Regulation Of NCC Research Articles

Sex differences in dietary sodium evoked NCC regulation and blood pressure in male and female Sprague-Dawley, Dahl salt-resistant, and Dahl salt-sensitive rats.

Hypertension affects approximately one in two United States adults and sex plays an important role in the pathogenesis of hypertension. The Na+-Cl- cotransporter (NCC), regulated by a kinase network including with-no-lysine kinase (WNK)1 and WNK4, STE20/SPS1-related proline alanine-rich kinase (SPAK), and oxidative stress response 1 (OxSR1), is critical to Na+ reabsorption and blood pressure regulation. Dietary salt differentially modulates NCC in salt-sensitive and salt-resistant rats, in part by modulation of WNK/SPAK/OxSR1 signaling. In this study, we tested the hypothesis that sex-dependent differences in NCC regulation contribute to the development of the salt sensitivity of blood pressure using male and female Sprague-Dawley (SD), Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats. In normotensive salt-resistant SD and DSR rats, a high-salt diet evoked significant decreases in NCC activity, expression, and phosphorylation. In males, these changes were associated with no change in WNK1 expression, a decrease in WNK4 levels, and suppression of SPAK/OxSR1 expression and phosphorylation. In contrast, in females, there was decreased NCC activity associated with suppression of SPAK/OxSR1 expression and phosphorylation. In hypertensive DSS rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension. Collectively, our findings support the existence of sex differences in male versus female rats with NCC regulation during dietary salt intake involving suppression of WNK4 expression in male rats only and the involvement of SPAK/OxSR1 signaling in both males and females.NEW & NOTEWORTHY NCC regulation is sex dependent. In normotensive male and female Sprague-Dawley and Dahl salt-resistant rats, which exhibit dietary Na+-evoked NCC suppression, male rats exhibit decreased WNK4 expression and decreased SPAK and OxSR1 levels, whereas female rats only suppress SPAK and OxSR1. In hypertensive Dahl salt-sensitive rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension.

The Total Denervation of the Ischemic Kidney Induces Differential Responses in Sodium Transporters' Expression in the Contralateral Kidney in Goldblatt Rats.

The Goldblatt model of hypertension (2K-1C) in rats is characterized by renal sympathetic nerve activity (rSNA). We investigated the effects of unilateral renal denervation of the clipped kidney (DNX) on sodium transporters of the unclipped kidneys and the cardiovascular, autonomic, and renal functions in 2K-1C and control (CTR) rats. The mean arterial pressure (MAP) and rSNA were evaluated in experimental groups. Kidney function and NHE3, NCC, ENaCβ, and ENaCγ protein expressions were assessed. The glomerular filtration rate (GRF) and renal plasma flow were not changed by DNX, but the urinary (CTR: 0.0042 ± 0.001; 2K-1C: 0.014 ± 0.003; DNX: 0.005 ± 0.0013 mL/min/g renal tissue) and filtration fractions (CTR: 0.29 ± 0.02; 2K-1C: 0.51 ± 0.06; DNX: 0.28 ± 0.04 mL/min/g renal tissue) were normalized. The Na+/H+ exchanger (NHE3) was reduced in 2K-1C, and DNX normalized NHE3 (CTR: 100 ± 6; 2K-1C: 44 ± 14, DNX: 84 ± 13%). Conversely, the Na+/Cl- cotransporter (NCC) was increased in 2K-1C and was reduced by DNX (CTR: 94 ± 6; 2K-1C: 144 ± 8; DNX: 60 ± 15%). In conclusion, DNX in Goldblatt rats reduced blood pressure and proteinuria independently of GRF with a distinct regulation of NHE3 and NCC in unclipped kidneys.

Does Uromodulin Play a Role in Tacrolimus-Mediated Upregulation of the Sodium Chloride Cotransporter?

Background: Tacrolimus, a calcineurin inhibitor (CNI), is a medication commonly prescribed to prevent organ transplant rejection. However, chronic CNI use has several adverse effects, including hypertension. Recent studies have revealed that the renal sodium chloride cotransporter (NCC) mediates tacrolimus-induced hypertension. However, the mechanisms underlying NCC upregulation are not fully understood. Previous studies indicate that uromodulin plays a key role in renal ion channel regulation. As such, we hypothesize that uromodulin mediates tacrolimus induced NCC upregulation. Experimental Design: To test this hypothesis, male C57BL/6 mice received tacrolimus (10 mg/kg/day) or vehicle (ethanol/DMSO/saline) for 21 consecutive days via intraperitoneal injection. The impact of tacrolimus on systolic blood pressure (BP) was monitored by tail-cuff plethysmography. To examine uromodulin and NCC expression, kidneys were harvested and processed for immunohistochemistry. Results: Compared to the BP of vehicle-treated mice (120 mm Hg ± 4.252), mice administered tacrolimus had significant increases in BP (170 mm Hg ± 3.528). Furthermore, tacrolimus treatment increased phosphorylated NCC (pNCC) and uromodulin expression. Conclusions: These results indicate that tacrolimus-induced hypertension is accompanied by pNCC and uromodulin upregulation in distal convoluted tubules. Significance: Overall, these findings highlight uromodulin as a possible regulator of NCC. Funding: R21 DK119879, R01 DK-133698, R25DK07838. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

