Regulation Of NCC Research Articles

Loss of WNK3 is compensated for by the WNK1/SPAK axis in the kidney of the mouse

WNK3 kinase is expressed throughout the nephron and acts as a positive regulator of NKCC2 and NCC in vitro. Here we addressed the in vivo relevance of WNK3 using WNK3-deficient mice. WNK3-/- mice were viable and showed no gross abnormalities. The net tubular function was similar in wild-type (WT) and WNK3-/- mice as assessed by determination of 24-h urine output (1.63 ± .06 in WT and 1.55 ± .1 ml in WNK3-/-, n=16; P=0.42) and ambient urine osmolarity (1,804 ± 62 in WT vs. 1,819 ± 61 mosmol/kg in WNK3-/-, n=40; P=0.86). Water restriction (48 h) increased urine osmolarity similarly in both genotypes to 3,440 ± 220 and 3,200 ± 180 mosmol/kg in WT and WNK3-/- mice, respectively (n=11; P=0.41). The glomerular filtration rate (343 ± 22 vs. 315 ± 13 ml/min), renal blood flow (1.35 ± 0.1 vs. 1.42 ± 0.04 ml), and plasma renin concentration (94 ± 18 vs. 80 ± 13 ng ANG I·ml(-1)·h(-1)) were similar between WT and WNK3-/- mice (n=13; P=0.54). WNK1 was markedly upregulated in WNK3-deficient mice, whereas the expression of WNK4 was similar in both genotypes. When the mice were fed a salt-restricted diet [0.02% NaCl (wt/wt)] the levels of pSPAK/OSR1, pNKCC2, and pNCC were enhanced in both genotypes compared with the baseline conditions, with the levels in WNK3-/- exceeding those in WT mice. The upregulation of pSPAK/OSR1, pNKCC2, and pNCC in WNK3-/- mice relative to the levels in WT mice when fed a low-salt diet was paralleled by an increased diuresis in response to hydrochlorothiazide. In summary, the overall relevance of WNK3 for the renal reabsorption of NaCl appears to be limited and can be largely compensated for by the activation of WNK3-independent pathways. Consequently, our data suggest that WNK3 may serve as a member of a kinase network that facilitates the fine-tuning of renal transepithelial NaCl transport.

A new model of the distal convoluted tubule

The Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na(+) balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl(-)-dependent (22)Na(+) uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.

Aldosterone does not require angiotensin II to activate NCC through a WNK4–SPAK–dependent pathway

We and others have recently shown that angiotensin II can activate the sodium chloride cotransporter (NCC) through a WNK4–SPAK-dependent pathway. Because WNK4 was previously shown to be a negative regulator of NCC, it has been postulated that angiotensin II converts WNK4 to a positive regulator. Here, we ask whether aldosterone requires angiotensin II to activate NCC and if their effects are additive. To do so, we infused vehicle or aldosterone in adrenalectomized rats that also received the angiotensin receptor blocker losartan. In the presence of losartan, aldosterone was still capable of increasing total and phosphorylated NCC twofold to threefold. The kinases WNK4 and SPAK also increased with aldosterone and losartan. A dose-dependent relationship between aldosterone and NCC, SPAK, and WNK4 was identified, suggesting that these are aldosterone-sensitive proteins. As more functional evidence of increased NCC activity, we showed that rats receiving aldosterone and losartan had a significantly greater natriuretic response to hydrochlorothiazide than rats receiving losartan only. To study whether angiotensin II could have an additive effect, rats receiving aldosterone with losartan were compared with rats receiving aldosterone only. Rats receiving aldosterone only retained more sodium and had twofold to fourfold increase in phosphorylated NCC. Together, our results demonstrate that aldosterone does not require angiotensin II to activate NCC and that WNK4 appears to act as a positive regulator in this pathway. The additive effect of angiotensin II may favor electroneutral sodium reabsorption during hypovolemia and may contribute to hypertension in diseases with an activated renin–angiotensin–aldosterone system.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-012-1104-0) contains supplementary material, which is available to authorized users.

