypertension

C57Bl/6 and BALB/c are different in the sensitivity of their blood pressure (BP) to NaCl. High NaCl diet increases BP in C57Bl/6J but not BALB/c mice. However, C57Bl/6J mice are less prone to hypertension-induced renal injury than other mouse strains. The goal of this study was to determine the role of H 2 O 2 in salt-sensitive hypertension. Basal renal levels of reactive oxygen species, including H 2 O 2, were higher in BALB/c renal proximal tubule cells (RPTC-BALB/c) than RPTCs from C57Bl/6J mice (RPTC-C57Bl/6J) (60.8 ± 5 % vs 41.8 ± 12 %, n=8, P&lt;0.03). Incubation media with 170 mM NaCl (HS) increased H 2 O 2 production in RPTC-BALB/c but not in RPTC-C57Bl/6J, compared with RPTCs incubated in low (90 mM) or normal (145 mM) NaCl (+68±43%, 90 vs 170 mM, n=7, P&lt;0.05). H 2 O 2 (10 μM) treatment of the basolateral side of RPTC-BALB/c in Transwells increased intracellular Na + 1.62-fold that of vehicle-treated cells (n=4, P&lt;0.05). Over-expression of catalase, abrogated the H 2 O 2 -induced increase in intracellular Na + in RPTC-BALB/c. Over-expression of catalase in the kidneys of BALB/c mice on normal (0.9%) NaCl diet did not alter their SBP. However, on high (4%) NaCl diet, SBP was increased in catalase over-expressing mice, relative to vehicle-treated controls (98±1.1 vs 112±1.4 mm Hg, n=3, P&lt;0.05). Decreasing H 2 O 2 by overexpression of catalase predisposes BALB/c mice to salt-sensitive hypertension, suggesting that high salt-induced H 2 O 2 negatively regulates renal sodium transport and provides resistance to salt-induced hypertension.