NCC regulation by WNK/SPAK pathway is influenced by alkalosis

The WNK/SPAK “potassium-switch” pathway provides a homeostatic mechanism that activates the sodium chloride cotransporter, NCC, to limit urinary potassium excretion in dietary potassium deficiency at the expense of increasing salt-sensitivity and blood pressure. Although it is well understood that high dietary potassium turns off the switch, and decreases NCC activity, several reports suggest the counter anion in potassium-rich diets may have an influence on the pathway. The goal of the present study was to determine if the chronic response to a high K diet depends on the counter anion. Wild-type C57BL6J mice were randomized to control (1%K+), potassium chloride (KCl: 5%K+), or potassium bicarbonate (KHCO3: 5%K+) diets, and physiological, molecular, and imaging analyses were performed at 4 and 10 days of diet consumption. In contrast to the classic response to high KCl diet, characterized by reduced NCC phosphorylation and membrane localization, NCC phosphorylation and apical localization increased after 10 days on the high KHCO3 diet. This response was paralleled by increased phosphorylation of the downstream potassium-switch kinase, SPAK, which in turn phosphorylates NCC. Immunofluorescence imaging revealed WNK4 bodies were more numerous and larger in the high KHCO3 group compared to the control, consistent with activation of upstream WNK signaling. Urine excretion studies revealed mice on the high KHCO3 retain more Na+ than mice on control or KCl diets, paralleling enhanced NCC activation. Unlike mice on the high KCl diet, mice on the high KHCO3 diet develop and maintain metabolic alkalosis and do not exhibit profound hyperkalemia, coincident with more robust activation of ENaC and ROMK than with the high KCl diet. In summary, these data identify an unexpected NCC activation mechanism that may limit distal sodium delivery to prevent excessive potassium losses when alkalosis super activates potassium secretion in more distal segments. NIDDK, LeDucq Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The serine-threonine protein phosphatases that regulate the thiazide-sensitive NaCl cotransporter.

The activity of the Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) is finely tuned by phosphorylation networks involving serine/threonine kinases and phosphatases. While much attention has been paid to the With-No-lysine (K) kinase (WNK)- STE20-related Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive kinase 1 (OSR1) signaling pathway, there remain many unanswered questions regarding phosphatase-mediated modulation of NCC and its interactors. The phosphatases shown to regulate NCC's activity, directly or indirectly, are protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), calcineurin (CN), and protein phosphatase 4 (PP4). PP1 has been suggested to directly dephosphorylate WNK4, SPAK, and NCC. This phosphatase increases its abundance and activity when extracellular K+ is increased, which leads to distinct inhibitory mechanisms towards NCC. Inhibitor-1 (I1), oppositely, inhibits PP1 when phosphorylated by protein kinase A (PKA). CN inhibitors, like tacrolimus and cyclosporin A, increase NCC phosphorylation, giving an explanation to the Familial Hyperkalemic Hypertension-like syndrome that affects some patients treated with these drugs. CN inhibitors can prevent high K+-induced dephosphorylation of NCC. CN can also dephosphorylate and activate Kelch-like protein 3 (KLHL3), thus decreasing WNK abundance. PP2A and PP4 have been shown in in vitro models to regulate NCC or its upstream activators. However, no studies in native kidneys or tubules have been performed to test their physiological role in NCC regulation. This review focuses on these dephosphorylation mediators and the transduction mechanisms possibly involved in physiological states that require of the modulation of the dephosphorylation rate of NCC.

The E3 ubiquitin-protein ligase Nedd4-2 regulates the sodium chloride cotransporter NCC but is not required for a potassium-induced reduction of NCC expression.