Activation of the renal Na + :Cl − cotransporter by angiotensin II is a WNK4-dependent process

Pseudohypoaldosteronism type II is a salt-sensitive form of hypertension with hyperkalemia in humans caused by mutations in the with-no-lysine kinase 4 (WNK4). Several studies have shown that WNK4 modulates the activity of the renal Na(+)Cl(-) cotransporter, NCC. Because the renal consequences of WNK4 carrying pseudoaldosteronism type II mutations resemble the response to intravascular volume depletion (promotion of salt reabsorption without K(+) secretion), a condition that is associated with high angiotensin II (AngII) levels, it has been proposed that AngII signaling might affect WNK4 modulation of the NCC. In Xenopus laevis oocytes, WNK4 is required for modulation of NCC activity by AngII. To demonstrate that WNK4 is required in the AngII-mediated regulation of NCC in vivo, we used a total WNK4-knockout mouse strain (WNK4(-/-)). WNK4 mRNA and protein expression were absent in WNK4(-/-) mice, which exhibited a mild Gitelman-like syndrome, with normal blood pressure, increased plasma renin activity, and reduced NCC expression and phosphorylation at T-58. Immunohistochemistry revealed normal morphology of the distal convoluted tubule with reduced NCC expression. Low-salt diet or infusion of AngII for 4 d induced phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and of NCC at S-383 and T-58, respectively, in WNK4(+/+) but not WNK4(-/-) mice. Thus, the absence of WNK4 in vivo precludes NCC and SPAK phosphorylation promoted by a low-salt diet or AngII infusion, suggesting that AngII action on the NCC occurs via a WNK4-SPAK-dependent signaling pathway. Additionally, stimulation of aldosterone secretion by AngII, but not by a high-K(+) diet, was impaired in WNK4(-/-) mice.

Loss of Wnk3 is Compensated for by ks‐Wnk1 in the Kidney of the Mouse

Wnk3 kinase is expressed throughout the nephron and acts as a positive regulator of NKCC2 and NCC in vitro. Here we addressed the in vivo relevance of Wnk3 by the use of Wnk3‐deficient mice. Wnk3−/− mice were viable and showed no gross abnormalities. Net tubular function was similar in WT and Wnk3−/− mice as assessed by determination of 24‐hr urine output (1.7±.2 in WT and 1.6±.2 mL in Wnk3−/−, p=.52) and ambient urine osmolarity (1680±170 vs. 1780±170 mosmol/kg; p=.11, n=10). Water restriction (48 hrs) increased urine osmolarity to 3440±220 and 3200±180 mosmol/kg in WT and Wnk3−/−, respectively (n=10, p=.41). During a salt‐restricted diet (7d, 0.02% NaCl) urine osmolarity tended to decrease in WT (1584±200 mosmol/kg; p=.34 vs. standard diet) whereas it was reduced significantly in Wnk3−/− mice (1183±130 mosmol/kg; p=.025 vs. standard diet). Glomerular filtration rate (343±22 vs. 315±13 mL/min), renal blood flow (1.35±.1 vs. 1.42±.04 mL), and plasma renin concentration (160±38 vs. 135±19 ng AngI/mL/hr) were similar in WT and Wnk3−/− (p>.05). The abundance of the kidney‐specific Wnk1 isoform (ks‐Wnk1) was markedly enhanced in Wnk3−/− compared to WT mice, whereas expression of full‐length Wnk1 and Wnk4 was unchanged. In summary, the function of Wnk3 can largely be compensated for by other kinases, most likely by ks‐Wnk1, and this may cause compensatory activation of downstream targets like eNaC.

WNK4 is required for the Angiotensin II activation of NCC in vivo

In Xenopus laevis oocytes and mpkDCT cells, WNK4 is required for modulation of NCC activity by angiotensin II (AngII). Additionally, in rats AngII promotes NCC phosphorylation independently of aldosterone. To define the requirement of WNK4 in AngII regulation of NCC in vivo, we used a complete WNK4‐knockout (WNK4−/−) mouse strain generated by targeted disruption in Pfizer Inc. Control littermates and WNK4−/− mice of 14 weeks were studied. WNK4 mRNA and protein expression were absent in WNK4−/− mice, which exhibited a mild Gitelman's like syndrome, with normal blood pressure, together with increased plasma renin activity, and reduction of NCC expression and phosphorylation at T‐58. Immunohistochemistry revealed normal morphology of distal convoluted tubule. Low salt diet or AngII infusion for four days induced SPAK and NCC phosphorylation at S‐383 and T‐58, respectively, in control littermates, but not in WNK4−/−. Low K+ diet aggravated hypokalemia, but induced SPAK phosphorylation in WNK4−/− mice. Thus, absence of WNK4 in vivo precludes the NCC and SPAK phosphorylation promoted by low salt diet or AngII infusion, but not by hypokalemia, suggesting that AngII action on NCC occurs by signaling through a WNK4‐SPAK dependent mechanism.

