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.     

WNK4 regulates apical and basolateral Cl- flux in extrarenal epithelia

Mutations in the serine-threonine kinase WNK4 [with no lysine (K) 4] cause pseudohypoaldosteronism type II, a Mendelian disease featuring hypertension with hyperkalemia. In the kidney, WNK4 regulates the balance between NaCl reabsorption and K(+) secretion via variable inhibition of the thiazide-sensistive NaCl cotransporter and the K(+) channel ROMK. We now demonstrate expression of WNK4 mRNA and protein outside the kidney. In extrarenal tissues, WNK4 is found almost exclusively in polarized epithelia, variably associating with tight junctions, lateral membranes, and cytoplasm. Epithelia expressing WNK4 include sweat ducts, colonic crypts, pancreatic ducts, bile ducts, and epididymis. WNK4 is also expressed in the specialized endothelium of the blood-brain barrier. These epithelia and endothelium all play important roles in Cl(-) transport. Because WNK4 is known to regulate renal Cl(-) handling, we tested WNK4's effect on the activity of mediators of epithelial Cl(-) flux whose extrarenal expression overlaps with WNK4. WNK4 proved to be a potent inhibitor of the activity of both the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and the Cl(-)/base exchanger SLC26A6 (CFEX) (>95% inhibition of NKCC1-mediated (86)Rb influx, P < 0.001; >80% inhibition of CFEX-mediated [(14)C] formate uptake, P < 0.001), mediators of Cl(-) flux across basolateral and apical membranes, respectively. In contrast, WNK4 showed no inhibition of pendrin, a related Cl(-)/base exchanger. These findings indicate a general role for WNK4 in the regulation of electrolyte flux in diverse epithelia. Moreover, they reveal that WNK4 regulates the activities of a diverse group of structurally unrelated ion channels, cotransporters, and exchangers.     

Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases

The Na(+):K(+):2Cl(-) cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus laevis oocytes, achieved by either coexpression of the K-Cl cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [Cl(-)](i). The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloride-sensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption.     

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.     

WNK3 modulates transport of Cl- in and out of cells: implications for control of cell volume and neuronal excitability

The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion cotransporters: Cl(-) influx is mediated by the Na-K-2Cl cotransporter NKCC1 and Cl(-) efflux via K-Cl cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.     

Function and expression of renal epithelial sodium transporters in rats with thyroid dysfunction

Thyroid disorders are accompanied by major changes in renal sodium handling and blood pressure. Sodium transporters play a crucial role in regulating sodium excretion. We determined the function and expression of type 3 Na/H (NHE3) exchanger, type 2 Na+K+2Cl co-transporter (NKCC2) co-transporter, NaCl co-transporter (NCC) cotransporter, and epithelial sodium channel (ENaC) in hypoand hyperthyroid rats at 6 weeks after each thyroid disorder induction. We measured the renal response to functional blockade of the tubular sodium transporters, using acetazolamide to inhibit the activity of NHE3, furosemide for NKCC2, hydrochlorotiazide for NCC, and amiloride for ENaC. Expression of sodium transporters was analyzed by measuring the protein abundance by Western blot. The responsiveness to NHE3 inhibition and NHE3 protein was lower in hypothyroid rats and higher in hyperthyroid rats vs controls. Hypothyroid rats showed greater diuretic and natriuretic responses to NKCC2 and ENaC blockade and higher protein abundance of NKCC2 vs controls. Hyperthyroid rats showed greater protein expression of NKCC2 and NCC vs controls. Groups did not differ in responsiveness to NCC blockade. The expression and activity of ENaC were lower in hyperthyroid rats. In conclusion, reduced NHE3 activity may participate in the low blood pressure of hypothyroid rats and elevated NHE3 activity in the high blood pressure of hyperthyroid rats. These proximal alterations are counter-balanced by functional upregulation of NKCC2 and ENaC in downstream nephron segments of hypothyroid rats and by downregulation of αENaC activity and expression in hyperthyroid rats.     

