Integrated renal and sympathetic mechanisms underlying the development of sex- and age-dependent hypertension and the salt sensitivity of blood pressure.

Aging is a non-modifiable understudied risk factor for hypertension. We hypothesized that sympathetically mediated activation of renal sodium reabsorption drives age-dependent hypertension and the salt sensitivity of blood pressure (BP). Using 3-, 8-, and 16-month-old male and female Sprague-Dawley rats as a model of normal aging, we assessed BP, indices of sympathetic tone, and the physiological responses to acute and chronic sodium challenge including sodium chloride cotransporter (NCC) regulation. The effects of renal nerve ablation and NCC antagonism were assessed in hypertensive male rats. We observed sex-dependent impaired renal sodium handling (24 h sodium balance (meq), male 3-month 0.36 ± 0.1 vs. 16-month 0.84 ± 0.2; sodium load excreted during 5% bodyweight isotonic saline volume expansion (%) male 3-month 77 ± 5 vs. 16-month 22 ± 8), hypertension (MAP (mmHg) male 3-month 123 ± 4 vs. 16-month 148 ± 6), and the salt sensitivity of BP in aged male, but not female, rats. Attenuated sympathoinhibitory afferent renal nerve (ARN) responses contributed to increased sympathetic tone and hypertension in male rats. Increased sympathetic tone contributes to renal sodium retention, in part through increased NCC activity via a dysfunctional with-no-lysine kinase-(WNK) STE20/SPS1-related proline/alanine-rich kinase signaling pathway, to drive hypertension and the salt sensitivity of BP in aged male rats. NCC antagonism and renal nerve ablation, which reduced WNK dysfunction and decreased NCC activity, attenuated age-dependent hypertension in male Sprague-Dawley rats. The contribution of an impaired sympathoinhibitory ARN reflex to sex- and age-dependent hypertension in an NCC-dependent manner, via an impaired WNK1/WNK4 dynamic, suggests this pathway as a mechanism-based target for the treatment of age-dependent hypertension.

Vitamin D is involved in the regulation of Cl- uptake in zebrafish (Danio rerio).

Cl- is a major anion in the bodily fluids of vertebrates, and maintaining its homeostasis is essential for normal physiological functions. Fishes inhabiting freshwater (FW) passively lose body fluid ions, including Cl-, to the external environment because of the electrochemical gradient of ions across the body surface. Therefore, FW fishes have to actively absorb Cl- from the surroundings to maintain ion homeostasis in their bodily fluids. Hormonal control is vital for modulating ion uptake in fish. Vitamin D is involved in the regulation of Ca2+ uptake and acid secretion in fish. In the present study, we found that the levels of bioactive vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), significantly increased in zebrafish embryos and adults after exposure to water containing low levels of Cl-. Moreover, the administration of 1α,25(OH)2D3 treatment (20 µg/L) in zebrafish embryos, and intraperitoneal (i.p.) injection of 1α,25(OH)2D3 (5 µg/kg body mass) in zebrafish adults, resulting the increased Cl- content in bodily fluid in zebrafish. Na+-Cl- cotransporter 2b (NCC2b) and Cl- channel 2c (CLC2c) are specifically expressed during Cl- uptake by ionocytes in zebrafish. Our results indicated that the mRNA and protein expression of NCC2b and CLC2c considerably increased in the zebrafish with exogenous 1α,25(OH)2D3 treatment. Additionally, exogenous 1α,25(OH)2D3 administration increased the number of NCC2b- and CLC2c-expressing cells in yolk skins of zebrafish embryos and the gill filaments of zebrafish adults. Transcript signals of vitamin D receptors (VDRs) were identified in NCC2b-expressing cells. Knockdown of VDRa and VDRb significantly reduced the expression of NCC2b and CLC2c and the number of NCC2b- and CLC2c-expressing cells. These results indicate that vitamin D can affect Cl- uptake in zebrafish and extend our knowledge of the role of vitamin D in fish physiology.

Chronic kidney disease increases the susceptibility to negative effects of low and high potassium intake.

