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Robert A Fenton - One of the best experts on this subject based on the ideXlab platform.

  • high dietary potassium causes ubiquitin dependent degradation of the kidney Sodium Chloride Cotransporter
    Journal of Biological Chemistry, 2021
    Co-Authors: Marleen L A Kortenoeven, Cristina Estevafont, Henrik Dimke, Soren Brandt Poulsen, Sathish K Murali, Robert A Fenton
    Abstract:

    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.

  • activation of the kidney Sodium Chloride Cotransporter by the β2 adrenergic receptor agonist salbutamol increases blood pressure
    Kidney International, 2021
    Co-Authors: Soren Brandt Poulsen, Marleen L A Kortenoeven, Johannes Loffing, Sathish K Murali, Lei Cheng, David Penton, Vladimir V Matchkov, Robert Little, Robert A Fenton
    Abstract:

    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 ex vivo 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.

  • The thiazide sensitive Sodium Chloride co-transporter NCC is modulated by site-specific ubiquitylation
    Scientific Reports, 2017
    Co-Authors: Lena L Rosenbaek, Gerardo Gamba, Federica Rizzo, Nanna Macaulay, Olivier Staub, Lorena Rojas-vega, Robert A Fenton
    Abstract:

    The renal Sodium Chloride Cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na^+ and K^+ homeostasis. Endogenous NCC is highly ubiquitylated, but the role of individual ubiquitylation sites is not established. Here, we assessed the role of 10 ubiquitylation sites for NCC function. Transient transfections of HEK293 cells with human wildtype (WT) NCC or various K to R mutants identified greater membrane abundance for K706R, K828R and K909R mutants. Relative to WT-NCC, stable tetracycline inducible MDCKI cell lines expressing K706R, K828R and K909R mutants had significantly higher total and phosphorylated NCC levels at the apical plasma membrane under basal conditions. Low Chloride stimulation increased membrane abundance of all mutants to similar or greater levels than WT-NCC. Under basal conditions K828R and K909R mutants had less ubiquitylated NCC in the plasma membrane, and all mutants displayed reduced NCC ubiquitylation following low Chloride stimulation. Thiazide-sensitive Sodium-22 uptakes were elevated in the mutants and internalization from the plasma membrane was significantly less than WT-NCC. K909R had increased half-life, whereas chloroquine or MG132 treatment indicated that K706 and K909 play roles in lysosomal and proteasomal NCC degradation, respectively. In conclusion, site-specific ubiquitylation of NCC plays alternative roles for NCC function.

  • functional assessment of Sodium Chloride Cotransporter ncc mutants in polarized mammalian epithelial cells
    American Journal of Physiology-renal Physiology, 2017
    Co-Authors: Federica Rizzo, Lena Lindtoft Rosenbaek, Nanna Macaulay, Olivier Staub, Robert A Fenton
    Abstract:

    The thiazide-sensitive Sodium Chloride Cotransporter NCC is important for maintaining serum Sodium (Na+) and, indirectly, serum potassium (K+) levels. Functional studies on NCC have used cell lines...

  • in primary aldosteronism mineralocorticoids influence exosomal Sodium Chloride Cotransporter abundance
    Journal of The American Society of Nephrology, 2017
    Co-Authors: Martin Wolley, Robert A Fenton, Richard D Gordon, Michael Stowasser
    Abstract:

    Distal tubular Sodium retention is a potent driver of hypertension, and the thiazide-sensitive Sodium-Chloride Cotransporter (NCC) has a key role in this process. In humans, factors regulating NCC are unclear, but in animal models, aldosterone is a potent regulator, possibly via effects on plasma potassium. We studied the effects of the mineralocorticoid fludrocortisone on the abundance of NCC and its phosphorylated form (pNCC) as well as WNK lysine deficient protein kinase 4 (WNK4) and STE20/SPS1-related, proline alanine-rich kinase (SPAK) in human urinary exosomes. We isolated exosomes from daily urine samples in 25 patients undergoing fludrocortisone suppression testing (100 μg every 6 hours for 4 days) to diagnose or exclude primary aldosteronism. Over the course of the test, NCC levels increased 3.68-fold (P<0.01) and pNCC levels increased 2.73-fold (P<0.01) relative to baseline. The ratio of pNCC/NCC dropped by 48% (P<0.01). The abundance of WNK4 increased 3.23-fold (P<0.01), but SPAK abundance did not change significantly (P=0.14). Plasma potassium concentration strongly and negatively correlated with pNCC, NCC, and WNK4 abundance (P<0.001 for all). This study shows that, in humans, mineralocorticoid administration is associated with a rapid increase in abundance of NCC and pNCC, possibly via the WNK pathway. These effects may be driven by changes in plasma potassium.

