NCC Phosphorylation Research Articles

Control of sodium and potassium homeostasis by renal distal convoluted tubules.

Distal convoluted tubules (DCT), which contain the Na-Cl cotransporter (NCC) inhibited by thiazide diuretics, undergo complex modulation to preserve Na+ and K+ homeostasis. The lysine kinases 1 and 4 (WNK1 and WNK4), identified as hyperactive in the hereditary disease pseudohypoaldosteronism type 2, are responsible for activation of NCC and consequent hypokalemia and hypertension. WNK4, highly expressed in DCT, activates the SPAK/OSR1 kinases, which phosphorylate NCC and other regulatory proteins and transporters in the distal nephron. WNK4 works as a chloride sensor through a Cl- binding site, which acts as an on/off switch at this kinase in response to changes of basolateral membrane electrical potential, the driving force of cellular Cl- efflux. High intracellular Cl- in hyperkalemia decreases NCC phosphorylation and low intracellular Cl- in hypokalemia increases NCC phosphorylation and activity, which makes plasma K+ concentration a central modulator of NCC and of K+ secretion. The WNK4 phosphorylation by cSrc or SGK1, activated by angiotensin II or aldosterone, respectively, is another relevant mechanism of NCC, ENaC, and ROMK modulation in states such as volume reduction, hyperkalemia, and hypokalemia. Loss of NCC function induces upregulation of electroneutral NaCl reabsorption by type B intercalated cells through the combined activity of pendrin and NDCBE, as demonstrated in double knockout mice (KO) animal models, Ncc/pendrin or Ncc/NDCBE. The analysis of ks-Nedd-4-2 KO animal models introduced the modulation of NEDD4-2 by intracellular Mg2+ activity as an important regulator of NCC, explaining the thiazide-induced persistent hypokalemia.

PS-BPC10-7: EFFECT OF ORAL POTASSIUM CHLORIDE SUPPLEMENTATION ON THE THIAZIDE-SENSITIVE SODIUM CHLORIDE COTRANSPORTER (NCC) IN HEALTHY HUMANS: A RANDOMISED CROSSOVER TRIAL

Objective: The putative “renal-K+ switch’’ mechanism links low K + intake with Na+-dependent Na+ retention and involves activation of NCC in the distal convoluted tubule in response to low K+ intake and suppression in response to high K+ intake. This study used urinary extracellular vesicles (uEVs) to examine NCC abundance and phosphorylation in healthy adults on a high Na+ diet to determine tubular responses to alterations in K+ (in the form of KCl) intake. Design and method: The study design was a double blind, randomised and placebo-controlled trial. Healthy adults maintained on a high Na+ (∼200mmol/day), low K+ (∼60mmol/day) diet underwent a run-in period of 1-week followed by a crossover study, with 5-day supplementary KCl (active phase, Span K, 24mmol thrice daily) or 5-day placebo administered in random order and separated by 2-day washout. After each phase, blood and 24-hour urine were collected for biochemistry, and spot urine for uEV analysis were collected for progressive ultracentrifugation. Participants who met dietary compliance (greater than 80%) and urinary response requirements were included in analyses. Results: Among the 23 (out of 28) participants who met inclusion criteria, supplementary KCl administration (verses placebo) in the active phase was associated with markedly higher levels of plasma K+ and 24-Hour urine levels of K+, Cl- and aldosterone, and lower levels of 24-Hour urinary Na+/K+ (Table 1). Among 18 participants who met inclusion criteria and whose uEV were successfully isolated, KCl intake was associated with less abundant NCC (fold change [FC](KCl/placebo) = 0.74 [0.30, 1.69], p < 0.01) and pNCC (FC = 0.81 [0.19, 1.75], p < 0.05) (Figure 1). Correlation analyses revealed an inverse correlation of plasma K+ with NCC (R2 = 0.11, p = 0.05) and delta urinary K+ with delta pNCC (R2 = 0.23, p = 0.04). Ambulatory systolic blood pressure positively correlated with timed urinary Na+/K+ (R2 = 0.08, p = 0.04) during interventions. Conclusions: In healthy adults on a high Na+ low K+ diet, supplementary KCl administration was associated with reductions in NCC and pNCC in uEVs, consistent with the “renal-K+ switch’’ mechanism, and offers a useful target for dietary intervention.