BACKGROUND AND HYPOTHESIS: Dietary potassium (K+) has emerged as a modifiable factor for cardiovascular and kidney health in the general population, but its role in people with chronic kidney disease (CKD) is unclear. Here, we hypothesize that CKD increases the susceptibility to negative effects of low and high K+ diets. METHODS: We compared the effects of low, normal, or high KChloride (KCl) diets and a high KCitrate diet for four weeks in male rats with normal kidney function and in male rats with CKD using the 5/6th nephrectomy model (5/6Nx). RESULTS: Compared to rats with normal kidney function, 5/6Nx rats on the low KCl diet developed more severe extracellular and intracellular hypokalemia and more severe kidney injury, characterized by nephromegaly, infiltration of T-cells and macrophages, decreased eGFR and increased albuminuria. The high KCl diet caused hyperkalemia, hyperaldosteronism, hyperchloremic metabolic acidosis and severe hypertension in 5/6Nx but not in sham rats. The high KCitrate diet caused hypochloremic metabolic alkalosis but attenuated hypertension despite higher abundance of the phosphorylated sodium chloride cotransporter (pNCC) and similar levels of plasma aldosterone and epithelial sodium channel (ENaC) abundance. All 5/6Nx groups had more collagen deposition than the sham groups and this effect was most pronounced in the high KCitrate group. Plasma aldosterone correlated strongly with kidney collagen deposition. CONCLUSIONS: CKD increases the susceptibility to negative effects of low and high K+ diets in male rats, although the injury patterns are different. The low K+ diet caused inflammation, nephromegaly and kidney function decline, whereas the high K+ diet caused hypertension, hyperaldosteronism and kidney fibrosis. High KCitrate attenuated the hypertensive but not the pro-fibrotic effect of high KCl, which may be attributable to K+-induced aldosterone secretion. Our data suggest that especially in people with CKD it is important to identify the optimal threshold of dietary K+ intake.

Randomized Trial on the Effect of Oral Potassium Chloride Supplementation on the Thiazide-Sensitive Sodium Chloride Cotransporter in Healthy Adults.

Introduction: The putative "renal-K switch" mechanism links dietary potassium intake with sodium retention and involves activation of the sodium chloride (NaCl) cotransporter (NCC) in the distal convoluted tubule in response to low potassium intake, and suppression in response to high potassium intake. This study examined NCC abundance and phosphorylation (phosphorylated NCC [pNCC]) in urinary extracellular vesicles (uEVs) isolated from healthy adults on a high sodium diet to determine tubular responses to alteration in potassium chloride (KCl) intake. Methods: Healthy adults maintained on a high sodium (∼4.5 g [200 mmol]/d) low potassium (∼2.3 g [60 mmol]/d) diet underwent a 5-day run-in period followed by a crossover study, with 5-day supplementary KCl (active phase, Span-K 3 tablets (potassium 24 mmol) thrice daily) or 5-day placebo administrated in random order and separated by 2-day washout. Ambulatory blood pressure (BP) and biochemistries were assessed, and uEVs were analyzed by western blotting. Results: Among the 18 participants who met analysis criteria, supplementary KCl administration (vs. placebo) was associated with markedly higher levels of plasma potassium and 24-hour urine excretion of potassium, chloride, and aldosterone. KCl supplementation was associated with lower uEV levels of NCC (median fold change (KCl/Placebo) = 0.74 [0.30-1.69], P < 0.01) and pNCC (fold change (KCl/Placebo) = 0.81 [0.19-1.75], P < 0.05). Plasma potassium inversely correlated with uEV NCC (R2 = 0.11, P = 0.05). Conclusions: The lower NCC and pNCC in uEVs in response to oral KCl supplementation provide evidence to support the hypothesis of a functional "renal-K switch" in healthy human subjects.

Regulation of Distal Nephron Transport by Intracellular Chloride and Potassium.