Decreased ENaC expression compensates the increased NCC activity following inactivation of the kidney-specific isoform of WNK1 and prevents hypertension

Mutations in WNK1 and WNK4 lead to familial hyperkalemic hypertension (FHHt). Because FHHt associates net positive Na(+) balance together with K(+) and H(+) renal retention, the identification of WNK1 and WNK4 led to a new paradigm to explain how aldosterone can promote either Na(+) reabsorption or K(+) secretion in a hypovolemic or hyperkalemic state, respectively. WNK1 gives rise to L-WNK1, an ubiquitous kinase, and KS-WNK1, a kinase-defective isoform expressed in the distal convoluted tubule. By inactivating KS-WNK1 in mice, we show here that this isoform is an important regulator of sodium transport. KS-WNK1(-/-) mice display an increased activity of the Na-Cl cotransporter NCC, expressed specifically in the distal convoluted tubule, where it participates in the fine tuning of sodium reabsorption. Moreover, the expression of the ROMK and BKCa potassium channels was modified in KS-WNK1(-/-) mice, indicating that KS-WNK1 is also a regulator of potassium transport in the distal nephron. Finally, we provide an alternative model for FHHt. Previous studies suggested that the activation of NCC plays a central role in the development of hypertension and hyperkalemia. Even though the increase in NCC activity in KS-WNK1(-/-) mice was less pronounced than in mice overexpressing a mutant form of WNK4, our study suggests that the activation of Na-Cl cotransporter is not sufficient by itself to induce a hyperkalemic hypertension and that the deregulation of other channels, such as the Epithelial Na(+) channel (ENaC), is probably required.     

Impaired phosphorylation of Na(+)-K(+)-2Cl(-) cotransporter by oxidative stress-responsive kinase-1 deficiency manifests hypotension and Bartter-like syndrome

Na(+)-K(+)-2Cl(-) cotransporters (NKCCs), including NKCC1 and renal-specific NKCC2, and the Na(+)-Cl(-) cotransporter (NCC) play pivotal roles in the regulation of blood pressure (BP) and renal NaCl reabsorption. Oxidative stress-responsive kinase-1 (OSR1) is a known upstream regulator of N(K)CCs. We generated and analyzed global and kidney tubule-specific (KSP) OSR1 KO mice to elucidate the physiological role of OSR1 in vivo, particularly on BP and kidney function. Although global OSR1(-/-) mice were embryonically lethal, OSR1(+/-) mice had low BP associated with reduced phosphorylated (p) STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase (SPAK) and p-NKCC1 abundance in aortic tissue and attenuated p-NKCC2 abundance with increased total and p-NCC expression in the kidney. KSP-OSR1(-/-) mice had normal BP and hypercalciuria and maintained significant hypokalemia on a low-K(+) diet. KSP-OSR1(-/-) mice exhibited impaired Na(+) reabsorption in the thick ascending loop on a low-Na(+) diet accompanied by remarkably decreased expression of p-NKCC2 and a blunted response to furosemide, an NKCC2 inhibitor. The expression of total SPAK and p-SPAK was significantly increased in parallel to that of total NCC and p-NCC despite unchanged total NKCC2 expression. These results suggest that, globally, OSR1 is involved in the regulation of BP and renal tubular Na(+) reabsorption mainly via the activation of NKCC1 and NKCC2. In the kidneys, NKCC2 but not NCC is the main target of OSR1 and the reduced p-NKCC2 in KSP-OSR1(-/-) mice may lead to a Bartter-like syndrome.     