SPAK, OSR1 and Cab39/MO25 form an Interdependent Signaling system which Regulates Thiazide‐Sensitive Salt‐transport, Distal Tubule Mass and Blood Pressure

STE20/SPS‐1‐related proline‐alanine‐rich protein kinase (SPAK) and, Oxidative Stress Related Kinase (OSR1), co‐localize at the apical membrane of the Thick ascending Limb (TAL) and Distal Convoluted Tubule (DCT) and both regulate the potassium‐dependent sodium‐chloride co‐transporter, NKCC2, thiazide‐sensitive sodium‐chloride cotransporter, NCC in vitro. Yet genetic ablation of SPAK in mice causes a salt‐wasting nephropathy that is restricted to the DCT, reminiscent of Gitelman's syndrome. Here, we explore why proper DCT function is especially SPAK‐dependent. In the TAL of SPAK−/− mice, OSR1 and Cab39/MO25, a newly described OSR1/SPAK regulatory protein, remain at the apical membrane where they function with a compensatory increase in the AMP‐activated kinase (AMPK) to hyper‐phosphorylate NKCC2. By contrast, the OSR1/SPAK/M025 signal transduction apparatus is completely disrupted in the DCT. OSR1 and MO25 become largely displaced from the thiazide‐sensitive sodium‐chloride cotransporter, NCC, and the apical membrane. OSR1 redistributes to dense punctate structures within the cytoplasm. These changes are paralleled by a dramatic decrease in NCC abundance and phosphorylation. Without SPAK and the proper localization of OSR1 and MO25, phosphorylation‐dependent regulation of NCC by dietary sodium restriction is lost. SPAK−/− mice also exhibit a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the interdependent nature of OSR1 and MO25 on SPAK in the DCT, SPAK−/− mice are highly sensitive to dietary salt‐restriction, displaying prolonged negative sodium balance and hypotension.

Nedd4-2 and the Regulation of Epithelial Sodium Transport

Nedd4-2 is a ubiquitin ligase previously demonstrated to regulate endocytosis and lysosomal degradation of the epithelial Na+ channel (ENaC) and other ion channels and transporters. Recent studies using Nedd4-2 knockout mice specifically in kidney or lung epithelia has revealed a critical role for this E3 ubiquitin ligase in regulating salt and fluid transport in these tissues/organs and in maintaining homeostasis of body blood pressure. Interestingly, the primary targets for Nedd4-2 may differ in these two organs: in the lung Nedd4-2 targets ENaC, and loss of Nedd4-2 leads to excessive ENaC function and to cystic fibrosis – like lung disease, whereas in the kidney, Nedd4-2 targets the Na+/Cl− cotransporter (NCC) in addition to targeting ENaC. In accord, loss of Nedd4-2 in the distal nephron leads to increased NCC abundance and function. The aldosterone-responsive kinase, Sgk1, appears to be involved in the regulation of NCC by Nedd4-2 in the kidney, similar to its regulation of ENaC. Collectively, these new findings underscore the physiological importance of Nedd4-2 in regulating epithelial salt and fluid transport and balance.

Dietary salt modulates the sodium chloride cotransporter expression likely through an aldosterone-mediated WNK4-ERK1/2 signaling pathway

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 (1mg/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 (0.1g/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 3h 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.

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

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.

Phosphorylation‐dependent regulation of NCC is blunted in SPAK null mice (SPAK −/− )