In Primary Aldosteronism Acute Potassium Chloride Supplementation Suppresses Abundance and Phosphorylation of the Sodium-Chloride Cotransporter.

Elevated abundance of sodium-chloride cotransporter (NCC) and phosphorylated NCC (pNCC) are potential markers of primary aldosteronism (PA), but these effects may be driven by hypokalemia. We measured plasma potassium in patients with PA. If potassium was <4.0 mmol/L, patients were given sufficient oral potassium chloride (KCl) over 24 hours to achieve as close to 4.0 mmol/L as possible. Clinical chemistries were assessed, and urinary extracellular vesicles (uEVs) were examined to investigate effects on NCC. Among 21 patients with PA who received a median total dose of 6.0 g (2.4-16.8 g) of KCl, increases were observed in plasma potassium (from 3.4 to 4.0 mmol/L; P<0.001), aldosterone (from 305 to 558 pmol/L; P=0.01), and renin (from 1.2 to 2.5 mIU/L; P<0.001), whereas decreases were detected in uEV levels of NCC (median fold change(post/basal) [FC]=0.71 [0.09-1.99]; P=0.02), pT60-NCC (FC=0.84 [0.06-1.66]; P=0.05), and pT55/60-NCC (FC=0.67 [0.08-2.42]; P=0.02). By contrast, in 10 patients with PA who did not receive KCl, there were no apparent changes in plasma potassium, NCC abundance, and phosphorylation status, but increases were observed in plasma aldosterone (from 178 to 418 pmol/L; P=0.006) and renin (from 2.0 to 3.0 mU/L; P=0.009). Plasma potassium correlated inversely with uEV levels of NCC (R 2=0.11; P=0.01), pT60-NCC (R 2=0.11; P=0.01), and pT55/60-NCC (R 2=0.11; P=0.01). Acute oral KCl loading replenished plasma potassium in patients with PA and suppressed NCC abundance and phosphorylation, despite a significant rise in plasma aldosterone. This supports the view that potassium supplementation in humans with PA overrides the aldosterone stimulatory effect on NCC. The increased plasma aldosterone in patients with PA without KCl supplementation may be due to aldosterone response to posture challenge.

Glucose/Fructose Delivery to the Distal Nephron Activates the Sodium-Chloride Cotransporter via the Calcium-Sensing Receptor.

The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.

REDUCED NACL COTRANSPORTER ABUNDANCE AND PHOSPHORYLATION IN URINARY EXTRACELLULAR VESICLES IN RESPONSE TO KCL SUPPLEMENTS IN HEALTHY ADULTS MAINTAINED ON A HIGH-NA+ LOW-K+ DIET: A

Objective: A renal-K switch mechanism has been established in rodent models and demonstrates that a low K+ intake acts as a trigger that links K+ and Na+-dependent blood pressure. This mechanism involves activation (phosphorylation) of the NaCl-cotransporter NCC in the distal convoluted tubule in response to low-K+ intake and suppression (dephosphorylation) after a high-K+ intake. As a first step to establishing a similar mechanism in humans, this study aimed to examine if the abundance of NCC and pNCC in urinary extracellular vesicles (uEVs) of healthy adults on a high-Na+ diet changes in response to alterations in K+ intake. Design and method: The study design was a double-blind, randomised and placebo-controlled trial. Healthy adults maintained on a high-Na+ (∼200mmol/day), low-K+ (∼60mmol/day) diet underwent a run-in period of 1-week followed by a cross-over study, with 5 days supplementary KCl (active phase, Span K, 24mmol thrice daily) or 5 days placebo administered in random order and separated by 2 days washout. NCC and pNCC were measured in uEVs after each phase. Participants who met dietary compliance (no less than 80%) and increased urinary K+ in the active phase were included in analyses. Results: Among the 18 (out of 28) participants who met inclusion criteria, supplementary KCl administration (versus placebo) in the active phase was associated with higher levels of plasma K+ (p &lt; 0.001) and 24-Hour urine levels of K+ (p &lt; 0.001), Cl- (p &lt; 0.001) and aldosterone (p &lt; 0.001), and lower levels of 24-Hour urinary Na+/K+ (p &lt; 0.001), NCC (p &lt; 0.01) and pNCC (p &lt; 0.05); whilst the ratio of pNCC/NCC remained unchanged. Correlation analyses revealed an inverse correlation of plasma K+ with NCC (R2 = 0.11, p = 0.05) and δurinary K+ with δpNCC (R2 = 0.23, p = 0.04). Ambulatory systolic blood pressure positively correlated with timed urinary Na+/K+ (R2 = 0.08, p = 0.04) during interventions.δ Conclusions: KCl supplementation in healthy adult subjects maintained on a high-Na+ low-K+ diet was associated with a fall in NCC and pNCC, consistent with the renal-K switch mechanism, and offers a useful target for dietary intervention.