BACKGROUND: Low potassium increases the phosphorylation and activity of the sodium chloride cotransporter (NCC) in the distal convoluted tubule of the nephron, which contributes to the hypertensive effect of the modern low potassium/high sodium diet. A central mediator of potassium regulation of NCC is the chloride-sensitive With No Lysine [K] (WNK) kinase. SUMMARY: Chloride directly inhibits WNKs by binding to the active site. The mechanisms underlying WNK regulation by extracellular potassium are reviewed, as well as the modulatory effect of kidney-specific-WNK1. WNK1, but not WNK1 kinase activity, is also required for the aldosterone-independent regulation of the epithelial sodium channel by potassium. Whether intracellular chloride could be involved in this process is discussed. Recent studies demonstrating direct regulation of WNKs by intracellular potassium are also reviewed, and the potential physiological relevance to renal epithelial ion transport is discussed. KEY MESSAGES: WNKs are sensors of the intracellular ionic milieu. In the nephron, changes in extracellular ion concentrations, resulting in changes in intracellular ion concentration, regulate WNK activity and downstream transporters and channels to maintain total body ion homeostasis.

Using human urinary extracellular vesicles to study physiological and pathophysiological states and regulation of the sodium chloride cotransporter.

The thiazide-sensitive sodium chloride cotransporter (NCC), expressed in the renal distal convoluted tubule, plays a major role in Na+, Cl- and K+ homeostasis and blood pressure as exemplified by the symptoms of patients with non-functional NCC and Gitelman syndrome. NCC activity is modulated by a variety of hormones, but is also influenced by the extracellular K+ concentration. The putative "renal-K+ switch" mechanism is a relatively cohesive model that links dietary K+ intake to NCC activity, and may offer new targets for blood pressure control. However, a remaining hurdle for full acceptance of this model is the lack of human data to confirm molecular findings from animal models. Extracellular vesicles (EVs) have attracted attention from the scientific community due to their potential roles in intercellular communication, disease pathogenesis, drug delivery and as possible reservoirs of biomarkers. Urinary EVs (uEVs) are an excellent sample source for the study of physiology and pathology of renal, urothelial and prostate tissues, but the diverse origins of uEVs and their dynamic molecular composition present both methodological and data interpretation challenges. This review provides a brief overview of the state-of-the-art, challenges and knowledge gaps in current uEV-based analyses, with a focus on the application of uEVs to study the "renal-K+ switch" and NCC regulation. We also provide recommendations regarding biospecimen handling, processing and reporting requirements to improve experimental reproducibility and interoperability towards the realisation of the potential of uEV-derived biomarkers in hypertension and clinical practice.

Acute Intravenous NaCl and Volume Expansion Reduces Sodium-Chloride Cotransporter Abundance and Phosphorylation in Urinary Extracellular Vesicles.

Background: Sodium chloride (NaCl) loading and volume expansion suppress the renin-angiotensin-aldosterone system to reduce renal tubular reabsorption of NaCl and water, but effects on the sodium-chloride cotransporter (NCC) and relevant renal transmembrane proteins that are responsible for this modulation in humans are less well investigated. Methods: We used urinary extracellular vesicles (uEVs) as an indirect readout to assess renal transmembrane proteins involved in NaCl and water homeostasis in 44 patients with hypertension who had repeatedly raised aldosterone/renin ratios undergoing infusion of 2 L of 0.9% saline over 4 hours. Results: When measured by mass spectrometry in 13 patients, significant decreases were observed in NCC (median fold change [FC]=0.70); pendrin (FC=0.84); AQP2 (FC=0.62); and uEV markers, including ALIX (FC=0.65) and TSG101 (FC=0.66). Immunoblotting reproduced the reduction in NCC (FC=0.54), AQP2 (FC=0.42), ALIX (FC=0.52), and TSG101 (FC=0.55) in the remaining 31 patients, and demonstrated a significant decrease in phosphorylated NCC (pNCC; FC=0.49). However, after correction for ALIX, the reductions in NCC (FC=0.90) and pNCC (FC=1.00) were no longer apparent, whereas the significant decrease in AQP2 persisted (FC=0.62). Conclusion: We conclude that (1) decreases in NCC and pNCC, induced by acute NaCl loading and volume expansion, may be due to diluted post-test urines; (2) the lack of change of NCC and pNCC when corrected for ALIX, despite a fall in plasma aldosterone, may be due to the lack of change in plasma K+; and (3) the decrease in AQP2 may be due to a decrease in vasopressin in response to volume expansion.