An SGK1 site in WNK4 regulates Na+ channel and K+ channel activity and has implications for aldosterone signaling and K+ homeostasis

The steroid hormone aldosterone is secreted both in the setting of intravascular volume depletion and hyperkalemia, raising the question of how the kidney maximizes NaCl reabsorption in the former state while maximizing K(+) secretion in the latter. Mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring increased renal NaCl reabsorption and impaired K(+) secretion. PHAII-mutant WNK4 achieves these effects by increasing activity of the Na-Cl cotransporter (NCC) and the Na(+) channel ENaC while concurrently inhibiting the renal outer medullary K(+) channel (ROMK). We now describe a functional state for WNK4 that promotes increased, rather than decreased, K(+) secretion. We show that WNK4 is phosphorylated by SGK1, a mediator of aldosterone signaling. Whereas wild-type WNK4 inhibits the activity of both ENaC and ROMK, a WNK4 mutation that mimics phosphorylation at the SGK1 site (WNK4(S1169D)) alleviates inhibition of both channels. The net result of these effects in the kidney would be increased K(+) secretion, because of both increased electrogenic Na(+) reabsorption and increased apical membrane K(+) permeability. Thus, modification at the PHAII and SGK1 sites in WNK4 impart opposite effects on K(+) secretion, decreasing or increasing ROMK activity and net K(+) secretion, respectively. This functional state for WNK4 would thus promote the desired physiologic response to hyperkalemia, and the fact that it is induced downstream of aldosterone signaling implicates WNK4 in the physiologic response to aldosterone with hyperkalemia. Together, the different states of WNK4 allow the kidney to provide distinct and appropriate integrated responses to intravascular volume depletion and hyperkalemia.     

Phosphatidylinositol 3-Kinase/Akt Signaling Pathway Activates the WNK-OSR1/SPAK-NCC Phosphorylation Cascade in Hyperinsulinemic db/db Mice

Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently identified with-no-lysine kinase (WNK)-oxidative stress-responsive kinase 1 (OSR1)/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter (NCC) phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both Spak(T243A/+) and Osr1(T185A/+) knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the phosphatidylinositol 3-kinase/Akt signaling cascade in the kidney in response to hyperinsulinemia. A phosphatidylinositol 3-kinase inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific phosphatidylinositol 3-kinase inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions, such as the metabolic syndrome.     

WNK1 and OSR1 regulate the Na+, K+, 2Cl- cotransporter in HeLa cells

Oxidative stress-responsive kinase (OSR) 1 and sterile20-related, proline-, alanine-rich kinase (SPAK) are Ste20p-related protein kinases that bind to the sodium, potassium, two chloride cotransporter, NKCC. Here we present evidence that the protein kinase with no lysine [K] (WNK) 1 regulates OSR1, SPAK, and NKCC activities. OSR1 exists in a complex with WNK1 in cells, is activated by recombinant WNK1 in vitro, and is phosphorylated in a WNK1-dependent manner in cells. Depletion of WNK1 from HeLa cells by using small interfering RNA reduces OSR1 kinase activity. In addition, depletion of either WNK1 or OSR1 reduces NKCC activity, indicating that WNK1 and OSR1 are both required for NKCC function. OSR1 and SPAK are likely links between WNK1 and NKCC in a pathway that contributes to volume regulation and blood pressure homeostasis in mammals.     

Serine-threonine kinase with-no-lysine 4 (WNK4) controls blood pressure via transient receptor potential canonical 3 (TRPC3) in the vasculature

Mutations in the serine-threonine kinase with-no-lysine 4 (WNK4) cause pseudohypoaldosteronism type 2 (PHAII), a Mendelian form of human hypertension. WNK4 regulates diverse ion transporters in the kidney, and dysregulation of renal transporters is considered the main cause of the WNK4 mutation-associated hypertension. Another determinant of hypertension is vascular tone that is regulated by Ca(2+)-dependent blood vessel constriction. However, the role of WNK4 in vasoconstriction as part of its function to regulate blood pressure is not known. Here, we report that WNK4 is a unique modulator of blood pressure by restricting Ca(2+) influx via the transient receptor potential canonical 3 (TRPC3) channel in the vasculature. Loss of WNK4 markedly augmented TRPC3-mediated Ca(2+) influx in vascular smooth muscle cells (VSMCs) in response to α-adrenoreceptor stimulation, which is the pathological hallmark of hypertension in resistance arteries. Notably, WNK4 depletion induced hypertrophic cell growth in VSMCs and increased vasoconstriction in small mesenteric arteries via TRPC3-mediated Ca(2+) influx. In addition, WNK4 mutants harboring the Q562E PHAII-causing or the D318A kinase-inactive mutation failed to mediate TRPC3 inhibition. These results define a previously undescribed function of WNK4 and reveal a unique therapeutic target to control blood pressure in WNK4-related hypertension.     