Gitelman‐like syndrome caused by pathogenic variants in mitochondrial DNA

Gitelman syndrome is the most frequent hereditary salt‐losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+‐Cl‐ cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants in CLCNKB, HNF1B, FXYD2 or KCNJ10 may result in renal phenocopies of Gitelman syndrome, as they can lead to reduced NCC activity. Nevertheless, ±10% of patients with a Gitelman syndrome phenotype remain genetically unsolved.After identification of mitochondrial DNA (mtDNA) variants in three families with Gitelman syndrome‐like electrolyte abnormalities, 156 families were investigated for variants in MT‐TI and MT‐TF, encoding the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. In NCC‐expressing HEK293 cells, mitochondrial dysfunction was induced to assess the effect on thiazide‐sensitive 22Na+ transport.Genetic investigations revealed four mtDNA variants in 13 families: m.591C&gt;T (n=7), m.616T&gt;C (n=1), m.643A&gt;G (n=1) (all in MT‐TF) and m.4291T&gt;C (n=4, in MT‐TI). Variants were near homoplasmic in affected individuals. Importantly, affected members of six families with an MT‐TF variant additionally suffered from progressive chronic kidney disease. Maximal mitochondrial respiratory capacity was reduced in patient fibroblasts, caused by dysfunction of oxidative phosphorylation complex IV. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, demonstrated an inhibitory effect on NCC phosphorylation and NCC‐mediated sodium uptake.Pathogenic mtDNA variants in MT‐TF and MT‐TI can cause a Gitelman syndrome‐like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome‐like tubulopathies.

KS‐WNK1 is Required to Translate the Response to Extreme Changes in Potassium Ingestion to NCC Activity and Expression

HypothesisKS‐WNK1 a shorter isoform of WNK1 that is exclusively expressed in the kidney, with abundance in the distal convoluted tubule (DCT), but its physiological role remains elusive. KS‐WNK1 stimulates NCC activity via WNK4‐SPAK pathway. Under low potassium diet, a known stimuli for NCC activity, WNK bodies are formed in DCT cells, and this requires the presence of KS‐WNK1. We recently shown that KS‐WNNK1 is highly sensitive to the CUL3‐KLHL3 complex (JCI 2020) and its expression under control conditions is negligible, but it is increased under low potassium diet (AJP Renal 2021). Thus, studies that have analyzed the role of KS‐WNK1 in mice exposed to normal K+ diet may not be the adequate approach. In wildlife, carnivorous mammals are exposed to cycles of no food and thus, zero K+ ingestion, followed by a vast meal in a few hours and thus, acute and high K+ ingestion. Due to the high sensitivity of KS‐WNK1 expression to K+ intake, we assessed NCC and KS‐WNK1 using a model from low K+ to high K+ diet to imitate what occurs in wildlife.MethodsWe exposed C57bl/6 (WT) and KS‐WNK1‐KO (KS‐KO) mice to 10 days of zero K+ diet (0KD) or normal K+ diet (NKD), followed by high K+ ingestion (HKD) (5%). Groups of mice were sacrificed before HKD and at 12 or 24 hours after HKD. We evaluated plasma K+ and by western blot we assessed NCC, phospho‐NCC, SPAK, phospho‐SPAK and KS‐WNK1 expression in kidney proteins.ResultsIn mice fed with NKD, there was no difference in plasma K+ or NCC phophorylation or expression between WT and KS‐KO mice. After 10 days of 0KD plasma K+ was significantly lower in KS‐KO mice than in WT mice (p&lt;0.05) and NCC phosphorylation was higher in WT mice (p&lt;0.05), consistently, KS‐WNK1 expression in WT mice was only observed in 0KD, but not in NKD and it is not present in KS‐KO mice. In WT mice pNCC/NCC dephosphorylation ratio at 12 hours after HKD was significantly higher in mice feed with 10 days of 0KD, than in mice fed with NKD, and accordingly, changes in plasma K+ were only observed in mice fed with 0KD and not in mice coming from NKD. The decrease in pNCC/tNCC dephosphorylation ratio after HKD, when mice were fed with 0KD was even lower in KS‐KO than in WT mice. Additionally, KS‐KO mice had higher plasma K+ after HKD coming from 0KD.ConclusionNo differences were observed between WT and KS‐KO in groups fed with NKD before the exposure to HKD. KS‐WNK1 was not expressed during NKD. In contrast, in mice fed with 0KD, our data show that activation of NCC was higher in WT that KS‐KO. Then, after HKD, down‐regulation of pNCC was more effective in WT than in KS‐KO mice. Therefore, our data suggest that KS‐WNK1 is not present and has no role when mice were fed with NKD, but is up‐regulated in 0KD and therefore, required to properly respond to extreme changes in potassium diet, as those that occurs in wildlife mammals.