Targeting Calcineurin Inhibitor-Induced Arterial Hypertension in Liver Transplanted Children Using Hydrochlorothiazide.

OBJECTIVE: Arterial hypertension (AH) is the most common toxic effect of calcineurin inhibitor (CNI)-based immunosuppression in children after liver transplantation (LT). Activation of the renal sodium chloride cotransporter (NCC) by CNIs has been described as a major cause of CNI-induced AH. Thiazides, for example, hydrochlorothiazide (HCTZ), can selectively block the NCC and may ameliorate CNI-induced AH after pediatric LT. METHODS: From 2005 thru 2015 we conducted a retrospective, single-center analysis of blood pressure in 2 pediatric cohorts (each n = 33) with or without HCTZ in their first year after LT. All patients received CNI-based immunosuppression. According to AAP guidelines, AH was defined as stage 1 and stage 2. Cohort 1 received an HCTZ-containing regimen to target the CNI-induced effect on the NCC, leading to AH. Cohort 2 received standard antihypertensive therapy without HCTZ. RESULTS: In children who have undergone LT and been treated with CNI, AH overall was observed less frequently in cohort 1 vs cohort 2 (31% vs 44%; ns). Moreover, severe AH (stage 2) was significantly lower in cohort 1 vs 2 (1% vs 18%; p < 0.001). Multivariate analysis revealed HCTZ as the only significant factor with a protective effect on occurrence of severe stage 2 AH. While monitoring safety and tolerability, mild asymptomatic hypokalemia was the only adverse effect observed more frequently in cohort 1 vs 2 (27% vs 3%; p = 0.013). CONCLUSIONS: Targeting NCC by HCTZ significantly improved control of severe CNI-induced AH and was well tolerated in children who underwent LT. This effect may reduce the risk of long-term end-organ damage and improve quality of life.

Polymorphisms in common antihypertensive targets: Pharmacogenomic implications for the treatment of cardiovascular disease.

The idea of personalized medicine came to fruition with sequencing the human genome; however, aside from a few cases, the genetic revolution has yet to materialize. Cardiovascular diseases are the leading cause of death globally, and hypertension is a common prelude to nearly all cardiovascular diseases. Thus, hypertension is an ideal candidate disease to apply tenants of personalized medicine to lessen cardiovascular disease. Herein is a survey that visually depicts the polymorphisms in the top eight antihypertensive targets. Although there are numerous genome-wide association studies regarding cardiovascular disease, few studies look at the effects of receptor polymorphisms on drug treatment. With 17,000+ polymorphisms in the combined target proteins examined, it is expected that some of the clinical variability in the treatment of hypertension is due to polymorphisms in the drug targets. Recent advances in techniques and technology, such as high throughput examination of single mutations, structure prediction, computational power for modeling, and CRISPR models of point mutations, allow for a relatively rapid and comprehensive examination of the effects of known and future polymorphisms on drug affinity and effects. As hypertension is easy to measure and has a plethora of clinically viable ligands, hypertension makes an excellent disease to study pharmacogenomics in the lab and the clinic. If the promises of personalized medicine are to materialize, a concerted effort to examine the effects polymorphisms have on drugs is required. A clinician with the knowledge of a patient's genotype can then prescribe drugs that are optimal for treating that specific patient.

Potassium and the kidney: a reciprocal relationship with clinical relevance.

By controlling urinary potassium excretion, the kidneys play a key role in maintaining whole-body potassium homeostasis. Conversely, low urinary potassium excretion (as a proxy for insufficient dietary intake) is increasingly recognized as a risk factor for the progression of kidney disease. Thus, there is a reciprocal relationship between potassium and the kidney: the kidney regulates potassium balance but potassium also affects kidney function. This review explores this relationship by discussing new insights into kidney potassium handling derived from recently characterized tubulopathies and studies on sexual dimorphism. These insights reveal a central but non-exclusive role for the distal convoluted tubule in sensing potassium and subsequently modifying the activity of the sodium-chloride cotransporter. This is another example of reciprocity: activation of the sodium-chloride cotransporter not only reduces distal sodium delivery and therefore potassium secretion but also increases salt sensitivity. This mechanism helps explain the well-known relationship between dietary potassium and blood pressure. Remarkably, in children, blood pressure is related to dietary potassium but not sodium intake. To explore how potassium deficiency can cause kidney injury, we review the mechanisms of hypokalemic nephropathy and discuss if these mechanisms may explain the association between low dietary potassium intake and adverse kidney outcomes. We discuss if potassium should be repleted in patients with kidney disease and what role dietary potassium plays in the risk of hyperkalemia. Supported by data and physiology, we reach the conclusion that we should view potassium not only as a potentially dangerous cation but also as a companion in the battle against kidney disease.