Paracellular Cl- permeability is regulated by WNK4 kinase: insight into normal physiology and hypertension

Paracellular ion flux across epithelia occurs through selective and regulated pores in tight junctions; this process is poorly understood. Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension and hyperkalemia. Whereas WNK4 is known to regulate several transcellular transporters and channels involved in NaCl and K+ homeostasis, its localization to tight junctions suggests it might also regulate paracellular flux. We performed electrophysiology on mammalian kidney epithelia with inducible expression of various WNK4 constructs. Induction of wild-type WNK4 reduced transepithelial resistance by increasing absolute chloride permeability. PHAII-mutant WNK4 produced markedly larger effects, whereas kinase-mutant WNK4 had no effect. The electrochemical and pharmacologic properties of these effects indicate they are attributable to the paracellular pathway. The effects of WNK4 persist when induction is delayed until after tight-junction formation, demonstrating a dynamic effect. WNK4 did not alter the flux of uncharged solutes, or the expression or localization of selected tight-junction proteins. Transmission and freeze-fracture electron microscopy showed no effect of WNK4 on tight-junction structure. These findings implicate WNK signaling in the coordination of transcellular and paracellular flux to achieve NaCl and K+ homeostasis, explain PHAII pathophysiology, and suggest that modifiers of WNK signaling may be potent antihypertensive agents.     

Metformin increases urinary sodium excretion by reducing phosphorylation of the sodium-chloride cotransporter

ObjectiveMetformin is an antidiabetic drug that is widely used to treat patients with diabetes mellitus. Recent studies have reported that treatment with metformin not only improved blood glucose levels but also reduced blood pressure. However, it remains unclear how metformin reduces blood pressure. We hypothesized that metformin affects sodium reabsorption in the kidneys.MethodsUrinary sodium excretion and expression of renal sodium transporters were examined in 8-week-old male C57BL/6 mice with acute and chronic treatment of metformin. In addition, we examined metformin effects using ex vivo preparations of mice kidney slices.ResultsIn this study, we demonstrated that metformin increased urinary sodium excretion by reducing phosphorylation of the thiazide-sensitive Na-Cl cotransporter (NCC) in acute and chronic metformin administration. We also confirmed reduction of phosphorylated NCC in an ex vivo study. The activity of other renal sodium transporters, such as NKCC2, ENaC, and NHE3 did not show significant changes. WNK-OSR1/SPAK kinase signals were not involved in this inactivation effect of metformin on NCC.ConclusionMetformin increased urinary sodium excretion by reducing phosphorylation of NCC, suggesting its role in improving hypertension.     

WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway

SLC12A cation/Cl- cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1-KCC4) mediate cellular Cl- efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular Cl- influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in hereditary hypertension, is coexpressed with several cation/Cl- cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1-KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage-induced inhibition of KCC1-KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the Cl- influx and efflux branches of the SLC12A cotransporter family.     

Regulation of diverse ion transport pathways by WNK4 kinase: a novel molecular switch.

Key components of complex physiological regulatory pathways can be uncovered through the molecular-genetic study of rare, inherited diseases. WNK kinases are a recently discovered class of serine-threonine kinases that are distinctive because of the substitution of cysteine for lysine in subdomain II of the catalytic domain. Mutations in PRKWNK1 and PRKWNK4, which encode WNK1 and WNK4, result in an inherited syndrome of hypertension and hyperkalemia. Recent physiological work has revealed that WNK4 alters the balance of NaCl reabsorption and K(+) secretion in the distal nephron by actions on both transcellular and paracellular ion-flux pathways. Additionally, WNK4 is expressed in extra-renal epithelia with prominent roles in Cl(-) handling, and it regulates transporters that are responsible for Cl(-) flux across apical and basolateral membranes. WNK kinases are components of a novel signaling pathway that is important for the control of blood pressure and electrolyte homeostasis.     

