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.     

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.     

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation

Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K⁺ reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K⁺ secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)—components of an E3 ubiquitin ligase complex that targets WNKs for degradation—cause constitutively increased renal salt reabsorption and impaired K⁺ secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3^S433 phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K⁺ secretion in the setting of volume depletion.Hypertension affects 1 billion people worldwide and is a major risk factor for death from stroke, myocardial infarction, and congestive heart failure. The study of Mendelian forms of hypertension has demonstrated the key role of increased renal salt reabsorption in disease pathogenesis (1–4). Observational and intervention trials (5, 6) also indicate that increased dietary K⁺ lowers blood pressure; however, the mechanism of this effect has been unclear.Pseudohypoaldosteronism type II (PHAII; Online Mendelian Inheritance in Man no. 145260), featuring hypertension and hyperkalemia, has revealed a previously unrecognized mechanism that regulates the balance between renal salt reabsorption and K⁺ secretion in response to aldosterone (7). Aldosterone is produced by the adrenal glomerulosa in volume depletion, in response to angiotensin II (AII), and in hyperkalemia via membrane depolarization (8). In volume depletion, aldosterone maximizes renal salt reabsorption, whereas in hyperkalemia, aldosterone promotes maximal renal K⁺ secretion. Volume depletion increases both the NaCl cotransporter (NCC) (9) and electrogenic Na⁺ reabsorption via the epithelial Na⁺ channel (ENaC) (10). The lumen-negative potential produced by ENaC activity provides the electrical driving force for paracellular Cl⁻ reabsorption (11). In hyperkalemia, the lumen-negative potential promotes K⁺ secretion via the K⁺ channel Kir1.1 (renal outer medullary K⁺ channel ROMK), reducing plasma K⁺ level (12, 13). Additionally, recent studies have implicated aldosterone signaling in intercalated cell transcellular Cl⁻ flux (14). In these cells, hyperkalemia induces phosphorylation of the mineralocorticoid receptor (MR) ligand-binding domain, making it incapable of ligand binding and activation. AII signaling induces dephosphorylation, and activation of the MR by aldosterone then induces transcellular Cl⁻ flux, which is required for defense of intravascular volume (14, 15). Because electrogenic Cl⁻ reabsorption and K⁺ secretion both dissipate the lumen-negative potential produced by ENaC, maximal Cl⁻ reabsorption inhibits K⁺ secretion and vice versa.Patients with PHAII have constitutive reabsorption of NaCl with concomitant inhibition of K⁺ secretion, resulting in hypertension and hyperkalemia, despite normal levels of aldosterone (7). Dominant mutations in the serine–threonine kinases with-no-lysine 4 (WNK4) or WNK1, or in CUL3 or KLHL3, elements of a ubiquitin ligase complex, cause this disease (2, 4). WNK4 modulates the activities of NCC, ENaC, Kir1.1, and MR (14, 16–21), and WNK4 function can be modulated by phosphorylation (21). CUL3/KLHL3 has been shown to target WNK4 and WNK1 for ubiquitination and degradation, and disease-causing mutations impair this binding and degradation (22–24). In particular, dominant mutations in the Kelch domain of KLHL3 prevent binding to WNKs; reciprocally, disease-causing point mutations in WNK4 also prevent WNK4–KLHL3 binding.These findings suggest that regulation of WNK degradation by CUL3/KLHL3 is highly regulated and that disease-causing mutations might phenocopy a state in which WNKs are normally turned off, producing constitutive salt reabsorption and inhibited K⁺ secretion. We now demonstrate that this inference is correct and implicate AII signaling in this process.     