Activation of the Thiazide-Sensitive Sodium-Chloride Cotransporter by Beta3-Adrenoreceptor in the Distal Convoluted Tubule.

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT.

Sex differences in prenatal programming of hypertension by dexamethasone.

Prenatal dexamethasone has been shown to increase blood pressure in male offspring but the mechanism for the increase in blood pressure is unclear. The present study examined if prenatal programming by maternal injection of dexamethasone on days 15 and 16 of gestation affected the blood pressure comparably in female and male offspring. Our hypothesis was that males would be affected by prenatal dexamethasone to a greater extent than females and that either an increase in renal tubular transporter abundance or an increase in renin or aldosterone system would be associated with hypertension with prenatal programming. Prenatal dexamethasone increased blood pressure at two months and six months of age and resulted in proteinuria and albuminuria at six months in male but not female rat offspring. There was no effect of prenatal dexamethasone on blood pressure and proteinuria at one month in male and in female offspring. While prenatal dexamethasone increased male renal thick ascending limb sodium potassium two chloride cotransporter protein abundance at two months, prenatal dexamethasone on days 15 and 16 of gestation did not affect transporter abundance in males at other ages, nor did it affect proximal tubule sodium/hydrogen exchanger or distal convoluted tubule sodium chloride cotransporter protein abundance at any age. There was no difference in systemic renin or aldosterone in the prenatal dexamethasone group compared to same sex controls. In conclusion, male but not female offspring have an increase in blood pressure and urinary protein excretion with prenatal dexamethasone. The increase in blood pressure with prenatal programming was not associated with a consistent increase in renal tubular transporter protein abundance, nor plasma renin activity and serum aldosterone.

Thiazide-Sensitive NCC (Sodium-Chloride Cotransporter) in Human Metabolic Syndrome: Sodium Sensitivity and Potassium-Induced Natriuresis.

The thiazide-sensitive sodium-chloride cotransporter (NCC;SLC12A3) is central to sodium and blood pressure regulation. Metabolic syndrome induces NCC upregulation generating sodium-sensitive hypertension in experimental animal models. We tested the role of NCC in sodium sensitivity in hypertensive humans with metabolic syndrome. Conversely, oral potassium induces NCC downregulation producing potassium-induced natriuresis. We determined the time course and magnitude of potassium-induced natriuresis compared with the natriuresis following hydrochlorothiazide (HCTZ) as a reference standard. We studied 19 obese hypertensive humans with metabolic syndrome during 13-day inpatient confinement. We determined sodium sensitivity by change in 24-hour mean systolic pressure by automated monitor from days 5 (low sodium) to 10 (high sodium). We determined NCC activity by standard 50 mg HCTZ sensitivity test (day 11). We determined potassium-induced natriuresis following 35 mmol KCl (day 13). We determined (1) whether NCC activity was greater in sodium-sensitive versus sodium-resistant participants and correlated with sodium sensitivity and (2) time course and magnitude of potassium-induced natriuresis following 35 mmol KCl directly compared with 50 mg HCTZ. NCC activity was not greater in sodium-sensitive versus sodium-resistant humans and did not correlate with sodium sensitivity. Thirty-five-millimoles KCl produced a rapid natriuresis approximately half that of 50 mg HCTZ with a greater kaliuresis. Our investigation tested a key hypothesis regarding NCC activity in human hypertension and characterized potassium-induced natriuresis following 35 mmol KCl compared with 50 mg HCTZ. In obese hypertensive adults with metabolic syndrome ingesting a high-sodium diet, 35 mmol KCl had a net natriuretic effect approximately half that of 50 mg HCTZ.