Dissecting the Effects of Aldosterone and Hypokalemia on the Epithelial Na+ Channel and the NaCl Cotransporter.

Primary hyperaldosteronism (PA) is characterized by aldosterone excess and hypertension. This may be linked to increased renal Na+ reabsorption via the epithelial Na+ channel (ENaC) and the NaCl cotransporter (NCC). The majority of PA patients have normal plasma K+ levels, but a subset of cases are associated with hypokalemia. High NCC levels observed in long-term studies with aldosterone-infused rodents have been attributed to direct effects of aldosterone. Aldosterone can also increase active phosphorylated NCC (pT58-NCC) acutely. However, direct effects of aldosterone on NCC have been contested by recent studies indicating that it is rather an indirect effect of hypokalemia. We therefore set out to determine isolated long-term aldosterone and K+ effects on ENaC and NCC using various in vivo and ex vivo approaches. In mice, aldosterone-induced hypokalemia was prevented by simultaneous amiloride infusion, coupled to increased cleavage of α- and γENaC but no effect on NCC. Regression analyses of in vivo data showed a positive correlation between aldosterone/K+ and αENaC but a negative correlation with NCC and pT58-NCC. Ex vivo, exposure of kidney tubules for 21 h to aldosterone increased cleavage of αENaC and γENaC, but no effects were observed on NCC or pT58-NCC. Exposure of tubules to low K+ media reduced αENaC but increased NCC and pT58-NCC. As hypokalemia can enhance cell proliferation markers in the distal convoluted tubule (DCT), we hypothesized that aldosterone infusion would increase proliferating cell nuclear antigen (PCNA) expression. Infusion of aldosterone in mice for 6 days greatly increased PCNA expression in the DCT. Collectively, in vivo and ex vivo data suggest that both aldosterone and K+ can increase ENaC directly. In contrast, the observed increase in abundance and phosphorylation of NCC in aldosterone-infused mice is likely an indirect effect of enhanced ENaC-mediated K+ secretion and subsequent hypokalemia. Thus, it is possible that NCC may only be increased in PA when the condition is associated with hypokalemia.

Does the early aldosterone-induced SGK1 play a role in early Kaliuresis?