WNK1-related Familial Hyperkalemic Hypertension results from an increased expression of L-WNK1 specifically in the distal nephron

Large deletions in the first intron of the With No lysine (K) 1 (WNK1) gene are responsible for Familial Hyperkalemic Hypertension (FHHt), a rare form of human hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis. We generated a mouse model of WNK1-associated FHHt to explore the consequences of this intronic deletion. WNK1^+/FHHt mice display all clinical and biological signs of FHHt. This phenotype results from increased expression of long WNK1 (L-WNK1), the ubiquitous kinase isoform of WNK1, in the distal convoluted tubule, which in turn, stimulates the activity of the Na–Cl cotransporter. We also show that the activity of the epithelial sodium channel is not altered in FHHt mice, suggesting that other mechanisms are responsible for the hyperkalemia and acidosis in this model. Finally, we observe a decreased expression of the renal outer medullary potassium channel in the late distal convoluted tubule of WNK1^+/FHHt mice, which could contribute to the hyperkalemia. In summary, our study provides insights into the in vivo mechanisms underlying the pathogenesis of WNK1-mediated FHHt and further corroborates the importance of WNK1 in ion homeostasis and blood pressure.Familial Hyperkalemic Hypertension (FHHt) is a rare disorder featuring hypertension, hyperkalemia, and hyperchloremic metabolic acidosis (Online Mendelian Inheritance in Man, OMIM, 145260) (1, 2). Twelve years ago, mutations in the With No lysine (K) 1 (WNK1) and WNK4 genes were shown to cause FHHt (3), initiating a field of extensive research on the regulation of blood pressure and ion homeostasis by these two serine-threonine kinases of the WNK family (review in ref. 4). Many questions, however, regarding their physiological roles and the mechanisms of WNK1-related FHHt still remain.The human mutations identified at the WNK1 locus do not modify the coding sequence but are large deletions in the 60-kb-long first intron, which result in an overexpression of WNK1 in the leukocytes of patients (3). The WNK1 gene generates two isoforms through alternative promoters. The long isoform, long WNK1 (L-WNK1), is expressed ubiquitously, whereas the shorter isoform, kidney-specific WNK1 (KS-WNK1), which lacks a functional kinase domain, is expressed specifically in the kidney (5). In the kidney, L-WNK1 is expressed at a low level in all nephron segments, whereas KS-WNK1 is expressed only in the distal nephron (6). We previously generated a transgenic mouse model that exhibited an ectopic expression of KS-WNK1 and an increased expression of L-WNK1 in the distal nephron on deletion of the first intron (7). This model, however, did not allow the study of the functional consequences of the deletion of WNK1 first intron, because a reporter gene was inserted under the control of each WNK1 promoter within the transgene.Several in vitro experiments suggest that an increase in L-WNK1 expression in the distal nephron could trigger the development of FHHt. The kinase can, indeed, stimulate the activity of the Na⁺–Cl⁻ cotransporter (NCC), which has been established as an essential component of the FHHt phenotype, through its interaction with either WNK4 and/or Ste20-related proline-alanine rich kinase (SPAK) (review in ref. 4). WNK4 inhibits NCC, and L-WNK1 relieves the cotransporter from this inhibition. L-WNK1 phosphorylates and thus, activates SPAK, which in turn, stimulates NCC membrane expression by phosphorylation. However, the characterization of L-WNK1 function in the distal nephron has been hampered by the absence of a valid mouse model, because L-WNK1 inactivation results in embryonic death caused by cardiovascular defects (8, 9).To understand how the intronic deletion leads to FHHt, we generated a mouse model harboring a heterozygous deletion in the endogenous first intron of WNK1 to reproduce the human genetic situation. These mice exhibit hyperkalemia, hypertension, and metabolic acidosis, which seem to result from NCC activation. This phenotype results from a twofold increase in L-WNK1 expression in the distal convoluted tubule (DCT) and a slightly increased expression of L-WNK1 in the connecting tubule (CNT), with no modification of KS-WNK1 expression. We also show that the activity of epithelial sodium (Na) channel (ENaC) is not altered in WNK1^+/FHHt mice, whereas the expression of renal outer medullary potassium (K) channel (ROMK) is decreased in the late DCT and CNT; this finding suggests that the hyperkalemia observed in WNK1^+/FHHt is not caused by decreased ENaC activity but, at least in part, by a decreased K⁺ excretion caused by the inhibition of ROMK by L-WNK1.     

Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK)

With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr¹⁰⁹², Tyr¹⁰⁹⁴, and Tyr¹¹⁴³, and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr¹⁰⁹² or Tyr¹¹⁴³ to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4^Y1092/1094F. Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser¹¹⁹⁶, an effect that was abrogated in the double mutant. WNK4^Y1143F inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4^Y1143F could not be reversed by SGK1. The failure to reverse WNK4^Y1143F-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4–PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4^Y1143F-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr¹⁰⁹² and Tyr¹¹⁴³ plays an important role in modulating the inhibitory effect of WNK4 on ROMK.With-no-lysine kinase 4 (WNK4) is expressed in the connecting tubule (CNT) and cortical collecting duct (CCD) (1, 2) and plays an important role in modulating the balance between renal K secretion and Na reabsorption (3–8). The effect of WNK4 on renal K secretion is partially mediated through inhibition of KCNJ1 (ROMK) channels in the CNT and in the CCD. ROMK inhibition is achieved by a stimulation of clathrin-mediated endocytosis (1), an effect that is dependent on intersectin, a scaffold protein containing two Eps15 homology domains (9).Serum/glucocorticoid-induced kinase 1 (SGK1), a downstream mediator of aldosterone signaling, suppresses the inhibitory effect of WNK4 on ROMK channels through phosphorylation of WNK4 at Ser¹¹⁶⁹ (2) and Ser¹¹⁹⁶ (5). Both volume depletion and high K intake increase aldosterone and SGK1 levels (10). However, it is not clear why a high K intake or volume depletion modulates differently the effect of SGK1 on ROMK channels.Candidate regulators of differential ROMK expression in hyperkalemia and hypovolemia should be regulated in a potassium-dependent manner. One such protein is the protein tyrosine kinase c-Src, whose expression in renal cortex is reduced in states of high potassium intake (11). We have previously demonstrated a key role of c-Src in determining the effect of SGK1 on WNK4 (12). C-Src abolishes SGK1-induced phosphorylation of WNK4 and restores the inhibitory effect of WNK4 on ROMK channels in the presence of SGK1 (13). This effect may play a role in preventing K secretion in the absence of hyperkalemia. High potassium intake, in contrast, will diminish c-Src levels, restore SGK1-induced phosphorylation of WNK4, and lead to increased renal potassium secretion via ROMK.Whereas protein phosphatase activity has been shown to be involved in c-Src–mediated modulation of the interaction between SGK1 and WNK4 (13), the molecular mechanism of c-Src’s interaction with WNK4 has been elusive. We here identify previously undescribed tyrosine phosphorylation sites in WNK4 that are targets of c-Src. We characterize the effects of tyrosine phosphorylation on the SGK1–WNK4 interaction, as well as WNK4-mediated ROMK inhibition.     

Disease-causing mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins

Mutations in the WNK4 gene cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder of hyperkalemia and hypertension. The target molecules of this putative kinase and the molecular mechanisms by which the mutations cause the phenotypes are currently unknown. Although recent reports found that expression of WNK4 in Xenopus oocytes causes inhibition of the thiazide-sensitive NaCl cotransporter and the renal K channel ROMK, there may be additional targets of WNK4. For example, an increase in paracellular chloride permeability has been postulated to be a mediator of PHAII pathogenesis, a possibility supported by the localization of WNK4 at tight junctions in vivo. To determine the validity of this hypothesis, we measured transepithelial Na and Cl permeability in Madin-Darby canine kidney II cells stably expressing wild-type or a pathogenic mutant of WNK4. We found that transepithelial paracellular Cl permeability was increased in cells expressing a disease-causing mutant WNK4 (D564A) but that Na permeability was decreased slightly. Furthermore, WNK4 bound and phosphorylated claudins 1-4, major tight-junction membrane proteins known to be involved in the regulation of paracellular ion permeability. The increases in phosphorylation of claudins were greater in cells expressing the mutant WNK4 than in cells expressing wild-type protein. These results clearly indicate that the pathogenic WNK4 mutant possesses a gain-of-function activity and that the claudins may be important molecular targets of WNK4 kinase. The increased paracellular "chloride shunt" caused by the mutant WNK4 could be the pathogenic mechanism of PHAII.     

WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl- cotransporters required for normal blood pressure homeostasis

WNK1 and WNK4 [WNK, with no lysine (K)] are serine-threonine kinases that function as molecular switches, eliciting coordinated effects on diverse ion transport pathways to maintain homeostasis during physiological perturbation. Gain-of-function mutations in either of these genes cause an inherited syndrome featuring hypertension and hyperkalemia due to increased renal NaCl reabsorption and decreased K(+) secretion. Here, we reveal unique biochemical and functional properties of WNK3, a related member of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is expressed throughout the nephron, predominantly at intercellular junctions. Because WNK4 is a potent inhibitor of members of the cation-cotransporter SLC12A family, we used coexpression studies in Xenopus oocytes to investigate the effect of WNK3 on NCC and NKCC2, related kidney-specific transporters that mediate apical NaCl reabsorption in the thick ascending limb and distal convoluted tubule, respectively. In contrast to WNK4's inhibitory activity, kinase-active WNK3 is a potent activator of both NKCC2 and NCC-mediated transport. Conversely, in its kinase-inactive state, WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane. Wild-type WNK3 increases and kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for the vasopressin-mediated plasmalemmal translocation and activation of NKCC2 in vivo. The effects of WNK3 on these transporters and their coexpression in renal epithelia implicate WNK3 in NaCl, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin.     

Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4

Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl(-) dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a "mirror image" of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na(+) influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ((22)Na-influx reduced 50%, P < 1 x 10(-9), surface expression reduced 75%, P < 1 x 10(-14) in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.     

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.     

WNK4 regulates activity of the epithelial Na+ channel in vitro and in vivo

Homeostasis of intravascular volume, Na(+), Cl(-), and K(+) is interdependent and determined by the coordinated activities of structurally diverse mediators in the distal nephron and the distal colon. The behavior of these flux pathways is regulated by the renin-angiotensin-aldosterone system; however, the mechanisms that allow independent modulation of individual elements have been obscure. Previous work has shown that mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension with hyperkalemia, due to altered activity of specific Na-Cl cotransporters, K(+) channels, and paracellular Cl(-) flux mediators of the distal nephron. By coexpression studies in Xenopus oocytes, we now demonstrate that WNK4 also inhibits the epithelial Na(+) channel (ENaC), the major mediator of aldosterone-sensitive Na(+) (re)absorption, via a mechanism that is independent of WNK4's kinase activity. This inhibition requires intact C termini in ENaC beta- and gamma-subunits, which contain PY motifs used to target ENaC for clearance from the plasma membrane. Importantly, PHAII-causing mutations eliminate WNK4's inhibition of ENaC, thereby paralleling other effects of PHAII to increase sodium balance. The relevance of these findings in vivo was studied in mice harboring PHAII-mutant WNK4. The colonic epithelium of these mice demonstrates markedly increased amiloride-sensitive Na(+) flux compared with wild-type littermates. These studies identify ENaC as a previously unrecognized downstream target of WNK4 and demonstrate a functional role for WNK4 in the regulation of colonic Na(+) absorption. These findings support a key role for WNK4 in coordinating the activities of diverse flux pathways to achieve integrated fluid and electrolyte homeostasis.     

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.     

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.     

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.     

WNK1, a kinase mutated in inherited hypertension with hyperkalemia,localizes to diverse Cl- -transporting epithelia