Renal Na-K-Cl cotransporter NKCC2 in Dahl salt-sensitive rats

BACKGROUND: Dahl salt-sensitive (DS) rats are characterized by enhanced NaCl reabsorption in the loop of Henle, but the responsible ion transport protein is unknown. OBJECTIVE: To investigate renal Na-K-Cl cotransporter NKCC2 function and expression in DS rats under a low-salt diet. METHODS : NKCC2 functioning was assessed in vitro by measuring bumetanide-sensitive rubidium uptake and cytosolic chloride concentration in isolated medullary thick ascending limb (mTAL) tubules, and in vivo by measuring the salidiuretic action of orally given bumetanide. NKCC2 expression was assessed by Western blot analysis of outer medullary proteins using T4 monoclonal antibody. RESULTS: mTAL tubules from DS rats exhibited significantly higher bumetanide-sensitive rubidium uptake (85.1 +/- 4.8 versus 66.2 +/- 4.4 nmol/min per mg protein in DS and DR, (Dahl salt-resistant) rats, respectively; P = 0.011) and significantly higher cytosolic chloride (32.8 +/- 1.7 versus 25.0 +/- 1.5 mmol/l in DS and DR rats, respectively). Moreover, DS rats showed a significantly higher (P < 0.001) natriuretic response to bumetanide (1.13 +/- 0.05 versus 0.64 +/- 0.09 mmole/3 h in DS and DR rats, respectively). Finally, Western blot analysis revealed less NKCC2 expression in DS rats. CONCLUSIONS: We conclude that DS rats have increased renal NKCC2 activity, thus explaining, at least in part, their genetic renal inability to excrete sodium. Moreover, DS rats have a decreased renal NKCC2 expression, which can be a compensatory phenomenon against NKCC2 hyperactivity.     

Regulation of Renal Na-K-Cl Cotransporter NKCC2 by Humoral Natriuretic Factors: Relevance in Hypertension

A furosemide-sensitive Na-K-Cl cotransporter (NKCC2 isoform) accounts for almost all luminal NaCl reabsorption in the thick ascending limb of Henle's loop (TALH). The activity of this transport protein is regulated by humoral factors (CIF: cotransport inhibitory factors). One family of CIF compounds is represented by the urinary phytoestrogens equol and genistein, which inhibit cotransport fluxes at similar concentrations as furosemide. Moreover, they possess similar salidiuretic potency as furosemide in the isolated perfused rat kidney, but are less potent than furosemide in vivo.Thus, dietary phytoestrogens can be responsible, at least in part, for the low blood pressure of vegetarians. A second type of CIF is represented by a circulating and urinary factor which is evoked by salt-loading. This, which is not a “ouabain-like” factor, appears to be a new retropituitary natriuretic compound. Endogenous CIF is increased in hypertensive Dahl salt-sensitive rats, probably as a compensatory mechanism against the enhanced NaCl reabsorption in the TALH, which characterizes this model of hypertension. Finally, chronic excess of circulating CIF inhibits and induces up-regulation of erythrocyte Na-K-Cl cotransporter NKCC1     

A novel protein kinase signaling pathway essential for blood pressure regulation in humans.

The discovery that mutations in WNK4 [encoding a member of the WNK family - so named because of the unique substitution of cysteine for lysine at a nearly invariant residue within subdomain II of its catalytic core: with no K (lysine)] cause pseudohypoaldosteronism type II, an autosomal dominant form of human hypertension, provided the initial clue that this serine/threonine kinase is a crucial part of a complex renal salt regulatory system. Recent findings from physiological studies of WNK4 in Xenopus laevis oocytes, mammalian cell systems and in vivo in mouse models have provided novel insights into the mechanisms by which the kidney regulates salt homeostasis, and therefore blood pressure, downstream of aldosterone signaling in mammals. The current evidence supports a model in which WNK4 coordinates the activities of diverse aldosterone-sensitive mediators of ion transport in the distal nephron to promote normal homeostasis in response to physiological perturbation.