Na+ and K+ balance is influenced by the activity of the sodium chloride cotransporter NCC in the distal convoluted tubule. NCC activity and abundance are reduced by high extracellular K+. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4–2 (Nedd4-2) has been proposed as a modulator of NCC abundance. Here, we examined the functional role of Nedd4-2 on NCC regulation and whether Nedd4-2 is important for the effects of high extracellular K+ on NCC. Total and plasma membrane levels of ubiquitylated NCC were lower in NCC-expressing MDCKI cells after Nedd4-2 deletion. NCC and phosphorylated NCC (pT58-NCC) levels were higher after Nedd4-2 deletion, and NCC levels on the plasma membrane were elevated. No significant changes were seen after Nedd4-2 knockdown in the levels of SPAK and phosphorylated SPAK (pS373-SPAK), the major NCC regulatory kinase. Nedd4-2 deficiency had no effect on the internalization rate of NCC from the plasma membrane, but NCC protein half-life was increased. In ex vivo experiments with kidney tubule suspensions from Nedd4-2 knockout (KO) mice, high K+ reduced total and pT58-NCC regardless of genotype. We conclude that Nedd4-2 is involved in ubiquitylation of NCC and modulating its plasma membrane levels and degradation. However, Nedd4-2 does not appear to be important for K+ induced reductions in NCC abundance.

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.

Role of inwardly rectifying K+ channel 5.1 (Kir5.1) in the regulation of renal membrane transport.

Kir5.1 interacts with Kir4.2 in proximal tubule and with Kir4.1 in distal convoluted tubule (DCT), connecting tubule (CNT) and cortical collecting duct (CCD) to form basolateral-K+-channels. Kir4.2/Kir5.1 and Kir4.1/Kir5.1 play an important role in regulating Na+/HCO3--transport of the proximal tubule and Na+/K+ -transport in the DCT/CNT/CCD. The main focus of this review is to provide an overview of the recent development in the field regarding the role of Kir5.1 regulating renal electrolyte transport in the proximal tubule and DCT. Loss-of-function-mutations of KCNJ16 cause a new form of tubulopathy, characterized by hypokalaemia, Na+-wasting, acid-base-imbalance and metabolic-acidosis. Abnormal bicarbonate transport induced by loss-of-function of KCNJ16-mutants is recapitulated in Kir4.2-knockout-(Kir4.2 KO) mice. Deletion of Kir5.1 also abolishes the effect of dietary Na+ and K+-intakes on the basolateral membrane voltage and NCC expression/activity. Long-term high-salt intake or high-K+-intake causes hyperkalaemic in Kir5.1-deficient mice. Kir4.2/Kir5.1 activity in the proximal tubule plays a key role in regulating Na+, K+ and bicarbonate-transport through regulating electrogenic-Na+-bicarbonate-cotransporter-(NBCe1) and type 3-Na+/H+-exchanger-(NHE3). Kir4.1/Kir5.1 activity of the DCT plays a critical role in mediating the effect of dietary-K+ and Na+-intakes on NCC activity/expression. As NCC determines the Na+ delivery rate to the aldosterone-sensitive distal nephron (ASDN), defective regulation of NCC during high-salt and high-K+ compromises renal K+ excretion and K+ homeostasis.

The role of mineralocorticoid receptor in the potassium switch: effect on NCC regulation+

Thiazide sensitive sodium‐chloride cotransporter (NCC) is a major salt transport pathway in the apical membrane of the nephron distal convoluted tubule. While we know the importance of its function in maintaining sodium‐potassium homeostasis, the regulation of NCC is complicated and the mechanisms are not well understood. The aim of this study is to evaluate the role of mineralocorticoid receptors (MRs) in the regulation of NCC. Here, we generated DCT‐specific MRs Knockout (KO) mice, in which MRs were deleted in cells expressing NCC, taking advantage of a cre recombinase under the control of the NCC gene. Under standard and Low‐Na+ diets, these DCT‐specific MRs‐KO mice display normal Na+/K+ balance but exhibit problems with Cl‐ regulation. These KO mice presented hypochloremia under normal diet but unexpectedly, hyperchloremia after 6d on low Na+ diet. The expression of NCC and phosphorylated NCC were both decreased in KO mice compared to that in control mice, under both standard and low‐Na+ diet. This decrease in NCC protein levels is related to a decrease in transcription levels. Under standard diet, decreased NCC expression is compensated by an increase in αENaC and pendrin expression, which is not the case under low‐salt diet. Under a low‐K+ diet, KO mice exhibit hypokalemia and volume depletion with increased blood CO2 levels and Renin mRNA levels. NCC expression and its phosphorylation are both increased in control and KO groups under low‐K+ diet compared to standard diet but, the levels of NCC protein and mRNA are still decreased in the KO group compared to the control group mice. Taken together, these results demonstrate that MRs is not necessary for the complete regulation of NCC under low‐Na+ and low‐K+ diet. On the other hand, MRs appears to be indispensable to assure proper NCC expression in adult DCT renal tubules.