Mutations in WNK1 and WNK4, genes encoding members of a novel family of serine-threonine kinases, have recently been shown to cause pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder featuring hypertension, hyperkalemia, and renal tubular acidosis. The localization of these kinases in the distal nephron and the Cl(-) dependence of these phenotypes suggest that these mutations increase renal Cl(-) reabsorption. Although WNK4 expression is limited to the kidney, WNK1 is expressed in many tissues. We have examined the distribution of WNK1 in these extrarenal tissues. Immunostaining using WNK1-specific antibodies demonstrated that WNK1 is not present in all cell types; rather, it is predominantly localized in polarized epithelia, including those lining the lumen of the hepatic biliary ducts, pancreatic ducts, epididymis, sweat ducts, colonic crypts, and gallbladder. WNK1 is also found in the basal layers of epidermis and throughout the esophageal epithelium. The subcellular localization of WNK1 varies among these epithelia. WNK1 is cytoplasmic in kidney, colon, gallbladder, sweat duct, skin, and esophagus; in contrast, it localizes to the lateral membrane in bile ducts, pancreatic ducts, and epididymis. These epithelia are all notable for their prominent role in Cl(-) flux. Moreover, these sites largely coincide with those involved in the pathology of cystic fibrosis, a disease characterized by deranged epithelial Cl(-) flux. Together with the known pathophysiology of PHAII, these findings suggest that WNK1 plays a general role in the regulation of epithelial Cl(-) flux, a finding that suggests the potential of new approaches to the selective modulation of these processes.     

A new kindred with pseudohypoaldosteronism type II and a novel mutation (564D>H) in the acidic motif of the WNK4 gene

We identified a new kindred with the familial syndrome of hypertension and hyperkalemia (pseudohypoaldosteronism type II or Gordon's syndrome) containing an affected father and son. Mutation analysis confirmed a single heterozygous G to C substitution within exon 7 (1690G>C) that causes a missense mutation within the acidic motif of WNK4 (564D>H). We confirmed the function of this novel mutation by coexpressing it in Xenopus oocytes with either the NaCl cotransporter (NCCT) or the inwardly rectifying K-channel (ROMK). Wild-type WNK4 inhibits 22Na+ flux in Xenopus oocytes expressing NCCT by approximately 90% (P<0.001), whereas the 564D>H mutant had no significantly inhibitory effect on flux through NCCT. In oocytes expressing ROMK, wild-type WNK4 produced >50% inhibition of steady-state current through ROMK at a +20-mV holding potential (P<0.001). The 564D>H mutant produced further inhibition with steady-state currents to some 60% to 70% of those seen with the wild-type WNK4. Using fluorescent-tagged NCCT (enhanced cyan fluorescent protein-NCCT) and ROMK (enhanced green fluorescent protein-ROMK) to quantify the expression of the proteins in the oocyte membrane, it appears that the functional effects of the 564D>H mutation can be explained by alteration in the surface expression of NCCT and ROMK. Compared with wild-type WNK4, WNK4 564D>H causes increased cell surface expression of NCCT but reduced expression of ROMK. This work confirms that the novel missense mutation in WNK4, 564D>H, is functionally active and highlights further how switching charge on a single residue in the acid motif of WNK4 affects its interaction with the thiazide-sensitive target NCCT and the potassium channel ROMK.     

A patient with pseudohypoaldosteronism type II caused by a novel mutation in WNK4 gene

Pseudohypoaldosteronism Type II (PHAII) is a very rare disorder characterized by hyperkalemia, hypertension, and slight hyper-chloremic metabolic acidosis. The index patient showed typical features of PHAII, including elevated blood pressure (140–150/90–100 mmHg), hyperkalemia in the range of 5.30–5.60 mmol/l (normal range is 3.50–5.10 mmol/l), accompanied by hyperchloremia of 109.5–112.0 mmol/l (normal 95.0–108.0 mmol/l) and acidosis with bicarbonate levels of 19.5–20.1 mmol/l (normal 22.0–27.0), GFR was 98.95 ml/min (normal > 90). However, these features were absent in his parents. Sequencing analysis found the patient with a WNK4 gene mutation, 1682 C > T in Exon 7, which resulted a missense mutation at codon 561 (P561L). The variation in codon 561 was not found in his parents and 100 unrelated control subjects. The identified WNK4 mutation which has not been described previously is the probable cause of PHAII.     