Mice lacking the STE20/SPS1-related proline-alanine-rich protein kinase (SPAK−/−) manifest a salt-wasting nephropathy similar to Gitelman's syndrome but distinguishable from Bartter's syndrome in that the animals maintain furosemide sensitivity (Yang et al, JASN. Nov 2010). Consistent with these observations, NCC expression was dramatically reduced in SPAK−/−. NCC and SPAK are co-expressed in the aldosterone sensitive distal nephron where NaCl reabsorption is tightly regulated to facilitate proper vascular volume. We hypothesized that the physiological regulation of NCC by NaCl availability/vascular volume is lost in SPAK−/−. Wildtype mice (WT) and littermate SPAK−/− were fed a Na deficient, control, or high Na diet for 10 days. Analysis of urine samples revealed that SPAK−/− have an elevated rate of naturiuresis but maintain a normal kaluretic response. Western blot analysis showed that total NCC abundance was reduced in SPAK−/− compared to WT under all dietary conditions, and NCC phosphorylation (@T58) was nearly absent in SPAK−/−. These results were confirmed by immunohistological studies, which also showed NKCC2 phosphorylation and expression are equal to or greater in SPAK−/− than in WT. These results suggest NCC, not NKCC2, is the primary target of SPAK-dependent regulation, and that SPAK is necessary to maintain the physiological regulation of NCC during changes in dietary NaCl availability.

Angiotensin II: a candidate for an aldosterone-independent mediator of potassium preservation during volume depletion

Two different stimulators of aldosterone secretion, high-potassium diet and low-sodium diet, have disparate effects on potassium secretion in the distal nephron. The mechanism by which the kidney preserves potassium in the face of a high-aldosterone, volume-depleted state has engendered much thought. Yue et al. now propose that angiotensin II inhibits the renal outer medullary potassium channel (ROMK1) through stimulation of the protein tyrosine kinase c-Src, perhaps acting as a signal to differentiate volume depletion from a high-potassium diet.

SPAK and WNK kinases: a new target for blood pressure treatment?

The regulation of sodium reabsorption by the distal kidney is fundamental to blood pressure control. The clinical success of thiazide diuretics as antihypertensive drugs underscores the importance of its target, the thiazide-sensitive sodium/chloride cotransporter (NCC), in this process. However, thiazides are often ineffective as monotherapy and have significant side-effects. An understanding of NCC regulation at a molecular level may allow the design of better tolerated and more effective antihypertensive agents. The aim of this review is to provide an overview of the recent developments in the regulation of NCC, highlighting a potential new therapeutic target for the treatment of hypertension. It has been appreciated for several years that WNK kinases affect NCC expression, following the discovery that mutations in WNK genes cause Gordon syndrome (pseudohypoaldosteronism type II), although the precise molecular mechanisms were unclear. What has emerged from further in-vitro work is a WNK signalling cascade with the STE20 kinases SPAK and OSR1 as the 'missing' intermediate kinases that are activated by WNKs. Confirmation that this WNK-SPAK cascade operates in vivo comes from work on the phenotype of a kinase-dead SPAK knockin mouse. This mouse is markedly hypotensive, salt wasting, and almost all of its NCC protein in the distal kidney is dephosphorylated. Finally, a genome-wide association study has identified an intronic SPAK polymorphism that associates with human blood pressure. SPAK is a recently identified regulator of NCC and, hence, sodium reabsorption in the distal nephron. SPAK gene variants may also be important players in essential hypertension. If the phenotype of the kinase-dead SPAK mouse mimics pharmacological inhibition of this kinase, then SPAK is a potentially very interesting new antihypertensive drug target.

Downregulation of NCC and NKCC2 cotransporters by kidney-specific WNK1 revealed by gene disruption and transgenic mouse models

WNK1 (with-no-lysine[K]-1) is a protein kinase of which mutations cause a familial hypertension and hyperkalemia syndrome known as pseudohypoaldosteronism type 2 (PHA2). Kidney-specific (KS) WNK1 is an alternatively spliced form of WNK1 kinase missing most of the kinase domain. KS-WNK1 downregulates the Na(+)-Cl(-) cotransporter NCC by antagonizing the effect of full-length WNK1 when expressed in Xenopus oocytes. The physiological role of KS-WNK1 in the regulation of NCC and potentially other Na(+) transporters in vivo is unknown. Here, we report that mice overexpressing KS-WNK1 in the kidney exhibited renal Na(+) wasting, elevated plasma levels of angiotensin II and aldosterone yet lower blood pressure relative to wild-type littermates. Immunofluorescent staining revealed reduced surface expression of total and phosphorylated NCC and the Na(+)-K(+)-2Cl(-) cotransporter NKCC2 in the distal convoluted tubule and the thick ascending limb of Henle's loop, respectively. Conversely, mice with targeted deletion of exon 4A (the first exon for KS-WNK1) exhibited Na(+) retention, elevated blood pressure on a high-Na(+) diet and increased surface expression of total and phosphorylated NCC and NKCC2 in respective nephron segments. Thus, KS-WNK1 is a negative regulator of NCC and NKCC2 in vivo and plays an important role in the control of Na(+) homeostasis and blood pressure. These results have important implications to the pathogenesis of PHA2 with WNK1 mutations.