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.

Zinc Deficiency‐Induced NFκB Activation Promotes Hypertension by Driving Renal Na + Reabsorption

Background : Zinc deficiency (ZnD) is a common comorbidity with chronic disorders including diabetes and kidney disease. Patients with these diseases also have a higher incidence of hypertension. Recently, we reported that a zinc deficient diet is sufficient to induce hypertension in mice. Changes in blood pressure were accompanied by increased Na+ reabsorption via the renal sodium chloride cotransporter (NCC). Although our novel results indicate that zinc plays a critical role in NCC regulation and its downstream effects on blood pressure, the specific mechanisms involved are unknown. Hypothesis : Since nuclear factor-κB (NFκB) is a key transcription factor implicated in ZnD-mediated effects, we tested the hypothesis that ZnD stimulates renal NFκB activation which drives NCC upregulation and thereby promotes hypertension. Experimental Design : Adult, male C57BL/6 mice were fed a zinc adequate (ZnA; 50ppm) or a ZnD (1ppm) diet for 6 weeks. To examine the role of NFκB in ZnD-induced hypertension, mice were switched from a ZnA-diet to a ZnD-diet for 5 weeks. After, ZnD mice were administered caffeic acid phenethyl ester (CAPE), an NFκB inhibitor, for 1 week. In mice, NCC expression (qRT-PCR) and blood pressure (tail-cuff) were measured. To further investigate the role of NFκB in NCC expression (qRT-PCR and Western blot), mouse distal convoluted tubule (mDCT) cells were treated with TPEN (a zinc chelator) or vehicle (DMSO) ± CAPE. Results : Compared to ZnA mice, NCC mRNA abundance was increased in ZnD mice (7.625 ± 1.747 vs 1.0 ± 0.127) and was accompanied by enhanced renal NFκB nuclear localization. In mDCT cells, TPEN-induced ZnD stimulated NCC mRNA expression (3.152 ± 0.966 vs 1.0 ± 0.174) and NFκB upregulation (1.573 ± 0.117 vs 1.0 ± 0). However, NFκB inhibition by CAPE prevented TPEN-induced NCC mRNA (0.636 ± 0.178 vs 3.152 ± 0.966) and protein (1.039 ± 0.018 vs 2.335 ± 0.062) expression in mDCT cells. Finally, CAPE administration lowered blood pressure (154.5 ± 5.42 vs 138.1 ± 1.97 mm Hg) in ZnD mice. Summary : These results demonstrate that 1) renal NFκB is a Zn2+-regulated transcription factor, 2) NFκB is involved in NCC regulation and 3) NFκB mediates ZnD-induced blood pressure increases. Significance : Taken together, these data indicate that ZnD stimulates NFκB activation thereby driving NCC upregulation and hypertension.

NFkB Mediates Renal Sodium Retention via the Sodium Chloride Cotransporter in Zinc Deficiency‐Induced Hypertension

Background : Zinc deficiency (ZnD) is comorbid with diseases such as kidney disease and diabetes. Individuals in these populations have a higher prevalence of hypertension. Recently, we reported that ZnD promotes hypertension in mice. The blood pressure elevations were accompanied with increased Na+ retention via the renal Sodium Chloride Cotransporter (NCC). Although our published results indicate that zinc plays a critical role in blood pressure and NCC regulation, the mechanisms involved were not investigated. Hypothesis : Since nuclear factor-kB (NFkB) mediates ZnD-induced detrimental effects, we tested the hypothesis that NFκB drives ZnD-induced NCC upregulation and subsequently Na+ retention and hypertension. Experimental Design : To determine the role of NFκB in ZnD-induced renal Na+ retention and hypertension, adult male C57Bl/6 mice were fed a ZnD diet (5 weeks) followed by administration of the NFκB inhibitor - caffeic acid phenethyl ester (CAPE; 1 week). Systolic blood pressure and urinary Na+ concentrations were examined. To examine if NFkB mediates ZnD-induced NCC upregulation, mouse distal convoluted tubule (mDCT) cells were treated with the zinc chelator N, N, N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) ± CAPE, a NFκB inhibitor. After 24 hours, NCC mRNA and protein expressions, as well as activation, were assessed by qRT-PCR, Western blot and immunofluorescence, respectively. Results : In mice, ZnD promoted hypertension and renal Na+ retention. Further, increased NCC expression was accompanied with enhanced NFkB expression in ZnD mice. However, CAPE administration lowered blood pressure and elevated urinary Na+ concentrations. In mDCT cells, TPEN-induced NCC upregulation was prevented by CAPE treatment. Conclusions : Together, these results demonstrate that 1) NFkB is a novel NCC regulator and 2) NFkB mediates ZnD-induced renal Na+ retention and hypertension. These novel findings indicate that ZnD drives renal NFkB activation thereby stimulating NCC-mediated Na+ retention and promoting hypertension. Significance : This study identifies NFkB as a possible pharmacological target to mitigate hypertension.

