NCC Activity Research Articles

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.

Angiotensin II induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter independent of aldosterone

We studied here the independent roles of angiotensin II and aldosterone in regulating the sodium chloride cotransporter (NCC) of the distal convoluted tubule. We adrenalectomized three experimental and one control group of rats. Following surgery, the experimental groups were treated with either a high physiological dose of aldosterone, a non-pressor, or a pressor dose of angiotensin II for 8 days. Aldosterone and both doses of angiotensin II lowered sodium excretion and significantly increased the abundance of NCC in the plasma membrane compared with the control. Only the pressor dose of angiotensin II caused hypertension. Thiazides inhibited the sodium retention induced by the angiotensin II non-pressor dose. Both aldosterone and the non-pressor dose of angiotensin II significantly increased phosphorylation of NCC at threonine-53 and also increased the intracellular abundance of STE20/SPS1-related, proline alanine-rich kinase (SPAK). No differences were found in other modulators of NCC activity such as oxidative stress responsive protein type 1 or with-no-lysine kinase 4. Thus, our in vivo study shows that aldosterone and angiotensin II independently increase the abundance and phosphorylation of NCC in the setting of adrenalectomy; effects are likely mediated by SPAK. These results may explain, in part, the hormonal control of renal sodium excretion and the pathophysiology of several forms of hypertension.

The activity of the thiazide-sensitive Na+–Cl–cotransporter is regulated by protein phosphatase PP4

Cation transport in the distal mammalian nephron relies on the SLC12 family of membrane cotransporters that include the thiazide-sensitive Na(+)-Cl⁻ cotransporter (NCC). NCC is regulated through a scaffold of interacting proteins, including the WNK kinases, WNK 1 and WNK 4, which are mutated in the hypertensive Gordon's syndrome. Dynamic regulation of NCC function by kinases must involve dephosphorylation by phosphatases, as illustrated by the role of PP1 and PP2B in the regulation of KCC members of the SLC12 family. There are 2 phosphorylation-controlled regulatory pathways for NCC: type 1, mediated by WNK4 and affecting trafficking to the surface membrane, and type 2, affecting intrinsic transporter kinetics by phosphorylation of conserved N-terminal S/T amino acids. Using the Xenopus oocyte expression system, we show that PP4 inhibits NCC activity - but not trafficking to the surface membrane - by a mechanism that requires phosphatase activity and a conserved N-terminal amino acid of NCC, threonine 58. This action is distinct from WNK4 regulation of membrane trafficking. In the mouse kidney, PP4 is selectively expressed in the distal nephron, including cells of the distal convoluted tubule cells, suggesting that PP4 may have a physiological role in regulating NCC and hence NaCl reabsorption in vivo.

A single residue in transmembrane domain 11 defines the different affinity for thiazides between the mammalian and flounder NaCl transporters

Little is known about the residues that control the binding and affinity of thiazide-type diuretics for their protein target, the renal Na(+)-Cl(-) cotransporter (NCC). Previous studies from our group have shown that affinity for thiazides is higher in rat (rNCC) than in flounder (flNCC) and that the transmembrane region (TM) 8-12 contains the residues that produce this difference. Here, an alignment analysis of TM 8-12 revealed that there are only six nonconservative variations between flNCC and mammalian NCC. Two are located in TM9, three in TM11, and one in TM12. We used site-directed mutagenesis to generate rNCC containing flNCC residues, and thiazide affinity was assessed using Xenopus laevis oocytes. Wild-type or mutant NCC activity was measured using (22)Na(+) uptake in the presence of increasing concentrations of metolazone. Mutations in TM11 conferred rNCC an flNCC-like affinity, which was caused mostly by the substitution of a single residue, S575C. Supporting this observation, the substitution C576S conferred to flNCC an rNCC-like affinity. Interestingly, the S575C mutation also rendered rNCC more active. Substitution of S575 in rNCC for other residues, such as alanine, aspartate, and lysine, did not alter metolazone affinity, suggesting that reduced affinity in flNCC is due specifically to the presence of a cysteine. We conclude that the difference in metolazone affinity between rat and flounder NCC is caused mainly by a single residue and that this position in the protein is important for determining its functional properties.

Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for renal electrolyte balance and its phosphorylation causes an increase in its transport activity and cellular localization. Here, we generated phospho-specific antibodies against two conserved N-terminal phosphorylation sites (Thr53, Thr58 and Thr53/Thr58) to assess the role of arginine vasopressin (AVP) in regulating NCC in rodent kidney in vivo. Immunohistochemistry showed distinct staining of phosphorylated NCC (pNCC) at the apical plasma membrane domain of distal convoluted tubule (DCT) cells. Unlike total NCC, pNCC was localized only to the apical plasma membrane as determined by immunogold electron microscopy. In AVP-deficient Brattleboro rats, acute deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) exposure significantly increased pNCC abundance at the apical plasma membrane by about threefold, whereas total NCC and its cellular distribution were not affected. dDAVP significantly increased the abundance of phosphorylated STE20/SPS1-related proline-alanine-rich kinase and oxidative stress-response kinase (SPAK and OSR1), kinases implicated in NCC phosphorylation. Intracellular calcium levels in early and late DCTs were increased in response to 1 min superfusion of dDAVP, confirming that these segments are AVP responsive. In rats fed a high-salt diet with angiotensin (ANG) type 1-receptor blockade, similar increases in pNCC and active SPAK and OSR1 were detected following chronic or acute dDAVP, thus indicating the effects of AVP are independent of ANGII. Our results show that AVP is a potent regulator of NCC activity.

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.

Insulin increases the activity of NCC through a PI3K dependent mechanism

Obesity and diabetes mellitus are associated with the development of arterial hypertension. One potential mechanism that has been advanced to explain the obesity‐induced hypertension is the effect that insulin may have upon renal salt reabsorption. In this study we assessed the effect of insulin on the activity of the renal NaCl cotransporter, NCC, using the heterologous expression system of Xenopus laevis oocytes. Three days after injection with NCC cRNA the effect of insulin was assessed. NCC activity was measured as the thiazide‐sensitive tracer 22Na+ uptake. We observed that NCC activity is increased by 3‐fold when oocytes were exposed to insulin for 1 to 15 minutes before the uptake. The positive effect was dose and time dependent, with significant effect of insulin as low as with one unit and, as fast as after one minute of exposure. The addition of wortmannin, an inhibitor of phosphatydil‐inositol 3‐kinase (PI3K) completely abrogated the positive effect of insulin on the transporter's activity, without affecting the basal function. The effect of insulin was also abolished by pI103, a PI3K specific inhibitor, and by the specific AKT inhibitor IV. MAP or mTOR kinases inhibitors had no effect. Coinjection of NCC cRNA with WNK4, WNK3, or SPAK cRNA did not affect the activation of NCC by insulin. We conclude that insulin is a powerful activator of NCC and that insulin's effect is translated through the PI3K‐AKT pathway, suggesting that is due to threonine/serine phosphorylation. These observations may have implications to explain the association between obesity and diabetes mellitus with arterial hypertension.

Mechanisms of Aldosterone Action on NCC

Aldosterone increases NCC function and protein expression in whole animal studies at 72 hours but more acute effects of aldosterone on NCC activity have not been studied. We have recently demonstrated that in a mammalian cell model with robust native NCC activity (mDCT15), aldosterone stimulated NCC activity by 26% at 6 hrs and by 52% at 24 hrs. However, the mechanism by which this acute stimulation was mediated is unknown. To examine this question, mDCT15 cells were incubated with either 100nM aldosterone or vehicle (DMSO) for 6 or 24 hours. Cells were then surface biotinylated and surface expression was assessed by Western blotting. Total NCC protein expression remained unchanged compared to control in both the 6 and 24 hour treatment groups. Furthermore, NCC surface expression did not change in the 6 or 24 hour aldosterone treated groups compared to control. This suggests that the effects of aldosterone upon NCC are not primarily mediated by increased surface expression, but instead due to an increase in individual transporter activity. Further work is needed to characterize this mechanism.Research support: NIH‐NIDDK K08 DK070668, DK081728.

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.

Vasopressin‐mediated signaling in the distal convoluted tubule regulates phosphorylation of the thiazide‐sensitive Na+‐Cl− cotransporter (NCC) at two conserved threonine residues

The Na+‐Cl− cotransporter (NCC) is important for renal electrolyte balance. Phosphorylation of NCC has been proposed to play a major role in its transport activity and cellular localization. Using recently generated phospho‐specific antibodies against two conserved N‐terminal phosphorylation sites (Thr53, Thr58 and Thr53/Thr58) we assessed the role of vasopressin (AVP) in regulating NCC in vivo. In AVP‐deficient Brattleboro rats, acute dDAVP exposure for 15 min significantly increased pNCC abundance at the apical plasma membrane approximately 300%. Acute dDAVP did not alter the abundance of the STE20/SPS1‐related proline/alanine‐rich kinase (SPAK) previously implicated in NCC phosphorylation. Early and late DCTs increased their intracellular Ca2+ levels in response to 1 min of superfusion of dDAVP (2 × 10−10 M), confirming that the DCT is an AVP responsive segment. In rats fed a high salt diet alongside AT1‐R blockade by candesartan, similar increases in pNCC abundance were observed following chronic or acute dDAVP. Candesartan treatment reduced the abundances of total SPAK and pSPAK, suggesting that this kinase can be regulated via the AT1‐R in vivo. Taken together our results indicate that AVP is a potent regulator of NCC activity.Funding is provided by the Danish Medical Research Council, the Novo Nordisk Fond and the Carlsberg Foundation.

WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway

WNK ((with no lysine (k)) kinase is a subfamily of serine/threonine kinases. Mutations in two members of this kinase family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia and metabolic acidosis. WNK1 and WNK4 were shown to modulate NCC activity through SPAK and OSR1. Activation of PKC by a phorbol ester inhibits NCC function via activation of ERK1/2 kinase. To investigate whether WNK4 affects NCC via MAPK signaling pathway besides SPAK and OSR1, we first determine whether WNK4 phosphorylates ERK1/2 kinase. We found that WNK4 increases the phosphorylation of ERK1/2 in a dose‐dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) do not increase the ERK1/2 phosphorylation. We next showed that hypertonic stimulation significantly increased ERK1/2 phosphorylation in mDCT cells. The ERK1/2 activation by hypertonic stimulation was significantly attenuated by WNK4 siRNA knock‐down expression (23.19 ± 8.21 % in siWNK4 group vs 100 % in control group, p <0.01, n=3). We further showed that WNK4 knock‐down significantly increases total NCC expression (100 ± 0 % vs 120.7 ± 6.2 %, p <0.05, n=4) as well as NCC surface expression (100 ± 0 % vs 256.8 ± 50.3 %, p <0.05, n=4). We conclude that the inhibitory effect of WNK4 on NCC is mediated by MAPK ERK 1/2 signaling pathway.Note: BZ and YZ contributed equally to this work.

Short-term stimulation of the thiazide-sensitive Na+-Cl−cotransporter by vasopressin involves phosphorylation and membrane translocation

Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na+-Cl- cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11beta-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V(2) receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT.

Abstract 5076: Differential Response of Renal Tubule Sodium Transporters in Salt Sensitive and Salt Resistant Mouse Strains to Salt Load

We have reported that C57BL6 and SJL mice from Jackson laboratory have different responses to a chronic salt load: blood pressure increases in C57BL6 but not in SJL mice. We have suggested that the increase in blood pressure with NaCl loading in C57BL6 mice is due to a dysfunction of renal dopamine receptors caused by increased expression and activity of G protein-coupled receptor kinase 4. In order to determine if the salt sensitivity in C57BL6 mice can be related to increased expression of renal sodium transporters, we studied the effects of chronic NaCl loading on renal sodium transporters in these two mouse strains. C57BL6 and SJL mice (10 weeks old, male) were fed high NaCl (6%) or normal NaCl (0.8%NaCl) (n =5/group) diet. The blood pressures (measured by telemetry) were similar at baseline but increased in C57BL6 not in SJL mice after 1 and 3 weeks of salt loading. The urinary sodium excretions were also similar at baseline (2.34±0.27 vs 2.3±0.18,mEq/mg Cr, respectively) and increased to a lesser extent in C57BL6 than in SJL mice (1 wk: 4.39±0.9 vs 7.96±0.59, P<0.05; 3 wks: 3.5±0.4 vs 5.0±0.9, P =0.16, respectively). On normal NaCl diet, the renal protein expression (immunoblotting) of sodium hydrogen exchanger 3 (NHE3, 287±33, % of SJL) sodium chloride cotransporter (NCC, 167±24, %) and β -epithelial sodium channel (ENaC,169±9, % ) was greater in C57BL6 than in SJL mice. Urinary aldosterone excretion and renal α -ENaC expression (an aldosterone-induced protein) were decreased by the salt load to a similar extent in the two mouse strains. In contrast, one and three weeks of salt loading decreased renal NCC, another aldosterone-induced protein, in SJL but not in C57BL6 mice. There was a difference in the molecular size of the NCC upper band (SJL = 160 kDa; C57BL6 = 175 kDa) while the 110 kDa band was similar in the two strains. The upper band is glycosylated NCC (proved by N-glycosidase F studies) which may be the active transporter; it remains to be determined if glycosylation affects NCC activity in mice. We conclude that the increased renal NCC expression, which is independent of aldosterone levels, may be responsible for the decreased sodium excretion in C57B6 mice. Our studies provide a renal molecular mechanism to the salt sensitivity in C57BL6 mice.

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.

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.