(Na+K+)-Activated Adenosinetriphosphatase and Hypertension in Dahl Salt-SEnsitive and -Resistant Rats

(Na⁺K⁺)-ATPase activity was compared in Dahl salt-sensitive (S) and salt-resistant (R) rats. When S and R rats were maintained on 1% NaCl diet their blood pressures at 5 weeks of age were similar and their renal micro-somal (Na⁺K⁺)-ATPase activities were also similar. At 6 months of age, on 1% NaCl diet, S rats have markedly elevated blood pressure compared to R and renal microsomal (Na⁺K⁺)-ATPase activity was suppressed in S compared to R. Feeding 8% NaCl diet for 5 weeks induced hypertension in young S rats but failed to alter renal or brain (Na⁺K⁺)-ATPase activity. Heart (Na⁺K⁺)-ATPase activity was elevated in S compared to R rats regardless of salt intake or blood pressure. It appears unlikely that mutations in the structural locus for the renal (Na⁺K⁺)-ATPase molecule are involved in the strain specific differences in susceptibility to salt-induced hypertension since the physical-chemical properties of the enzyme from the two strains were found to be similar. Since renal (Na⁺K⁺)-ATPase activities were unchanged by salt feeding and resultant blood pressure changes in young S rats, the suppressed renal (Na⁺K⁺)-ATPase activity seen only in old S rats is probably a response to prolonged renal damage and not a response to “natriuretic factors” Elevated heart (Na⁺K⁺)-ATPase in S-rat hearts is unexplained.     

New Therapeutic Approaches for the Treatment of Hyperkalemia in Patients Treated with Renin-Angiotensin-Aldosterone System Inhibitors

Hyperkalemia (serum potassium >?5.5 mEq/L) is a common clinical problem in patients with chronic kidney disease, hypertension, diabetes, and heart failure. It can result from increased K⁺ intake, impaired distribution between intracellular and extracellular spaces, and most frequently, decreased renal excretion. Patients at the highest risk of hyperkalemia are treated with renin-angiotensin-aldosterone system inhibitors (RAASIs) as they improve cardiovascular and renal outcomes and are strongly recommended in clinical guidelines. However, RAASIs cause or increase the risk of hyperkalemia, a key limitation to fully titrate RAASIs in patients who are most likely to benefit from treatment. Until recently, drugs for the treatment of hyperkalemia presented limited efficacy and/or safety concerns and there was an unmet need of new drugs to control hyperkalemia while maintaining RAASI therapy. We provide an overview of the mechanisms involved in K⁺ homeostasis and the epidemiology and management of hyperkalemia as a complication in cardiovascular patients and, finally, analyze the efficacy and safety of two new polymer-based, non-systemic agents, patiromer calcium and sodium zirconium cyclosilicate (ZS-9), designed to increase fecal K⁺ loss and to normalize elevated serum K⁺ levels and chronically maintain K⁺ homeostasis in hyperkalemic patients treated with RAASIs.     

KLHL3 regulates paracellular chloride transport in the kidney by ubiquitination of claudin-8