RasGRP1 stimulation enhances ubiquitination and endocytosis of the sodium-chloride cotransporter

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.

Role of SPAK binding sites in SLC12 cotransporters basal activity and regulation by WNK3

With‐no‐lysine kinase 3 (WNK3) is a powerful activator of NKCC1/2 and NCC while is an inhibitor of KCC1 ‐ KCC4. WNKs lie upstream of the STE‐20 kinases SPAK/OSR1. It is known that interaction with SPAK occurs through the SPAK‐binding motif, RFx(V/I). WNK3 contains three such motifs, NCC and NKCC2 one; while KCC4 lacks this motif. In the present study we examined the relevance of SPAK binding sites for NCC, NKCC2, and KCC4 basal activity and regulation by WNK3. The sites of WNK3 (F242A, F745A, or F1337A; one at a time), NCC (F19A), and NKCC2 (F17A) were eliminated. Basal activity and effect of WNK3 was assessed by measuring the tracer 22Na+ or 86Rb+uptake in Xenopus oocytes injected with wild type or mutant cRNAs. Basal activity of NCC‐F19A and NKCC2‐F17A was reduced by ~50%. However, the response to WNK3 and to intracellular chloride depletion was still present. The effect of WNK3 toward NKCC2, but not NCC or KCC4, was eliminated by WNK3‐F1337A. Interestingly, the WNK3 effect towards NCC or KCC4, but not NKCC2, was prevented in WNK3‐F242A. Biochemical analysis revealed that catalytic activity is preserved in both, WNK3‐F242A and WNK3‐F1337A. Thus, we conclude that basal activity of NCC and NKCC2, but not KCC4 requires the presence of an SPAK binding site on the cotransporter. Modulation of NCC, NKCC2, and KCC4 by WNK3 requires SPAK binding sites in the kinase, however, different sites are used for regulation of NKCC2 in contrast to NCC or KCC4.

Molecular physiology of the thiazide-sensitive sodium–chloride cotransporter

This review summarizes recent advances in the understanding of the molecular physiology and regulation of the thiazide-sensitive sodium-chloride cotransporter (NCC). Mutations of with-no-lysine (WNK) kinases 1 and 4 result in hyperactivity of NCC and familial hyperkalemic hypertension, a genetic syndrome of hypertension. Recent studies have shown that WNK1 and WNK4 activate the STE20 family protein kinases Ste20-related proline/alanine-rich kinase and odd-skipped-related 1, resulting in phosphorylation and activation of NCC. Additionally, a mouse knock-in model for a WNK4 familial hyperkalemic hypertension mutant demonstrated increased Ste20-related proline/alanine-rich kinase/odd-skipped-related 1 and NCC phosphorylation. It is unclear how these studies fit with the data indicating that WNK4 inhibits NCC, and the familial hyperkalemic hypertension mutations of WNK4 are loss-of-function mutations. Another WNK kinase, WNK3, also regulates NCC, activating NCC and antagonizing the effect of WNK4. Extracellular signal-related kinase 1/2 mitogen-activated protein kinase activation by Ras guanyl nucleotide-releasing protein 1 is another kinase pathway that appears to be a potent regulator of NCC. Other studies have described a role for angiotensin II in pressure natriuresis via actions on NCC. Recent studies examining the hormonal regulation of NCC have implicated angiotensin II and aldosterone in regulation of the WNK4-Ste20-related proline/alanine-rich kinase-NCC pathway. NCC is subject to a complex regulatory network of kinases, which appear quite sensitive to alterations of the hormonal and physiologic milieu.