Urinary K+ potassium excretion rapidly increases after a potassium‐rich meal. The early aldosterone‐induced sgk1 gene (encoding serum and glucocorticoid‐induced kinase 1), activates potassium clearance, but the role of this kinase in the early activation of K+ secretion has not been clearly defined. Here, we challenged inducible renal‐tubule‐specific Sgk1Pax8 / LC1 knockout mice with an acute high‐potassium load (HK:5%K+) and compared the physiological and molecular responses to control mice. We observe that urinary excretion after a K+ load over the first 3 h is not dependent on SGK1 but is coincident with the rapid dephosphorylation of the Na+,Cl−‐cotransporter (NCC) to increase distal salt delivery. Molecular analyses indicate that whereas SGK1‐mediated phosphorylation of the ubiquitin‐protein ligase NEDD4‐2 begins to increase by 3h, SGK1‐dependent proteolytic activation of ENaC only becomes detectable after 6 h of HK intake. Consistent with SGK1‐dependent ENaC activation via inhibition of NEDD4‐2‐mediated ubiquitylation, Sgk1Pax8 / LC1 mice are unable to efficiently inhibit NEDD4‐2 or increase ENaC cleavage after 6 h of HK. Nevertheless, no defect in acute K+ balance was detected in the mutant mice after 6 h of HK. Moreover, we found that Sgk1Pax8 / LC1 mice reduce NCC phosphorylation and NCC‐mediated salt absorption to a greater extent than control mice after a K+ load, promoting increased amiloride‐sensitive Na+‐reabsorption via ENaC to maintain adequate kaliuresis. Together, these data indicate that: (a) during the early 3 h of HK intake, K+ excretion is SGK1‐independent even under an extreme K+ challenge, (b) shortly after, SGK1 inhibits NEDD4‐2 and activates ENaC to stimulate K+‐secretion, (c) SGK1‐dependent phosphorylation of NCC occurs, acting more likely as a brake pedal to prevent excessive K+ loss.

Gitelman-Like Syndrome Caused by Pathogenic Variants in mtDNA.

Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl- cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB, HNF1B, FXYD2, or KCNJ10 may result in the same renal phenotype of Gitelman syndrome, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a Gitelman syndrome phenotype, the genotype is unknown. We identified mitochondrial DNA (mtDNA) variants in three families with Gitelman-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF, which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced in NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22Na+ transport. Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF), and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV and reduced maximal mitochondrial respiratory capacity were found in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake. Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies.

Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3-Ubiquitin Ligases.

The thiazide-sensitive sodium chloride cotransporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which is controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of cullins (Cul1, 3, 4, and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect cullin neddylation levels in ex vivo renal tubules. In the short term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan-cullin inhibitor), but the response to low extracellular K+ was absent. In the long term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation, and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

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.

High dietary potassium causes ubiquitin-dependent degradation of the kidney sodium-chloride cotransporter

The thiazide-sensitive sodium-chloride cotransporter (NCC) in the renal distal convoluted tubule (DCT) plays a critical role in regulating blood pressure (BP) and K+ homeostasis. During hyperkalemia, reduced NCC phosphorylation and total NCC abundance facilitate downstream electrogenic K+ secretion and BP reduction. However, the mechanism for the K+-dependent reduction in total NCC levels is unknown. Here, we show that NCC levels were reduced in ex vivo renal tubules incubated in a high-K+ medium for 24–48 h. This reduction was independent of NCC transcription, but was prevented using inhibitors of the proteasome (MG132) or lysosome (chloroquine). Ex vivo, high K+ increased NCC ubiquitylation, but inhibition of the ubiquitin conjugation pathway prevented the high K+-mediated reduction in NCC protein. In tubules incubated in high K+ media ex vivo or in the renal cortex of mice fed a high K+ diet for 4 days, the abundance and phosphorylation of heat shock protein 70 (Hsp70), a key regulator of ubiquitin-dependent protein degradation and protein folding, were decreased. Conversely, in similar samples the expression of PP1α, known to dephosphorylate Hsp70, was also increased. NCC coimmunoprecipitated with Hsp70 and PP1α, and inhibiting their actions prevented the high K+-mediated reduction in total NCC levels. In conclusion, we show that hyperkalemia drives NCC ubiquitylation and degradation via a PP1α-dependent process facilitated by Hsp70. This mechanism facilitates K+-dependent reductions in NCC to protect plasma K+ homeostasis and potentially reduces BP.

Phosphorylation of NCC and its Association with The Actin Cytoskeleton Linker Protein Ezrin in the Diabetic Kidney is Reduced by Dapagliflozin Treatment