WNK-SPAK/OSR1-NCC kinase signaling pathway as a novel target for the treatment of salt-sensitive hypertension.

Hypertension is the most prevalent health condition worldwide, affecting ~1 billion people. Gordon's syndrome is a form of secondary hypertension that can arise due to a number of possible mutations in key genes that encode proteins in a pathway containing the With No Lysine [K] (WNK) and its downstream target kinases, SPS/Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1). This pathway regulates the activity of the thiazide-sensitive sodium chloride cotransporter (NCC), which is responsible for NaCl reabsorption in the distal nephron. Therefore, mutations in genes encoding proteins that regulate the NCC proteins disrupt ion homeostasis and cause hypertension by increasing NaCl reabsorption. Thiazide diuretics are currently the main treatment option for Gordon's syndrome. However, they have a number of side effects, and chronic usage can lead to compensatory adaptations in the nephron that counteract their action. Therefore, recent research has focused on developing novel inhibitory molecules that inhibit components of the WNK-SPAK/OSR1-NCC pathway, thereby reducing NaCl reabsorption and restoring normal blood pressure. In this review we provide an overview of the currently reported molecular inhibitors of the WNK-SPAK/OSR1-NCC pathway and discuss their potential as treatment options for Gordon's syndrome.

Distal convoluted tubule sexual dimorphism revealed by advanced 3D imaging.

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is more abundant in kidneys of female subjects than of male subjects. Because morphological remodeling of the distal convoluted tubule (DCT) is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in the structure of the DCT, leading to a larger female DCT. Until now, this has never been directly examined. Here, optical clearing techniques were combined with antibody labeling of DCT segment markers, state-of-the-art high-speed volumetric imaging, and analysis tools to visualize and quantify DCT morphology in male and female mice and study the DCT remodeling response to furosemide. We found an unexpected sex difference in the structure of the DCT. Compared with the male mice, female mice had a shorter DCT, a higher cellular density of NCC, and a greater capacity to elongate in response to loop diuretics. Our study revealed a sexual dimorphism of the DCT. Female mice expressed a greater density of NCC transporters in a shorter structure to protect Na+ balance in the face of greater basal distal Na+ delivery yet have a larger reserve and structural remodeling capacity to adapt to unique physiological stresses. These observations provide insight into mechanisms that may drive sex differences in the therapeutic responses to diuretics.

Sympathetic Regulation of the NCC (Sodium Chloride Cotransporter) in Dahl Salt-Sensitive Hypertension.

Increased sympathoexcitation and renal sodium retention during high salt intake are hallmarks of the salt sensitivity of blood pressure. The mechanism(s) by which excessive sympathetic nervous system release of norepinephrine influences renal sodium reabsorption is unclear. However, studies demonstrate that norepinephrine can stimulate the activity of the NCC (sodium chloride cotransporter) and promote the development of SSH (salt-sensitive hypertension). The adrenergic signaling pathways governing NCC activity remain a significant source of controversy with opposing studies suggesting a central role of upstream α1- and ß-adrenoceptors in the canonical regulatory pathway involving WNKs (with-no-lysine kinases), SPAK (STE20/SPS1-related proline alanine-rich kinase), and OxSR1 (oxidative stress response 1). In our previous study, α1-adrenoceptor antagonism in norepinephrine-infused male Sprague-Dawley rats prevented the development of norepinephrine-evoked SSH in part by suppressing NCC activity and expression. In these studies, we used selective adrenoceptor antagonism in male Dahl salt-sensitive rats to test the hypothesis that norepinephrine-mediated activation of the NCC in Dahl SSH occurs via an α1-adrenoceptor dependent pathway. A high-salt diet evoked significant increases in NCC activity, expression, and phosphorylation in Dahl salt-sensitive rats that developed SSH. Increases were associated with a dysfunctional WNK1/4 dynamic and a failure to suppress SPAK/OxSR1 activity. α1-adrenoceptor antagonism initiated before high-salt intake or following the establishment of SSH attenuated blood pressure in part by suppressing NCC activity, expression, and phosphorylation. Collectively, our findings support the existence of a norepinephrine-activated α1-adrenoceptor gated pathway that relies on WNK/SPAK/OxSR1 signaling to regulate NCC activity in SSH.