Chih Jen Cheng - One of the best experts on this subject based on the ideXlab platform.

  • reply to farfel et al is enhanced Chloride reabsorption in proximal tubule a possible mechanism of metabolic acidosis in phaii
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Jen Chi Chen, Chou Long Huang, Shih-hua Lin, Chih Jen Cheng
    Abstract:

    Hyperchloremic metabolic acidosis along with hypertension and hyperkalemia are features of pseudohypoaldosteronism type II (PHAII). Increased activity of Sodium Chloride Cotransporter (NCC) is believed to be an important mechanism of these phenotypic features (1). Gain-of-function mutations of WNK4 in PHAII activate NCC in the distal convoluted tubule, which leads to enhanced Sodium and Chloride reabsorption causing hypertension and diminished Sodium delivery to the downstream cortical collecting duct, resulting in hyperkalemia. Our recent report (2) that Chloride-insensitive WNK4 knockin mouse exhibits increased WNK4 kinase activity and fully recapitulates PHAII phenotype supports the notion. Still, as mentioned by Farfel et al. (3), there remain debates on the … [↵][1]1To whom correspondence may be addressed. Email: laurence1234kimo{at}yahoo.com.tw. [1]: #xref-corresp-1-1

  • WNK4 kinase is a physiological intracellular Chloride sensor.
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Jen Chi Chen, Chou Long Huang, Yi Fen Lo, Chih Jen Cheng
    Abstract:

    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.

  • chronic metabolic acidosis activates renal tubular Sodium Chloride Cotransporter through angiotension ii dependent wnk4 spak phosphorylation pathway
    Scientific Reports, 2016
    Co-Authors: Yuwei Fang, Sungsen Yang, Chih Jen Cheng, Shih-hua Lin, Minhua Tseng, Huimin Hsu
    Abstract:

    The mechanism by which chronic metabolic acidosis (CMA) regulates Sodium (Na(+))-Chloride (Cl(-)) Cotransporter (NCC) in the renal distal convoluted tubules remains unexplored. We examined the role of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and with-no-lysine kinase 4 (WNK4) on expression of NCC in mouse models of CMA. CMA was induced by NH4Cl in wild type mice (WTA mice), SPAK, and WNK4 knockout mice. The quantities of Ncc mRNA, expression of total NCC, phosphorylated (p)-NCC, SPAK and WNK4 in the kidneys as well as NCC inhibition with hydrochlorothiazide and Na(+) balance were evaluated. Relative to WT mice, WTA mice had similar levels of Ncc mRNA, but increased expression of total and p-NCC, SPAK, and WNK4 and an exaggerated response to hydrochlorothiazide which could not be observed in SPAK or WNK4 knockout mice with CMA. In WTA mice, increased plasma renin activity, aldosterone and angiotensin II concentrations accompanied by a significantly negative Na(+) balance. High Na(+) diet abolished the enhanced NCC expression in WTA mice. Furthermore, an angiotensin II type 1 receptor blocker rather than a mineralocorticoid receptor antagonist exerted a marked inhibition on Na(+) reabsorption and NCC phosphorylation in WTA mice. CMA increases WNK4-SPAK-dependent NCC phosphorylation and appears to be secondary to previous natriuresis with volume-dependent angiotensin II activation.

  • A unifying mechanism for WNK kinase regulation of Sodium-Chloride Cotransporter
    Pflügers Archiv - European Journal of Physiology, 2015
    Co-Authors: Chou Long Huang, Chih Jen Cheng
    Abstract:

    Mammalian with-no-lysine [K] (WNK) kinases are a family of four serine-threonine protein kinases, WNK1-4. Mutations of WNK1 and WNK4 in humans cause pseudohypoaldosteronism type II (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. Increased Na^+ reabsorption through Na^+–Cl^− Cotransporter (NCC) in the distal convoluted tubule plays an important role in the pathogenesis of hypertension in patients with PHA2. However, how WNK1 and WNK4 regulate NCC and how mutations of WNKs cause activation of NCC have been controversial. Here, we review current state of literature supporting a compelling model that WNK1 and WNK4 both contribute to stimulation of NCC. The precise combined effects of WNK1 and WNK4 on NCC remain unclear but likely are positive rather than antagonistic. The recent discovery that WNK kinases may function as an intracellular Chloride sensor adds a new dimension to the physiological role of WNK kinases. Intracellular Chloride-dependent regulation of WNK’s may underlie the mechanism of regulation of NCC by extracellular K^+. Definite answer yet will require future investigation by tubular perfusion in mice with altered WNK kinase expression.