Ezrin, an actin cytoskeleton linker protein is known to regulate various membrane proteins in the kidney. The Sodium Chloride Cotransporter (NCC) is responsible for 5-10% of salt reabsorption from the filtrate in the distal convoluted tubules. Dapagliflozin, an SGLT2 inhibitor, is a novel drug in the treatment of type 2 diabetes mellitus. We hypothesize that dapagliflozin decreases sodium reabsorption and blood pressure by attenuating the association between NCC and ezrin in the distal convoluted tubules in the hypertensive diabetic db/db mice. Twelve week-old male db/db mice with a blood glucose of more than 300 mg/dl were salt-loaded (4% NaCl) for 10 days to induce hypertension. The mice were subjected to metabolic cage studies for 24-hour urine collections in order to measure urinary electrolytes, creatinine, and albumin. Blood pressure was measured by the tail-cuff method to evaluate the effect of dapagliflozin or vehicle given by oral gavage (10 mg/kg of body weight per day for 2 weeks). At the end of the study the mice were euthanized, and the left kidney was formalin-fixed and paraffin-embedded for 3,3′-Diaminobenzidine (DAB) double staining for phospho-NCC and ezrin proteins while the right kidney was homogenized for Western blotting. Western blot analysis showed attenuation of total NCC and ezrin proteins in the diabetic db/db mice given an oral gavage of dapagliflozin compared to vehicle. DAB analysis showed reduced expression of the actin cytoskeleton linker protein ezrin and phosphorylated NCC in the dapagliflozin treated group compared to the control group. Taken together, we show dapagliflozin decreases sodium retention and blood pressure by reducing the density of renal NCC at the luminal membrane and the association between NCC and the actin cytoskeleton.

Effects of Chronic Potassium Supplementation and the Accompanying Anion on Blood Pressure

Increased dietary NaCl is associated with higher blood pressure (BP), but dietary potassium supplementation can reduce BP and reduce ‘salt sensitivity’ of BP, improving cardiovascular (CV) outcomes. Rodents fed a high K+ diet often exhibit reduced 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. Recently, the effect of extreme K+ diets on rodent BP seemed inconsistent with patterns of BP modulation seen in humans. Therefore, the aim of this study was to establish how BP is altered during chronic alteration of dietary potassium intake alongside alternative anions and/or NaCl supplementation. 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+) (HS) 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 K+ diet with either control or high NaCl level. Throughout the experiment BP, heart rate, activity, urinary ion excretion and plasma Na+/K+/Cl- levels were monitored. 2 weeks 0 K+ diet significantly increased BP, an effect augmented under HS diet. A trend to reduce BP was apparent under control and HS diet during chronic KCl feeding (> effect during resting period), whereas chronic KCit feeding only tended to reduce BP during HS diet. Plasma K+ levels inversely correlated with BP, with chronic KCl inducing a significant increase of 0.5mM, whereas chronic KCit feeding had no significant effect. Plasma aldosterone was also significantly increased 3.5 fold by chronic KCl feeding but not significantly by KCit diet. Total and phosphorylated NCC levels were significantly increased on 0 K+ diet. KCl and KCit feeding during control NaCl or HS intake reduced total NCC levels, whereas phosphorylated NCC was only reduced during chronic KCl intake. All dietary conditions had minimal effect on levels of WNK/SPAK pathway proteins, but αENaC (cleaved/uncleaved) levels were increased, substantially more during KCit feeding. Pendrin abundance was also significantly elevated by KCit feeding. In summary, we show a negative correlation of BP with plasma K+, consistent with human interventional studies. Chronic KCl has a greater ability to lower BP than KCit feeding. This may be related to the ability of KCl to lower NCC phosphorylation, or that large increases in αENaC and pendrin levels during KCit feeding oppose the BP lowering effects of reduced total NCC. As aldosterone levels were increased more with KCl, a differential role of aldosterone also needs to be considered.

Activation of the kidney sodium chloride cotransporter by the β2-adrenergic receptor agonist salbutamol increases blood pressure

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the β2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to β1-adrenergic receptors, the β2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In exvivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 μM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, β2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

Renal Natriuretic Peptide Receptor-C Deficiency Attenuates NaCl Cotransporter Activity in Angiotensin II-Induced Hypertension.