Two Mineralocorticoid Receptor-Mediated Mechanisms of Pendrin Activation in Distal Nephrons.

BACKGROUND: Regulation of sodium chloride transport in the aldosterone-sensitive distal nephron is essential for fluid homeostasis and BP control. The chloride-bicarbonate exchanger pendrin in ß-intercalated cells, along with sodium chloride cotransporter (NCC) in distal convoluted tubules, complementarily regulate sodium chloride handling, which is controlled by the renin-angiotensin-aldosterone system. METHODS: Using mice with mineralocorticoid receptor deletion in intercalated cells, we examined the mechanism and roles of pendrin upregulation via mineralocorticoid receptor in two different models of renin-angiotensin-aldosterone system activation. We also used aldosterone-treated NCC knockout mice to examine the role of pendrin regulation in salt-sensitive hypertension. RESULTS: Deletion of mineralocorticoid receptor in intercalated cells suppressed the increase in renal pendrin expression induced by either exogenous angiotensin II infusion or endogenous angiotensin II upregulation via salt restriction. When fed a low-salt diet, intercalated cell-specific mineralocorticoid receptor knockout mice with suppression of pendrin upregulation showed BP reduction that was attenuated by compensatory activation of NCC. In contrast, upregulation of pendrin induced by aldosterone excess combined with a high-salt diet was scarcely affected by deletion of mineralocorticoid receptor in intercalated cells, but depended instead on hypokalemic alkalosis through the activated mineralocorticoid receptor-epithelial sodium channel cascade in principal cells. In aldosterone-treated NCC knockout mice showing upregulation of pendrin, potassium supplementation corrected alkalosis and inhibited the pendrin upregulation, thereby lowering BP. CONCLUSIONS: In conjunction with NCC, the two pathways of pendrin upregulation, induced by angiotensin II through mineralocorticoid receptor activation in intercalated cells and by alkalosis through mineralocorticoid receptor activation in principal cells, play important roles in fluid homeostasis during salt depletion and salt-sensitive hypertension mediated by aldosterone excess.

Sympathetic regulation of NCC in norepinephrine-evoked salt-sensitive hypertension in Sprague-Dawley rats.

Salt sensitivity of blood pressure is characterized by inappropriate sympathoexcitation and renal Na+ reabsorption during high salt intake. In salt-resistant animal models, exogenous norepinephrine (NE) infusion promotes salt-sensitive hypertension and prevents dietary Na+-evoked suppression of the Na+-Cl- cotransporter (NCC). Studies of the adrenergic signaling pathways that modulate NCC activity during NE infusion have yielded conflicting results implicating α1- and/or ß-adrenoceptors and a downstream kinase network that phosphorylates and activates NCC, including with no lysine kinases (WNKs), STE20/SPS1-related proline-alanine-rich kinase (SPAK), and oxidative stress response 1 (OxSR1). In the present study, we used selective adrenoceptor antagonism in NE-infused male Sprague-Dawley rats to investigate the differential roles of α1- and ß-adrenoceptors in sympathetically mediated NCC regulation. NE infusion evoked salt-sensitive hypertension and prevented dietary Na+-evoked suppression of NCC mRNA, protein expression, phosphorylation, and in vivo activity. Impaired NCC suppression during high salt intake in NE-infused rats was paralleled by impaired suppression of WNK1 and OxSR1 expression and SPAK/OxSR1 phosphorylation and a failure to increase WNK4 expression. Antagonism of α1-adrenoceptors before high salt intake or after the establishment of salt-sensitive hypertension restored dietary Na+-evoked suppression of NCC, resulted in downregulation of WNK4, SPAK, and OxSR1, and abolished the salt-sensitive component of hypertension. In contrast, ß-adrenoceptor antagonism attenuated NE-evoked hypertension independently of dietary Na+ intake and did not restore high salt-evoked suppression of NCC. These findings suggest that a selective, reversible, α1-adenoceptor-gated WNK/SPAK/OxSR1 NE-activated signaling pathway prevents dietary Na+-evoked NCC suppression, promoting the development and maintenance of salt-sensitive hypertension.