  • thiazide sensitive na cl Cotransporter ncc gene inactivation results in increased duodenal ca2 absorption enhanced osteoblast differentiation and elevated bone mineral density
    Journal of Bone and Mineral Research, 2015
    Co-Authors: Yujuei Hsu, Sungsen Yang, Chih Jen Cheng, Shuting Liu, Shihming Huang, Tom Chau, Pauling Chu, Donald Salter, Herngsheng Lee, Shih-hua Lin
    Abstract:

    Inactivation of the thiazide-sensitive Sodium Chloride Cotransporter (NCC) due to genetic mutations in Gitelman's syndrome (GS) or pharmacological inhibition with thiazide diuretics causes hypocalciuria and increased bone mineral density (BMD) with unclear extrarenal calcium (Ca(2+) ) regulation. We investigated intestinal Ca(2+) absorption and bone Ca(2+) metabolism in nonsense Ncc Ser707X (S707X) homozygous knockin mice (Ncc(S707X/S707X) mice). Compared to wild-type and heterozygous knockin littermates, Ncc(S707X/S707X) mice had increased intestinal absorption of (45) Ca(2+) and expression of the active Ca(2+) transport machinery (transient receptor potential vanilloid 6, calbindin-D9K , and plasma membrane Ca(2+) ATPase isoform 1b). Ncc(S707X/S707X) mice had also significantly increased Ca(2+) content accompanied by greater mineral apposition rate (MAR) in their femurs and higher trabecular bone volume, cortical bone thickness, and BMD determined by μCT. Their osteoblast differentiation markers, such as bone alkaline phosphatase, procollagen I, osteocalcin, and osterix, were also significantly increased while osteoclast activity was unaffected. Analysis of marrow-derived bone cells, either treated with thiazide or directly cultured from Ncc S707X knockin mice, showed that the differentiation of osteoblasts was associated with increased phosphorylation of mechanical stress-induced focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). In conclusion, NCC inhibition stimulates duodenal Ca(2+) absorption as well as osteoblast differentiation and bone Ca(2+) storage, possibly through a FAK/ERK dependent mechanism.

Johannes Loffing - One of the best experts on this subject based on the ideXlab platform.

  • activation of the kidney Sodium Chloride Cotransporter by the β2 adrenergic receptor agonist salbutamol increases blood pressure
    Kidney International, 2021
    Co-Authors: Soren Brandt Poulsen, Marleen L A Kortenoeven, Johannes Loffing, Sathish K Murali, Lei Cheng, David Penton, Vladimir V Matchkov, Robert Little, Robert A Fenton
    Abstract:

    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 ex vivo 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.

  • Uromodulin is expressed in the distal convoluted tubule, where it is critical for regulation of the Sodium Chloride Cotransporter NCC.
    Kidney international, 2018
    Co-Authors: Natsuko Tokonami, Johannes Loffing, Olivier Devuyst, Tomoaki Takata, Jan Beyeler, Iris Ehrbar, Ayumi Yoshifuji, Erik Ilsø Christensen, Eric Olinger
    Abstract:

    Uromodulin, the most abundant protein in normal urine, is essentially produced by the cells lining the thick ascending limb. There it regulates the activity of the Cotransporter NKCC2 and is involved in Sodium Chloride handling and blood pressure regulation. Conflicting reports suggested that uromodulin may also be expressed in the distal convoluted tubule (DCT) where its role remains unknown. Using microdissection studies combined with fluorescent in situ hybridization and co-immunostaining analyses, we found a significant expression of uromodulin in mouse and human DCT at approximately 10% of thick ascending limb expression levels, but restricted to the early part of the DCT (DCT1). Genetic deletion of Umod in mouse was reflected by a major shift in NCC activity from the DCT1 to the downstream DCT2 segment, paralleled by a compensatory expansion of DCT2. By increasing the distal Sodium Chloride and calcium ion load with chronic furosemide administration, an intrinsic compensatory defect in the DCT from Umod-/- compared to wild type mice was found manifested as Sodium wasting and hypercalciuria. In line, co-expression studies in HEK cells suggested a facilitating role for uromodulin in NCC phosphorylation, possibly via SPAK-OSR1 modulation. These experiments demonstrate a significant expression of uromodulin in the early part of mouse and human DCT. Thus, biosynthesis of uromodulin in the DCT1 is critical for its function, structure and plasticity, suggesting novel links between uromodulin, blood pressure control and risk of kidney stones.

  • acetazolamide attenuates lithium induced nephrogenic diabetes insipidus
    Journal of The American Society of Nephrology, 2016
    Co-Authors: Theun De Groot, Marleen L A Kortenoeven, Johannes Loffing, Anne P Sinke, Ruben Baumgarten, Jack F M Wetzels, Mohammad Alsady, Olivier Devuyst, Peter M T Deen
    Abstract:

    To reduce lithium-induced nephrogenic diabetes insipidus (lithium-NDI), patients with bipolar disorder are treated with thiazide and amiloride, which are thought to induce antidiuresis by a compensatory increase in prourine uptake in proximal tubules. However, thiazides induced antidiuresis and alkalinized the urine in lithium-NDI mice lacking the Sodium-Chloride Cotransporter, suggesting that inhibition of carbonic anhydrases (CAs) confers the beneficial thiazide effect. Therefore, we tested the effect of the CA-specific blocker acetazolamide in lithium-NDI. In collecting duct (mpkCCD) cells, acetazolamide reduced the cellular lithium content and attenuated lithium-induced downregulation of aquaporin-2 through a mechanism different from that of amiloride. Treatment of lithium-NDI mice with acetazolamide or thiazide/amiloride induced similar antidiuresis and increased urine osmolality and aquaporin-2 abundance. Thiazide/amiloride-treated mice showed hyponatremia, hyperkalemia, hypercalcemia, metabolic acidosis, and increased serum lithium concentrations, adverse effects previously observed in patients but not in acetazolamide-treated mice in this study. Furthermore, acetazolamide treatment reduced inulin clearance and cortical expression of Sodium/hydrogen exchanger 3 and attenuated the increased expression of urinary PGE2 observed in lithium-NDI mice. These results show that the antidiuresis with acetazolamide was partially caused by a tubular-glomerular feedback response and reduced GFR. The tubular-glomerular feedback response and/or direct effect on collecting duct principal or intercalated cells may underlie the reduced urinary PGE2 levels with acetazolamide, thereby contributing to the attenuation of lithium-NDI. In conclusion, CA activity contributes to lithium-NDI development, and acetazolamide attenuates lithium-NDI development in mice similar to thiazide/amiloride but with fewer adverse effects.

  • hydrochlorothiazide attenuates lithium induced nephrogenic diabetes insipidus independently of the Sodium Chloride Cotransporter
    American Journal of Physiology-renal Physiology, 2014
    Co-Authors: Anne P Sinke, Marleen L A Kortenoeven, Johannes Loffing, Theun De Groot, Ruben Baumgarten, Jack F M Wetzels, Olivier Devuyst, Peter M T Deen
    Abstract:

    Lithium is the most common cause of nephrogenic diabetes insipidus (Li-NDI). Hydrochlorothiazide (HCTZ) combined with amiloride is the mainstay treatment in Li-NDI. The paradoxical antidiuretic action of HCTZ in Li-NDI is generally attributed to increased Sodium and water uptake in proximal tubules as a compensation for increased volume loss due to HCTZ inhibition of the Na-Cl Cotransporter (NCC), but alternative actions for HCTZ have been suggested. Here, we investigated whether HCTZ exerted an NCC-independent effect in Li-NDI. In polarized mouse cortical collecting duct (mpkCCD) cells, HCTZ treatment attenuated the Li-induced downregulation of aquaporin-2 (AQP2) water channel abundance. In these cells, amiloride reduces cellular Li influx through the epithelial Sodium channel (ENaC). HCTZ also reduced Li influx, but to a lower extent. HCTZ increased AQP2 abundance on top of that of amiloride and did not affect the ENaC-mediated transcellular voltage. MpkCCD cells did not express NCC mRNA or protein. These data indicated that in mpkCCD cells, HCTZ attenuated lithium-induced downregulation of AQP2 independently of NCC and ENaC. Treatment of Li-NDI NCC knockout mice with HCTZ revealed a significantly reduced urine volume, unchanged urine osmolality, and increased cortical AQP2 abundance compared with Li-treated NCC knockout mice. HCTZ treatment further resulted in reduced blood Li levels, creatinine clearance, and alkalinized urinary pH. Our in vitro and in vivo data indicate that part of the antidiuretic effect of HCTZ in Li-NDI is NCC independent and may involve a tubuloglomerular feedback response-mediated reduction in glomerular filtration rate due to proximal tubular carbonic anhydrase inhibition.