Expression and phosphorylation of the Na+-Cl− cotransporter NCC in vivo is regulated by dietary salt, potassium, and SGK1

The Na-Cl cotransporter NCC is expressed in the distal convoluted tubule, activated by phosphorylation, and has been implicated in renal NaCl and K(+) homeostasis. The serum and glucocorticoid inducible kinase 1 (SGK1) contributes to renal NaCl retention and K(+) excretion, at least in part, by stimulating the epithelial Na(+) channel and Na(+)-K(+)-ATPase in the downstream segments of aldosterone-sensitive Na(+)/K(+) exchange. In this study we confirmed in wild-type mice (WT) that dietary NaCl restriction increases renal NCC expression and its phosphorylation at Thr(53), Thr(58), and Ser(71), respectively. This response, however, was attenuated in mice lacking SGK1 (Sgk1(-/-)), which may contribute to impaired NaCl retention in those mice. Total renal NCC expression and phosphorylation at Thr(53), Thr(58), and Ser(71) in WT were greater under low- compared with high-K(+) diet. This finding is consistent with a regulation of NCC to modulate Na(+) delivery to downstream segments of Na(+)/K(+) exchange, thereby modulating K(+) excretion. Dietary K(+)-dependent variation in renal expression of total NCC and phosphorylated NCC were not attenuated in Sgk1(-/-) mice. In fact, high-K(+) diet-induced NCC suppression was enhanced in Sgk1(-/-) mice. The hyperkalemia induced in Sgk1(-/-) mice by a high-K(+) diet may have augmented NCC suppression, thereby increasing Na(+) delivery and facilitating K(+) excretion in downstream segments of impaired Na(+)/K(+) exchange. In summary, changes in NaCl and K(+) intake altered NCC expression and phosphorylation, an observation consistent with a role of NCC in NaCl and K(+) homeostasis. The two maneuvers dissociated plasma aldosterone levels from NCC expression and phosphorylation, implicating additional regulators. Regulation of NCC expression and phosphorylation by dietary NaCl restriction appears to involve SGK1.

Phorbol Esters Stimulate Ubiquitination of NCC By Activating ERK1/2

The sodium chloride co‐transporter (NCC) is the principal salt‐absorptive pathway in the distal convoluted tubule (DCT). We have described a pathway of NCC regulation in which phorbol esters (PE) stimulate Ras guanyl‐releasing protein 1 (RasGRP1), activating ERK1/2 MAPK and decreasing NCC activity. We have also shown that PEs trigger the ubiquitination and internalization of NCC. To test whether these findings are related, we examined the ability of the PE TPA to increase NCC ubiquitination while inhibiting the RasGRP1/ERK1/2 pathway. mDCT cells were treated with 100nM TPA and the MEK1/2 inhibitor U0126 for 15 minutes. Lysis, immunoprecipitation for NCC, and immunoblotting for ubiquitin showed that U0126 prevented any PE‐induced increase in ubiquitination (no change compared to a 493±4% increase in ubiquitination with TPA alone). To examine RasGRP1's role, its expression was silenced using siRNA. These cells showed no increase in ubiquitination compared to RasGRP1 knockdown alone. These data indicate that TPA's ability to enhance NCC ubiquitination is dependent upon activation of RasGRP1 and ERK1/2.

PTH suppresses sodium chloride cotransporter activity by a Mapk‐dependent process

Recently, we described that Diacylglycerol (DAG) analogues act through Ras guanyl‐releasing protein1 (RasGRP1) stimulation of ERK1/2 MAPK to decrease function and surface expression of the thiazide‐sensitive sodium chloride cotransporter (NCC). However, upstream hormonal mediators of DAG‐dependent NCC suppression have not been identified. Although PTH activates DAG through G‐protein coupled receptor activation of Phospholipase C, PTH regulation of NCC has not been demonstrated. Utilizing a mouse DCT (mDCT) cell line with native NCC activity, we investigated whether PTH may mediate suppression of NCC function through MAPK activation. Thiazide‐sensitive 22Na+ uptakes were measured by incubating mDCT cells for 15 minutes in a Cl−free medium before exposure to a 22Na+containing medium with or without thiazide. Uptakes were determined after 15 minute incubations with vehicle, PTH (10‐7M) alone, PTH + U0126 (MEK 1/2 MAPK inhibitor), and PTH + bisindolylmaleimide (BIM, PKC inhibitor). PTH decreased NCC activity by 43.6 +/− 4.5% (p<0.05 for PTH vs. vehicle). U0126 blocked the PTH‐induced suppression while BIM had no effect on the suppression. This is consistent with our previous data that RasGRP1 and not PKC is the mediator of DAG effects on NCC activity. These studies indicate that PTH decreases NCC function via downstream activation of the MAPK pathway. This work was supported by NIH Grant K08 DK070668.