A rare Mendelian syndrome—pseudohypoaldosteronism type II (PHA-II)—features hypertension, hyperkalemia, and metabolic acidosis. Genetic linkage studies and exome sequencing have identified four genes—with no lysine kinase 1 (wnk1), wnk4, Kelch-like 3 (KLHL3), and Cullin 3 (Cul3)—mutations of which all caused PHA-II phenotypes. The previous hypothesis was that the KLHL3–Cul3 ubiquitin complex acted on the wnk4–wnk1 kinase complex to regulate Na⁺/Cl⁻ cotransporter (NCC) mediated salt reabsorption in the distal tubules of the kidney. Here, we report the identification of claudin-8 as a previously unidentified physiologic target for KLHL3 and provide an alternative explanation for the collecting duct’s role in PHA-II. Using a tissue-specific KO approach, we have found that deletion of claudin-8 in the collecting duct of mouse kidney caused hypotension, hypokalemia, and metabolic alkalosis, an exact mirror image of PHA-II. Mechanistically, the phenotypes in claudin-8 KO animals were caused by disruption of the claudin-8 interaction with claudin-4, the paracellular chloride channel, and delocalization of claudin-4 from the tight junction. In mouse collecting duct cells, knockdown of KLHL3 profoundly increased the paracellular chloride permeability. Mechanistically, KLHL3 was directly bound to claudin-8, and this binding led to the ubiquitination and degradation of claudin-8. The dominant PHA-II mutation in KLHL3 impaired claudin-8 binding, ubiquitination, and degradation. These findings have attested to the concept that the paracellular pathway is physiologically regulated through the ubiquitination pathway, and its deregulation may lead to diseases of electrolyte and blood pressure imbalances.Gordon’s syndrome, also known as pseudohypoaldosteronism II (PHA-II) or familial hyperkalemic hypertension, features several metabolic derangements, including hypertension, hyperkalemia, and hyperchloremic metabolic acidosis (1). Mutations in four genes have been found to cause Gordon’s syndrome. Two encode the serine-threonine kinases with no lysine kinases (WNKs) (2). The other two encode proteins important in the cullin-really interesting new gene E3 ubiquitin ligase (CRL) complex—Kelch-like 3 (KLHL3) and Cullin 3 (CUL3) (3, 4). Disease-causing mutations in WNKs are dominant and confer gain of function to augment NaCl reabsorption in the distal convoluted tubule (DCT) by a signaling cascade of SPAK/OSR1 to NCC (5, 6). Mutations in KLHL3 are either recessive or dominant. Recessive mutations include premature termination, frameshift, and splicing alternatives, consistent with loss of function, whereas dominant mutations cluster in the β-propeller domain important in target recognition (3, 4). CUL3 mutations are all dominant and de novo and result in the skipping of exon 9 and in-frame deletion of a 57-aa segment important in maintaining the CRL architecture (3). KLHL3 was found to interact with WNK4 and regulate its ubiquitination and degradation (7, 8). Presumably, loss of KLHL3 function may lead to increases in WNK4 protein levels that, in turn, promote NCC phosphorylation and NaCl reabsorption in the DCT. Compatible with this notion, a knock-in mouse model harboring a dominant mutation (R528H) allele in the KLHL3 gene developed PHA-II–related phenotypes and concomitant increases in WNK4 protein abundance and NCC phosphorylation levels (9).All four PHA-II genes have significant expression in renal tubules, including the DCT, the connecting tubule (CNT), and the collecting duct (CD) (2, 3). Although the PHA-II mechanism in the DCT has been extensively studied, the CNT/CD’s role remains largely elusive. The predominant NaCl transport pathway in the CNT/CD is through epithelial Na⁺ channel (ENaC) mediated Na⁺ reabsorption and electrically coupled paracellular Cl⁻ reabsorption (also known as the chloride shunt) (10, 11). WT WNK4 inhibits ENaC conductivity, whereas PHA-II–causing mutations eliminate WNK4’s inhibition of ENaC, thereby promoting Na⁺ reabsorption in the CNT/CD (12). The disease-causing mutation in WNK4 has also been found to augment paracellular Cl⁻ conductance in renal epithelial cells (13, 14). The hypothesis of unopposed chloride shunt is particularly important to explain hyperchloremia and hyperkalemia in PHA-II. The shunt conductance would favor Cl⁻ reabsorption and decrease the lumen-negative transepithelial voltage as the driving force for K⁺ secretion (15). To reveal the molecular nature of chloride shunt and its potential role in PHA-II pathophysiology, we have generated the KO mouse models for two claudin molecules—claudin-4 and claudin-8—both of which are required to generate paracellular Cl⁻ conductance in vitro (16). The claudin-4 KO mice developed hypotension, hypochloremia, and metabolic alkalosis because of renal loss of Cl⁻ (11). The claudin-8 KO phenocopied claudin-4 KO, which can be mechanistically attributed to their interaction and coassembly into the tight junction (TJ). Most intriguingly, KLHL3 directly interacted with claudin-8 and regulated its ubiquitination and degradation. Loss of KLHL3 in the CD cells increased the TJ conductance to chloride. The dominant PHA-II mutation in KLHL3 abolished its interaction and regulation of claudin-8. These findings have revealed a previously unidentified physiological substrate for KLHL3 and provided important insights of the TJ’s role in PHA-II pathogenesis.