  • The Sodium Chloride Cotransporter SLC12A3: new roles in Sodium, potassium, and blood pressure regulation
    Pflügers Archiv - European Journal of Physiology, 2014
    Co-Authors: Arthur D. Moes, Nils Lubbe, Robert Zietse, Johannes Loffing, Ewout J. Hoorn
    Abstract:

    SLC12A3 encodes the thiazide-sensitive Sodium Chloride Cotransporter (NCC), which is primarily expressed in the kidney, but also in intestine and bone. In the kidney, NCC is located in the apical plasma membrane of epithelial cells in the distal convoluted tubule. Although NCC reabsorbs only 5 to 10 % of filtered Sodium, it is important for the fine-tuning of renal Sodium excretion in response to various hormonal and non-hormonal stimuli. Several new roles for NCC in the regulation of Sodium, potassium, and blood pressure have been unraveled recently. For example, the recent discoveries that NCC is activated by angiotensin II but inhibited by dietary potassium shed light on how the kidney handles Sodium during hypovolemia (high angiotensin II) and hyperkalemia. The additive effect of angiotensin II and aldosterone maximizes Sodium reabsorption during hypovolemia, whereas the inhibitory effect of potassium on NCC increases delivery of Sodium to the potassium-secreting portion of the nephron. In addition, great steps have been made in unraveling the molecular machinery that controls NCC. This complex network consists of kinases and ubiquitinases, including WNKs, SGK1, SPAK, Nedd4-2, Cullin-3, and Kelch-like 3. The pathophysiological significance of this network is illustrated by the fact that modification of each individual protein in the network changes NCC activity and results in salt-dependent hypotension or hypertension. This review aims to summarize these new insights in an integrated manner while identifying unanswered questions.

H Cai - One of the best experts on this subject based on the ideXlab platform.

  • dietary salt modulates the Sodium Chloride Cotransporter expression likely through an aldosterone mediated wnk4 erk1 2 signaling pathway
    Pflügers Archiv: European Journal of Physiology, 2012
    Co-Authors: Lingyun Lai, H Cai, Yiqian Zhang, Xiuyan Feng, Defeng Liu, Jing Chen, Bowen Niu
    Abstract:

    WNK is a serine/threonine kinase. Mutation in WNK1 or WNK4 kinase results in pseudohypoaldosteronism type II (PHA II) featuring hypertension, hyperkalemia and metabolic acidosis. Sodium Chloride Cotransporter (NCC) is known to be regulated by phosphorylation and trafficking. Dietary salt and hormonal stimulation, such as aldosterone, also affect the regulation of NCC. We have previously reported that WNK4 inhibits NCC protein expression. To determine whether dietary salt affects NCC abundance through WNK4-mediated mechanism, we investigated the effects of dietary salt change with or without aldosterone infusion (1 mg/kg/day) on NCC and WNK4 expression in rats. We found that high-salt (HS, 4% NaCl) diet significantly inhibits NCC mRNA expression and protein abundance while enhancing WNK4 mRNA and protein expression, whereas low-salt (LS, 0.07% NaCl) diet increases NCC mRNA expression and protein abundance while reducing WNK4 expression. We also found that aldosterone infusion in HS-fed rats increases NCC mRNA expression and protein abundance, but decreases WNK4 expression. Administration with spironolactone (0.1 g/kg/day) in LS-fed rats decreases NCC mRNA expression and protein abundance while increasing WNK4 expression. We further showed that ERK1/2 phosphorylation was increased in HS-fed rats, but decreased in LS-fed rats. In HEK293 cells, over-expressed WNK4 increases ERK1/2 phosphorylation, whereas knockdown of WNK4 expression decreases ERK1/2 phosphorylation. Aldosterone treatment for 3 h decreases ERK1/2 phosphorylation. These data suggest that dietary salt change affects NCC protein abundance in an aldosterone-dependent mechanism likely via the WNK4-ERK1/2-mediated pathway.