Distal convoluted tubule Cl- concentration is modulated via K+ channels and transporters.

Cl--sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT). Cl- enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl- channel ClC-K2 or K+-Cl- cotransporter (KCC). While KCC is electroneutral, Cl- exit via ClC-K2 is electrogenic. Therefore, an alteration in DCT basolateral K+ channel activity is expected to influence Cl- movement across the basolateral membrane. Although a role for intracellular Cl- in the regulation of WNK and NCC has been established, intracellular Cl- concentrations ([Cl-]i) have not been directly measured in the mammalian DCT. Therefore, to measure [Cl-]i in DCT cells, we generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl- fluorescent imaging in the isolated DCT. Basal measurements indicated that the mean [Cl-]i was ~7 mM. Stimulation of Cl- exit with low-Cl- hypotonic solutions decreased [Cl-]i, whereas inhibition of KCC by DIOA or inhibition of ClC-K2 by NPPB increased [Cl-]i, suggesting roles for both KCC and ClC-K2 in the modulation of [Cl-]i . Blockade of basolateral K+ channels (Kir4.1/5.1) with barium significantly increased [Cl-]i. Finally, a decrease in extracellular K+ concentration transiently decreased [Cl-]i, whereas raising extracellular K+ transiently increased [Cl-]i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl-]i. We conclude that the alteration in ClC-K2, KCC, and Kir4.1/5.1 activity influences [Cl-]i in the DCT.

Zinc Deficiency‐Induced NFkB Activation Promotes Hypertension by Driving Renal Na+ Reabsorption

BackgroundZinc deficiency (ZnD) is a common comorbidity with chronic disorders including diabetes and kidney disease. Patients with these diseases also have a higher incidence of hypertension. Recently, we reported that a zinc deficient diet is sufficient to induce hypertension in mice. Changes in blood pressure were accompanied by increased Na+ reabsorption via the renal sodium chloride cotransporter (NCC). Although our novel results indicate that zinc plays a critical role in NCC regulation and its downstream effects on blood pressure, the specific mechanisms involved are unknown.HypothesisSince nuclear factor‐kB (NFkB) is a key transcription factor implicated in ZnD‐mediated effects, we tested the hypothesis that ZnD stimulates renal NFκB activation which drives NCC upregulation and thereby promotes hypertension.Experimental DesignAdult, male C57BL/6 mice were fed a zinc adequate (ZnA; 50ppm) or a ZnD (1ppm) diet for 6 weeks. To examine the role of NFkB in ZnD‐induced hypertension, mice were switched from a ZnA‐diet to a ZnD‐diet for 5 weeks. After, ZnD mice were administered caffeic acid phenethyl ester (CAPE), an NFkB inhibitor, for 1 week. In mice, NCC expression (qRT‐PCR) and blood pressure (tail‐cuff) were measured. To further investigate the role of NFkB in NCC expression (qRT‐PCR and Western blot), mouse distal convoluted tubule (mDCT) cells were treated with TPEN (a zinc chelator) or vehicle (DMSO) ± CAPE.ResultsCompared to ZnA mice, NCC mRNA abundance was increased in ZnD mice (7.625 ± 1.747 vs 1.0 ± 0.127) and was accompanied by enhanced renal NFkB nuclear localization. In mDCT cells, TPEN‐induced ZnD stimulated NCC mRNA expression (3.152 ± 0.966 vs 1.0 ± 0.174) and NFkB upregulation (1.573 ± 0.117 vs 1.0 ± 0). However, NFkB inhibition by CAPE prevented TPEN‐induced NCC mRNA (0.636 ± 0.178 vs 3.152 ± 0.966) and protein (1.039 ± 0.018 vs 2.335 ± 0.062) expression in mDCT cells. Finally, CAPE administration lowered blood pressure (154.5 ± 5.42 vs 138.1 ± 1.97 mm Hg) in ZnD mice.SummaryThese results demonstrate that 1) renal NFkB is a Zn2+‐regulated transcription factor, 2) NFkB is involved in NCC regulation and 3) NFkB mediates ZnD‐induced blood pressure increases.SignificanceTaken together, these data indicate that ZnD stimulates NFkB activation thereby driving NCC upregulation and hypertension.Support or Funding InformationAHA‐16SDG27080009 and NIH‐R21DK119879