Angiotensin II signaling increases activity of the renal Na-Cl cotransporter through a WNK4-SPAK-dependent pathway

Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum K(+) levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and K(+) secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the K(+) channel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (AngII) levels, AngII signaling might modulate WNK4 activity. We show that AngII signaling in Xenopus oocytes increases NCC activity by abrogating WNK4's inhibition of NCC but does not alter WNK4's inhibition of ROMK. This effect requires AngII, its receptor AT1R, and WNK4, and is prevented by the AT1R inhibitor losartan. NCC activity is also increased by WNK4 harboring mutations found in PHAII, and this activity cannot be further augmented by AngII signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. AngII's effect on NCC is also dependent on the kinase SPAK because dominant-negative SPAK or elimination of the SPAK binding motif in NCC prevent activation of NCC by AngII signaling. These effects extend to mammalian cells. AngII increases phosphorylation of specific sites on SPAK and NCC that are necessary for activation of each in mpkDCT cells. These findings place WNK4 in the signaling pathway between AngII and NCC, and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing K(+) secretion.

The regulation of salt transport and blood pressure by the WNK-SPAK/OSR1 signalling pathway

It has recently been shown that the WNK [with-no-K(Lys)] kinases (WNK1, WNK2, WNK3 and WNK4) have vital roles in the control of salt homeostasis and blood pressure. This Commentary focuses on recent findings that have uncovered the backbone of a novel signal-transduction network that is controlled by WNK kinases. Under hyperosmotic or hypotonic low-Cl- conditions, WNK isoforms are activated, and subsequently phosphorylate and activate the related protein kinases SPAK and OSR1. SPAK and OSR1 phosphorylate and activate ion co-transporters that include NCC, NKCC1 and NKCC2, which are targets for the commonly used blood-pressure-lowering thiazide-diuretic and loop-diuretic drugs. The finding that mutations in WNK1, WNK4, NCC and NKCC2 cause inherited blood-pressure syndromes in humans highlights the importance of these enzymes. We argue that these new findings indicate that SPAK and OSR1 are promising drug targets for the treatment of hypertension, because inhibiting these enzymes would reduce NCC and NKCC2 activity and thereby suppress renal salt re-absorption. We also discuss unresolved and controversial questions in this field of research.

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.

Cotransporters, WNKs and hypertension: an update

Studies of inherited conditions characterized by high or low blood pressure reveal the importance of a new signalling cascade, With no Lysine kinases (WNK) --> ste20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase-1 (OSR1) --> Cation-Chloride Cotransporters (CCC), in regulating blood pressure and in the pathogenesis of essential hypertension. This review explores how these molecules interact to co-ordinate sodium homeostasis and how errors in these interactions may result in hypertension. Studies using transgenic animals and gene knockins have clarified the role of mutant WNK4 in hypertension, by revealing its main action to be increasing the expression and activity of sodium-chloride cotransporter (NCC) in the kidney. Functional studies show how phosphorylation of WNK1 regulates both its activity and ability to interact with SPAK/OSR1, and clearly place it upstream of SPAK/OSR1 in the cascade. The structural basis for the interactions between SPAK/OSR1 and targets has been identified. WNKs, activated by upstream kinases or autophosphorylation, bind and phosphorylate SPAK/OSR1, which in turn phosphorylate and activate NCCs and Na-K-Cl cotransporters (NKCCs). This increases sodium retention in the kidney (NKCC2, NCC) and vascular resistance (NKCC1), but decreases renin release (NKCC1). Hypertension-associated mutant WNKs increase surface expression and activation of renal tubular NKCC2 and NCC. Whether this adequately explains the hypertension awaits studies of these mutants in other tissues.

Activation of the thiazide-sensitive Na+-Cl– cotransporter by the WNK-regulated kinases SPAK and OSR1

Mutations increasing WNK1 kinase expression in humans cause the pseudohypoaldosteronism type II hypertension syndrome. This condition is treated effectively by thiazide diuretics, which exert their effects by inhibiting the Na+-Cl(-) cotransporter (NCC), suggesting a link between WNK1 and NCC. Here, we demonstrate that the SPAK and OSR1 kinases that are activated by WNK1 phosphorylate human NCC at three conserved residues (Thr46, Thr55 and Thr60). Activation of the WNK1-SPAK/OSR1 signalling pathway by treatment of HEK293 or mpkDCT kidney distal-convoluted-tubule-derived cells with hypotonic low-chloride conditions induced phosphorylation of NCC at residues phosphorylated by SPAK/OSR1. Efficient phosphorylation of NCC was dependent upon a docking interaction between an RFXI motif in NCC and SPAK/OSR1. Mutation of Thr60 to Ala in NCC markedly inhibited phosphorylation of Thr46 and Thr55 as well as NCC activation induced by hypotonic low-chloride treatment of HEK293 cells. Our results establish that the WNK1-SPAK/OSR1 signalling pathway plays a key role in controlling the phosphorylation and activity of NCC. They also suggest a mechanism by which increased WNK1 overexpression could lead to hypertension and that inhibitors of SPAK/OSR1 might be of use in reducing blood pressure by suppressing phosphorylation and hence activity of NCC.