Glucocorticoid receptor activation stimulates the sodium-chloride cotransporter and influences the diurnal rhythm of its phosphorylation.

The sodium-chloride cotransporter (NCC) in the distal convoluted tubule contributes importantly to sodium balance and blood pressure (BP) regulation. NCC phosphorylation determines transport activity and has a diurnal rhythm influenced by glucocorticoids. Disturbing this rhythm induces "nondipping" BP, an abnormality that increases cardiovascular risk. The receptor through which glucocorticoids regulate NCC is not known. In this study, we found that acute administration of corticosterone to male C57BL6 mice doubled NCC phosphorylation without affecting total NCC abundance in both adrenalectomized and adrenal-intact mice. Corticosterone also increased the whole kidney expression of canonical clock genes: period circadian protein homolog 1 (Per1), Per2, cryptochrome 1, and aryl hydrocarbon receptor nuclear translocator-like protein 1. In adrenal-intact mice, chronic blockade of glucocorticoid receptor (GR) with RU486 did not change total NCC but prevented corticosterone-induced NCC phosphorylation and activation of clock genes. Blockade of mineralocorticoid receptor (MR) with spironolactone reduced the total pool of NCC but did not affect stimulation by corticosterone. The diurnal rhythm of NCC phosphorylation, measured at 6-h intervals, was blunted by chronic GR blockade, and a similar dampening of diurnal variation was seen in GR heterozygous null mice. These effects on NCC phosphorylation did not reflect altered rhythmicity of plasma corticosterone or serum and glucocorticoid-induced kinase 1 activity. Both mineralocorticoids and glucocorticoids emerge as regulators of NCC, acting via distinct receptor pathways. MR activation provides maintenance of the NCC protein pool; GR activation dynamically regulates NCC phosphorylation and establishes the diurnal rhythm of NCC activity. This study has implications for circadian BP homeostasis, particularly in individuals with abnormal glucocorticoid signaling as is found in chronic stress and corticosteroid therapy.

Dietary Na+ intake in healthy humans changes the urine extracellular vesicle prostasin abundance while the vesicle excretion rate, NCC, and ENaC are not altered.

Low Na+ intake activates aldosterone signaling, which increases renal Na+ reabsorption through increased apical activity of the NaCl cotransporter (NCC) and the epithelial Na+ channel (ENaC). Na+ transporter proteins are excreted in urine as an integral part of cell-derived extracellular vesicles (uEVs). It was hypothesized that Na+ transport protein levels in uEVs from healthy humans reflect their physiological regulation by aldosterone. Urine and plasma samples from 10 healthy men (median age: 22.8 yr) were collected after 5 days on a low-Na+ (70 mmol/day) diet and 5 days on a high-Na+ (250 mmol/day) diet. uEVs were isolated by ultracentrifugation and analyzed by Western blot analysis for EV markers (CD9, CD63, and ALIX), transport proteins (Na+-K+-ATPase α1-subunit, NCC, ENaC α- and γ-subunits, and aquaporin 2), and the ENaC-cleaving protease prostasin. Plasma renin and aldosterone concentrations increased during the low-Na+ diet. uEV size and concentration were not different between diets by tunable resistive pulse sensing. EV markers ALIX and CD9 increased with the low-Na+ diet, whereas CD63 and aquaporin 2 excretion were unchanged. Full-length ENaC γ-subunits were generally not detectable in uEVs, whereas ENaC α-subunits, NCC, and phosphorylated NCC were consistently detected but not changed by Na+ intake. Prostasin increased with low Na+ in uEVs. uEV excretion of transporters was not correlated with blood pressure, urinary Na+ and K+ excretion, plasma renin, or aldosterone. In conclusion, apical Na+ transporter proteins and proteases were excreted in uEVs, and while the excretion rate and size of uEVs were not affected, EV markers and prostasin increased in response to the low-Na+ diet.