NFkB Mediates Renal Sodium Retention via the Sodium Chloride Cotransporter in Zinc Deficiency‐induced Hypertension

BackgroundZinc deficiency (ZnD) is comorbid with diseases such as kidney disease and diabetes. Individuals in these populations have a higher prevalence of hypertension. Recently, we reported that ZnD promotes hypertension in mice. The blood pressure elevations were accompanied with increased Na+ retention via the renal Sodium Chloride Cotransporter (NCC). Although our published results indicate that zinc plays a critical role in blood pressure and NCC regulation, the mechanisms involved were not investigated.HypothesisSince nuclear factor‐kB (NFκB) mediates ZnD‐induced detrimental effects, we tested the hypothesis that NFκB drives ZnD‐induced NCC upregulation and subsequently renal Na+ retention and hypertension.Experimental DesignTo determine the role of NFκB in ZnD‐induced renal Na+ retention and hypertension, adult male C57Bl/6 mice were fed a ZnD diet (5 weeks) followed by administration of the NFκB inhibitor ‐ caffeic acid phenethyl ester (CAPE; 1 week). Systolic blood pressure and urinary Na+ concentrations were examined. To examine if NFκB mediates ZnD‐induced NCC upregulation, mouse distal convoluted tubule (mDCT) cells were treated with the zinc chelator N, N, N′, N′‐tetrakis(2‐pyridinylmethyl)‐1,2‐ethanediamine (TPEN) ± CAPE, a NFκB inhibitor. After 24 hours, NCC mRNA and protein expressions, as well as activation, were assessed by qRT‐PCR, Western blot and immunofluorescence, respectively.ResultsIn mice, ZnD promoted hypertension and renal Na+ retention. Further, increased NCC expression was accompanied with enhanced NFκB expression in ZnD mice. However, CAPE administration lowered blood pressure and elevated urinary Na+ concentrations. In mDCT cells, TPEN‐induced NCC upregulation was prevented by CAPE treatment.ConclusionsTogether, these results demonstrate that 1) NFκB is a novel NCC regulator and 2) NFκB mediates ZnD‐induced renal Na+ retention and hypertension. These novel findings indicate that ZnD drives renal NFκB activation thereby stimulating NCC‐mediated Na+ retention and promoting hypertension.SignificanceThis study identifies NFκB as a possible pharmacological target to mitigate hypertension.Support or Funding InformationAmerican Heart Association ‐ Scientist Development Grant #16SDG27080009 National Institutes of Health ‐ R21 Exploratory/Development Grant #R21DK119879

Impaired NCC Activity and Regulation on Dietary High Dietary Salt Intake in Aged Sprague Dawley Rats Is Associated with Increased Salt Sensitivity of Blood Pressure

BackgroundHypertension (HTN) is one of the leading causes of death worldwide and its prevalence increases with age. Studies revealed that enhanced global sympathetic tone is related to increases in blood pressure (BP) in aged human subjects. Dietary sodium intake is associated with the regulation of blood pressure, as increased sodium retention is a well‐established precursor of hypertension risk. An excessive dietary sodium intake leads to increases in BP in individuals that demonstrate the salt sensitivity of BP and evoke the development of salt sensitive HTN (SSH). Recent studies have demonstrated that excessive release of norepinephrine by increased sympathetic nerve activity upregulates renal sodium chloride cotransporter (NCC) activity to promote sodium reabsorption. However, the underlying mechanisms of the regulation of sodium homeostasis and blood pressure in response to increase in sympathetic tone with age remain unclear.HypothesisNCC‐mediated sodium retention driven by increased sympathetic tone with age contributes to age‐related HTN. Furthermore, the activity and regulation of NCC are impaired on a high salt (HS) diet and this impaired NCC activity and regulation drives the development of age‐dependent SSH.MethodsThree different age groups (3, 8, 16 month old (MO)) of male Sprague‐Dawley (SD) rats were fed a normal salt (NS; 0.6% NaCl) or HS (4% NaCl) diet for 21 days respectively. On day 21, basal MAP, NCC activity (peak natriuresis to IV hydrochlorothiazide (2mg/kg) infusion), and vascular sympathetic tone (peak MAP change in response to ganglionic blockade (IV hexamethonium; 30mg/kg)) were measured in vivo. Plasma and renal NE concentrations were also assessed via ELISA. The expression of total NCC, phosphorylated NCC, with‐no‐lysine [K] kinases (WNK) 1, WNK4, STE20/SPS1‐related proline‐alanine‐rich protein kinase (SPAK), oxidative stress responsive kinase 1 (OxSR1), and phosphorylated SPAK/OxSR1 were assessed via immunoblotting (N=6/gp).ResultsFirst, 3MO SD rats are normotensive in both diet groups and show dietary sodium‐evoked suppression of NCC activity, expression, and phosphorylation and upstream regulatory kinases including WNK1, SPAK, and OxSR1 on a HS diet relative to a NS diet. Second, SD rats show age‐dependent HTN, increases in renal sympathetic tone, NCC activity and expression, and WNK1 expression. Last, SD rats show aged‐dependent SSH, increased sympathetic tone, and impaired suppression of NCC activity, phosphorylation, and the expression of kinases SPAK and OxSR1.ConclusionSD rats show age‐dependent HTN that is coupled with increases in sympathetic tone, NCC activity and expression, which strongly suggests that the NCC contributes to the development of age‐dependent HTN. Moreover, the NCC may play a pivotal role in the development of age‐dependent SSH as aged SD rats show impaired dietary‐sodium evoked suppression of NCC activity and regulation. Collectively, these findings suggest that increased sympathetic tone may drive NCC activity that evokes age‐dependent HTN and SSH. MO, month old; NS, 0.6% NaCl; HS, 4% NaCl. Variables 3 MO 8 MO 16 MO NS HS NS HS NS HS MAP (mmHg) 124 ± 2 126 ± 3 135 ± 4 # 143 ± 5 # 149 ± 3 # † 169 ± 1 * # † ΔUNaV to HCTZ (μeq/min) 9 ± 1 7 ± 1 * 18 ± 2 # 16 ± 1 # 15 ± 5 # 16 ± 6 # Plasma NE (nmol/L) 44 ± 4 28 ± 4 * 55 ± 3 # 42 ± 4 * # 54 ± 4 # 41 ± 4 * # Renal NE (pg/mg) 612 ± 36 368 ± 32 * 835 ± 48 # 722 ± 44 * # 974 ± 13 # † 1019 ± 134 # † ΔMAP to hexamethonium (mmHg) −33 ± 4 −24 ± 3 * −64 ± 5 # −65 ± 6 # −60 ± 3 # −67 ± 7 # Total NCC expression (fold change) 1.00 ± 0.22 0.32 ± 0.09 * 1.91 ± 0.25 # 1.13 ± 0.15 * # 1.82 ± 0.23 # 0.75 ± 0.11 * Total pNCCT53 expression (fold change) 1.00 ± 0.20 0.19 ± 0.05 * 1.24 ± 0.46 0.87 ± 0.22 0.49 ± 0.18 1.73 ± 0.22 * # WNK1 expression (fold change) 1.00 ± 0.20 0.49 ± 0.06 * 1.07 ± 0.34 1.36 ± 0.15 2.15 ± 0.38 # † 1.60 ± 0.29 # WNK4 expression (fold change) 1.00 ± 0.11 0.86 ± 0.07 1.95 ± 0.14 # 1.28 ± 0.10 # 1.05 ± 0.07 † 1.17 ± 0.09 SPAK expression (fold change) 1.00 ± 0.12 0.48 ± 0.09 * 1.25 ± 0.09 1.30 ± 0.23 # 0.42 ± 0.04 # † 0.41 ± 0.13 † OxSR1 expression (fold change) 1.00 ± 0.12 0.62 ± 0.05 * 1.09 ± 0.12 1.10 ± 0.06 # 1.19 ± 0.20 1.17 ± 0.08 # pSPAK/OxSR1 expression (fold change) 1.00 ± 0.24 0.84 ± 0.30 0.55 ± 0.23 1.42 ±0.24 * 1.12 ± 0.17 2.86 ± 0.43 * # † *p<0.05 vs. respective NS group; #p<0.05 vs. respective 3 MO group; † p<0.05 vs. respective 8 MO group.

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 β-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 β-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, β-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.