  • wnk4 kinase regulates surface expression of the human Sodium Chloride Cotransporter in mammalian cells
    Kidney International, 2006
    Co-Authors: H Cai, Valeriu Cebotaru, Yinghong Wang, X M Zhang, Liudmila Cebotaru, Sandra E Guggino, William B Guggino
    Abstract:

    Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of Sodium Chloride Cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9±6.8% vs 100% in control, P n =6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9±5.3% vs 100%, P =NS, n =6). Another mutant R1185C still partially reduces surface expression of NCC (76.2±11.8% vs 100%, P n =6). The reduction of NCC surface expression by WNK4 WT (62.9±3.3% of control group) is not altered by WT dynamin ((61.8±3.7% ( P =NS)) or its mutant K44A ((65.4±14.1% ( P =NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.

  • wnk4 kinase regulates surface expression of the human Sodium Chloride Cotransporter in mammalian cells commentary
    Kidney International, 2006
    Co-Authors: J B Peng, H Cai, Valeriu Cebotaru, Yinghong Wang, X M Zhang, Liudmila Cebotaru, Sandra E Guggino, P D Bell, William B Guggino
    Abstract:

    Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of Sodium Chloride Cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9±6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9±5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2 ± 11.8% vs 100%, P < 0.05, n = 6). The reduction of NCC surface expression by WNK4 WT (62.9±3.3% of control group) is not altered by WT dynamin ((61.8±3.7% (P=NS)) or its mutant K44A ((65.4±14.1% (P= NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.

Robert S Hoover - One of the best experts on this subject based on the ideXlab platform.

  • wnk3 kinase enhances the Sodium Chloride Cotransporter expression via an erk 1 2 signaling pathway
    Nephron, 2016
    Co-Authors: Dexuan Wang, Robert S Hoover, Yiqian Zhang, Jinhua Han, Shufang Pan, Xiuyan Feng, Zhizhi Zhuang, Courtney M Caroti, Jieqiu Zhuang, Qiyi Zeng
    Abstract:

    Background: WNK kinase is a serine/threonine kinase that plays an important role in normal blood pressure homeostasis. WNK3 was previously found to enhance the activity of Sodium Chloride Cotransporter (NCC) in Xenopus oocyte. However, the mechanism through which it works remains unclear. Methods: Using overexpression and siRNA knock-down techniques, the effects of WNK3 on NCC in both Cos-7 and mouse distal convoluted cells were analyzed by Western blot. Results: We found that WNK3 significantly increased NCC protein expression in a dose-dependent manner. NCC protein expression in Cos-7 cells was markedly decreased after 2 h treatment with protease inhibitor, cycloheximide (CHX) in the NCC alone group, but was significantly decreased after 8 h treatment of CHX in the WNK3 + NCC group. WNK3 significantly increased NCC protein expression in both NCC alone and WNK3 + NCC groups regardless the overnight treatments of bafilomycin A1, a proton pump inhibitor, suggesting that WNK3-mediated increased NCC expression is not dependent on the lysosomal pathway. We further found that WNK3 group had a quicker NCC recovery than the control group using CHX pulse assay, suggesting that WNK3 increases NCC protein synthesis. WNK3 enhanced NCC protein level while reducing ERK 1/2 phosphorylation. In addition, knock-down of ERK 1/2 expression reversed WNK3-mediated increase of NCC expression. Conclusion: These results suggest that WNK3 enhances NCC protein expression by increasing NCC synthesis via an ERK 1/2-dependent signaling pathway.