Genome-wide association studies have identified that NPR-C (natriuretic peptide receptor-C) variants are associated with elevation of blood pressure. However, the mechanism underlying the relationship between NPR-C and blood pressure regulation remains elusive. Here, we investigate whether NPR-C regulates Ang II (angiotensin II)-induced hypertension through sodium transporters activity. Wild-type mice responded to continuous Ang II infusion with an increased renal NPR-C expression. Global NPR-C deficiency attenuated Ang II-induced increased blood pressure both in male and female mice associated with more diuretic and natriuretic responses to a saline challenge. Interestingly, Ang II increased both total and phosphorylation of NCC (NaCl cotransporter) abundance involving in activation of WNK4 (with-no-lysine kinase 4)/SPAK (Ste20-related proline/alanine-rich kinase) which was blunted by NPR-C deletion. NCC inhibitor, hydrochlorothiazide, failed to induce natriuresis in NPR-C knockout mice. Moreover, low-salt and high-salt diets-induced changes of total and phosphorylation of NCC expression were normalized by NPR-C deletion. Importantly, tubule-specific deletion of NPR-C also attenuated Ang II-induced elevated blood pressure, total and phosphorylation of NCC expression. Mechanistically, in distal convoluted tubule cells, Ang II dose and time-dependently upregulated WNK4/SPAK/NCC kinase pathway and NPR-C/Gi/PLC/PKC signaling pathway mediated NCC activation. These results demonstrate that NPR-C signaling regulates NCC function contributing to sodium retention-mediated elevated blood pressure, which suggests that NPR-C is a promising candidate for the treatment of sodium retention-related hypertension.

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 β-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.

Geniposide in Gardenia jasminoides var. radicans Makino modulates blood pressure via inhibiting WNK pathway mediated by the estrogen receptors.

To investigate the effects of geniposide in an iridoid found in Gardenia jasminoides var. radicans Makino (GJRM) in spontaneous hypertensive rat (SHR) and explore the possible mechanisms. In this study, we detected the content of geniposide in GJRM by high-performance liquid chromatography (HPLC). Then, we used acute diuretic experiments to determine whether geniposide has diuretic effect. Moreover, we carried out experiments on SHR to further study the mechanism of hypertension, while real-time PCR, Western blot and immunohistochemistry were used for the experiments in vivo test. Hypotonic model was used for in vitro test. Our data showed that the content of geniposide in the extract of GJRM is 27.54%. Meanwhile, 50mg/kg geniposide showed the strongest effect on promoting urine volume. Further study indicated that the extract of GJRM and geniposide could significantly reduce blood pressure and promote the excretion of urine and Na+ in SHR. In addition, geniposide significantly inhibited the activation of the with-no-lysine kinase (WNK) signalling pathway and significantly increases the protein expressions of estrogen receptor α (ERα), estrogen receptor β (ERβ) and G protein-coupled receptor 30 (GPR30) in SHR. In hypotonic model, geniposide significantly inhibits the phosphorylation of NKCC and NCC and could be antagonistic to estrogen receptor antagonists. Collectively, we would suggest that geniposide may potentially be utilized as an adjunct to existing thiazide and thiazide-like diuretics to control hypertension, mainly through inhibiting the activation of the WNK signalling pathway mediated by the estrogen receptor.

Sodium-calcium exchanger 1 is the key molecule for urinary potassium excretion against acute hyperkalemia.

The sodium (Na+)-chloride cotransporter (NCC) expressed in the distal convoluted tubule (DCT) is a key molecule regulating urinary Na+ and potassium (K+) excretion. We previously reported that high-K+ load rapidly dephosphorylated NCC and promoted urinary K+ excretion in mouse kidneys. This effect was inhibited by calcineurin (CaN) and calmodulin inhibitors. However, the detailed mechanism through which high-K+ signal results in CaN activation remains unknown. We used Flp-In NCC HEK293 cells and mice to evaluate NCC phosphorylation. We analyzed intracellular Ca2+ concentration ([Ca2+]in) using live cell Ca2+ imaging in HEK293 cells. We confirmed that high-K+-induced NCC dephosphorylation was not observed without CaN using Flp-In NCC HEK29 cells. Extracellular Ca2+ reduction with a Ca2+ chelator inhibited high-K+-induced increase in [Ca2+]in and NCC dephosphorylation. We focused on Na+/Ca2+ exchanger (NCX) 1, a bidirectional regulator of cytosolic Ca2+ expressed in DCT. We identified that NCX1 suppression with a specific inhibitor (SEA0400) or siRNA knockdown inhibited K+-induced increase in [Ca2+]in and NCC dephosphorylation. In a mouse study, SEA0400 treatment inhibited K+-induced NCC dephosphorylation. SEA0400 reduced urinary K+ excretion and induced hyperkalemia. Here, we identified NCX1 as a key molecule in urinary K+ excretion promoted by CaN activation and NCC dephosphorylation in response to K+ load.