Inhibition of Protein Phosphatase 1 prevents high potassium mediated downregulation of the thiazide‐sensitive sodium chloride cotransporter NCC

The “aldosterone paradox” is a poorly understood physiological mechanism in which increases in the hormone aldosterone either serve to stabilize blood pressure by increasing sodium‐chloride (NaCl) reabsorption or to excrete potassium (K+). Altered NaCl transport via the NaCl cotransporter NCC in the renal distal convoluted tubule (DCT) has been suggested to play a central role in switching from one response to the other. Under low NaCl intake, NCC abundance and activity is high, whereas it is low during high K+ intake ‐ leading to increased delivery of NaCl to downstream segments and increased electrogenic K+ secretion. Despite its essential role, little is known about the molecular alterations in the DCT during the aldosterone paradox, and the proteins involved in the regulation of NCC during this response.To define the proteome of the DCT and how it is modified by increased circulating aldosterone levels subsequent to long term low dietary NaCl or high K+ intake, transgenic mice with expression of enhanced green fluorescent protein (eGFP) under the control of the parvalbumin promoter were used. These mice have specific eGFP‐labeling of DCT cells, allowing high purity fluorescence‐activated cell sorting (FACS) of DCT cells. Mice were maintained on low NaCl or high K+ diets for 4 days, which resulted in increased aldosterone levels and a change in NCC abundance. Following FACS, the DCT proteome was determined by liquid chromatography‐coupled tandem mass spectrometry. 3078 unique proteins were identified in the DCT. 210 proteins were significantly altered in abundance following a low NaCl diet. 625 were significantly altered in abundance following a high K+ diet. Protein Phosphatase 1α (Pp1α) abundance was only increased by high K+ diet. In freshly isolated mouse tubules from the renal cortex, a higher K+ concentration in the medium for 1–4 days resulted in a significant decrease in NCC expression and increased expression of PP1α, mimicking the in vivo situation. The K+‐induced decrease of total NCC levels could be inhibited by treating the cells with the PP1‐blocker tautomycetin, suggesting that PP1 is involved in the K+‐induced downregulation of NCC. In summary, these results suggest that PP1α is involved in the decreased NCC abundance seen during long term high potassium intake.Support or Funding InformationDanish Medical Research Council, Fondation LeDuq, Novo Nordisk Foundation, Aarhus University Research Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

WNK4 kinase is a physiological intracellular chloride sensor

With-no-lysine (WNK) kinases regulate renal sodium-chloride cotransporter (NCC) to maintain body sodium and potassium homeostasis. Gain-of-function mutations of WNK1 and WNK4 in humans lead to a Mendelian hypertensive and hyperkalemic disease pseudohypoaldosteronism type II (PHAII). X-ray crystal structure and in vitro studies reveal chloride ion (Cl-) binds to a hydrophobic pocket within the kinase domain of WNKs to inhibit its activity. The mechanism is thought to be important for physiological regulation of NCC by extracellular potassium. To test the hypothesis that WNK4 senses the intracellular concentration of Cl- physiologically, we generated knockin mice carrying Cl--insensitive mutant WNK4. These mice displayed hypertension, hyperkalemia, hyperactive NCC, and other features fully recapitulating human and mouse models of PHAII caused by gain-of-function WNK4. Lowering plasma potassium levels by dietary potassium restriction increased NCC activity in wild-type, but not in knockin, mice. NCC activity in knockin mice can be further enhanced by the administration of norepinephrine, a known activator of NCC. Raising plasma potassium by oral gavage of potassium inactivated NCC within 1 hour in wild-type mice, but had no effect in knockin mice. The results provide compelling support for the notion that WNK4 is a bona fide physiological intracellular Cl- sensor and that Cl- regulation of WNK4 underlies the mechanism of regulation of NCC by extracellular potassium.

Dietary salt regulates ubiquitination and urinary excretion of Na-Cl cotransporter

Background: Membranous localization of NCC is mandatory for its function at distal convoluted tubule. Post-translational modification like phosphorylation and ubiquitination are important for various membrane proteins for their membranous localization. It has previously been reported that phosphorylation of NCC increased its membranous localization. We previously reported, low salt diet increased and high salt diet decreased expression and phosphorylation of NCC in kidney.Objectives: In this study, we investigated whether ubiquitination is involved in the mechanisms of NCC regulation by dietary salt.Materials & Methods: We examined ubiquitination and urinary excretion of NCC from mice fed with low-, regular- and high-salt diet. Study was done at Tokyo Medical and Dental University,during the period of January, 2013 to March, 2013.Results: We found that, high salt diet increased and low salt diet decreased ubiquitination of NCC, correlated well with their total NCC abundance in kidney. Urinary excretion of exosomal NCC was increased both under low and high salt diet.Conclusion: These results clearly indicated that dietary salt regulates ubiquitination of Na-Cl cotransporter and its urinary excretion.KYAMC Journal Vol. 9, No.-1, April 2018, Page 2-5