  • parathyroid hormone pth regulates the Sodium Chloride Cotransporter via ras guanyl releasing protein 1 ras grp1 and extracellular signal regulated kinase erk 1 2 mitogen activated protein kinase mapk pathway
    Translational Research, 2011
    Co-Authors: Leslie L Cooke, Robert S Hoover
    Abstract:

    The Sodium Chloride Cotransporter (NCC) is the principal salt absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of thiazide diuretics. Using a mammalian cell model system to assess NCC function, we demonstrated previously that Ras guanyl releasing protein 1 (Ras-GRP1) mediates phorbol ester-induced suppression of the function and surface expression of NCC in a protein kinase C (PKC)-independent and extracellular signal-regulated kinase (ERK)1/2-dependent manner. Given that phorbol esters are functional analogs of diacylglycerol (DAG), this finding suggested a potential physiologic regulation of NCC by DAG. The parathyroid hormone (PTH) receptor is a G-protein-coupled receptor that is expressed in the DCT and activates PLC resulting in the generation of DAG. In this article, we demonstrate that PTH suppresses NCC function via a PLC/Ras-GRP1/ERK pathway. A functional assessment of NCC measuring thiazide-sensitive 22 Na + flux revealed that PTH suppresses NCC function. The inhibition of PLC prevented the suppression of NCC, indicating that PLC was necessary for this effect. Inhibitors of PKC and protein kinase A (PKA) had no effect on this suppression, but mitogen-activated protein kinase (MAPK) inhibitors prevented the PTH effect completely. Ras-GRP1 activates the MAPK pathway though activation of the small G-protein Ras. Gene silencing of Ras-GRP1 prevented the PTH-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of Ras-GRP1 in mediating the PTH-induced suppression of NCC activity through stimulation of the MAPK pathway.

  • rasgrp1 stimulation enhances ubiquitination and endocytosis of the Sodium Chloride Cotransporter
    American Journal of Physiology-renal Physiology, 2010
    Co-Authors: Erikjan Kamsteeg, Peter M T Deen, Leslie L Cooke, Lauren Moddes, Robert S Hoover
    Abstract:

    The Sodium-Chloride Cotransporter (NCC) is the principal salt-absorptive pathway in the distal convoluted tubule. Recently, we described a novel pathway of NCC regulation in which phorbol esters (PE) stimulate Ras guanyl-releasing protein 1 (RasGRP1), triggering a cascade ultimately activating ERK1/2 MAPK and decreasing NCC cell surface expression (Ko B, Joshi LM, Cooke LL, Vazquez N, Musch MW, Hebert SC, Gamba G, Hoover RS. Proc Natl Acad Sci USA 104: 20120-20125, 2007). Little is known about the mechanisms which underlie these effects on NCC activity. Regulation of NCC via changes in NCC surface expression has been reported, but endocytosis of NCC has not been demonstrated. In this study, utilizing biotinylation, internalization assays, and a dynamin dominant-negative construct, we demonstrate that the regulation of NCC by PE occurs via an enhancement in internalization of NCC and is dynamin dependent. In addition, immunoprecipitation of NCC and subsequent immunoblotting for ubiquitin showed increased ubiquitination of NCC with phorbol ester treatment. MEK1/2 inhibitors and gene silencing of RasGRP1 indicated that this effect was dependent on RasGRP1 and ERK1/2 activation. Inhibition of ubiquitination prevents any PE-mediated decrease in NCC surface expression as measured by biotinylation or NCC activity as measured by radiotracer uptake. These findings confirmed that the PE effect on NCC is mediated by endocytosis of NCC. Furthermore, ubiquitination of NCC is essential for this process and this ubiquitination is dependent upon RasGRP1-mediated ERK1/2 activation.

  • molecular physiology of the thiazide sensitive Sodium Chloride Cotransporter
    Current Opinion in Nephrology and Hypertension, 2009
    Co-Authors: Robert S Hoover
    Abstract:

    Purpose of reviewThis review summarizes recent advances in the understanding of the molecular physiology and regulation of the thiazide-sensitive Sodium–Chloride Cotransporter (NCC).Recent findingsMutations of with-no-lysine (WNK) kinases 1 and 4 result in hyperactivity of NCC and familial hyperkale

  • phorbol ester stimulation of rasgrp1 regulates the Sodium Chloride Cotransporter by a pkc independent pathway
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: B R Ko, Steven C Hebert, Gerardo Gamba, Leslie L Cooke, Leena M Joshi, Norma Vazquez, Mark W Musch, Robert S Hoover
    Abstract:

    The Sodium-Chloride Cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRP1), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive 22Na+ uptakes revealed that TPA completely suppresses NCC function. Biotinylation experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway.