Search

Calcineurin is a calcium/calmodulin-regulated phosphatase known for its role in activation of T cells following engagement of the T cell receptor. Calcineurin inhibitors (CNIs) are widely used as immunosuppressive agents; common adverse effects of CNIs are hypertension and hyperkalemia. While previous studies have implicated activation of the Na-Cl cotransporter (NCC) in the renal distal convoluted tubule (DCT) in this toxicity, the molecular mechanism of this effect is unknown. The renal effects of CNIs mimic the hypertension and hyperkalemia that result from germ-line mutations in with-no-lysine (WNK) kinases and the Kelch-like 3 (KLHL3)-CUL3 ubiquitin ligase complex. WNK4 is an activator of NCC and is degraded by binding to KLHL3 followed by WNK4's ubiquitylation and proteasomal degradation. This binding is prevented by phosphorylation of KLHL3 at serine 433 (KLHL3 S433-P ) via protein kinase C, resulting in increased WNK4 levels and increased NCC activity. Mechanisms mediating KLHL3 S433-P dephosphorylation have heretofore been unknown. We now demonstrate that calcineurin expressed in DCT is a potent KLHL3 S433-P phosphatase. In mammalian cells, the calcium ionophore ionomycin, a calcineurin activator, reduces KLHL3 S433-P levels, and this effect is reversed by the calcineurin inhibitor tacrolimus and by siRNA-mediated knockdown of calcineurin. In vivo, tacrolimus increases levels of KLHL3 S433-P , resulting in increased levels of WNK4, phosphorylated SPAK, and NCC. Moreover, tacrolimus attenuates KLHL3-mediated WNK4 ubiquitylation and degradation, while this effect is absent in KLHL3 with S433A substitution. Additionally, increased extracellular K + induced calcineurin-dependent dephosphorylation of KLHL3 S433-P These findings demonstrate that KLHL3 S433-P is a calcineurin substrate and implicate increased KLHL3 phosphorylation in tacrolimus-induced pathologies., Competing Interests: Conflict of interest statement: R.P.L. is a nonexecutive director of Roche and its subsidiary Genentech., (Copyright © 2019 the Author(s). Published by PNAS.)

Midaortic syndrome (MAS) is a rare cause of severe childhood hypertension characterized by narrowing of the abdominal aorta in children and is associated with extensive vascular disease. It may occur as part of a genetic syndrome, such as neurofibromatosis, or as consequence of a pathological inflammatory disease. However, most cases are considered idiopathic. We hypothesized that in a high percentage of these patients, a monogenic cause of disease may be detected by evaluating whole exome sequencing data for mutations in 1 of 38 candidate genes previously described to cause vasculopathy. We studied a cohort of 36 individuals from 35 different families with MAS by exome sequencing. In 15 of 35 families (42.9%), we detected likely causal dominant mutations. In 15 of 35 (42.9%) families with MAS, whole exome sequencing revealed a mutation in one of the genes previously associated with vascular disease ( NF1 , JAG1 , ELN , GATA6 , and RNF213 ). Ten of the 15 mutations have not previously been reported. This is the first report of ELN , RNF213 , or GATA6 mutations in individuals with MAS. Mutations were detected in NF1 (6/15 families), JAG1 (4/15 families), ELN (3/15 families), and one family each for GATA6 and RNF213 Eight individuals had syndromic disease and 7 individuals had isolated MAS. Whole exome sequencing can provide conclusive molecular genetic diagnosis in a high fraction of individuals with syndromic or isolated MAS. Establishing an etiologic diagnosis may reveal genotype/phenotype correlations for MAS in the future and should, therefore, be performed routinely in MAS., (© 2018 American Heart Association, Inc.)

Kelch-like 3 (KLHL3) is a component of an E3 ubiquitin ligase complex that regulates blood pressure by targeting With-No-Lysine (WNK) kinases for degradation. Mutations in KLHL3 cause constitutively increased renal salt reabsorption and impaired K + secretion, resulting in hypertension and hyperkalemia. Although clinical studies have shown that dietary K + intake affects blood pressure, the mechanisms have been obscure. In this study, we demonstrate that the KLHL3 ubiquitin ligase complex is involved in the low-K + -mediated activation of Na-Cl cotransporter (NCC) in the kidney. In the distal convoluted tubules of mice eating a low-K + diet, we found increased KLHL3 phosphorylation at S433 (KLHL3 S433-P ), a modification that impairs WNK binding, and also reduced total KLHL3 levels. These changes are accompanied by the accumulation of the target substrate WNK4, and activation of the downstream kinases SPAK (STE20/SPS1-related proline-alanine-rich protein kinase) and OSR1 (oxidative stress-responsive 1), resulting in NCC phosphorylation and its accumulation at the plasma membrane. Increased phosphorylation of S433 was explained by increased levels of active, phosphorylated protein kinase C (but not protein kinase A), which directly phosphorylates S433. Moreover, in HEK cells expressing KLHL3 and WNK4, we showed that the activation of protein kinase C by phorbol 12-myristate 13-acetate induces KLHL3 S433-P and increases WNK4 levels by abrogating its ubiquitination. These data demonstrate the role of KLHL3 in low-K + -mediated induction of NCC; this physiologic adaptation reduces distal electrogenic Na + reabsorption, preventing further renal K + loss but promoting increased blood pressure.

Many Mendelian traits are likely unrecognized owing to absence of traditional segregation patterns in families due to causation by de novo mutations, incomplete penetrance, and/or variable expressivity. Genome-level sequencing can overcome these complications. Extreme childhood phenotypes are promising candidates for new Mendelian traits. One example is early onset hypertension, a rare form of a global cause of morbidity and mortality. We performed exome sequencing of 40 unrelated subjects with hypertension due to primary aldosteronism by age 10. Five subjects (12.5%) shared the identical, previously unidentified, heterozygous CACNA1H(M1549V) mutation. Two mutations were demonstrated to be de novo events, and all mutations occurred independently. CACNA1H encodes a voltage-gated calcium channel (CaV3.2) expressed in adrenal glomerulosa. CACNA1H(M1549V) showed drastically impaired channel inactivation and activation at more hyperpolarized potentials, producing increased intracellular Ca(2+), the signal for aldosterone production. This mutation explains disease pathogenesis and provides new insight into mechanisms mediating aldosterone production and hypertension.

We recently implicated two recurrent somatic mutations in an adrenal potassium channel, KCNJ5, as a cause of aldosterone-producing adrenal adenomas (APAs) and one inherited KCNJ5 mutation in a Mendelian form of early severe hypertension with massive adrenal hyperplasia. The mutations identified all altered the channel selectivity filter, producing increased Na(+) conductance and membrane depolarization, the signal for aldosterone production and proliferation of adrenal glomerulosa cells. We report herein members of four kindreds with early onset primary aldosteronism of unknown cause. Sequencing of KCNJ5 revealed that affected members of two kindreds had KCNJ5(G151R) mutations, identical to one of the prevalent recurrent mutations in APAs. These individuals had severe progressive aldosteronism and hyperplasia requiring bilateral adrenalectomy in childhood for blood pressure control. Affected members of the other two kindreds had KCNJ5(G151E) mutations, which are not seen in APAs. These subjects had easily controlled hypertension and no evidence of hyperplasia. Surprisingly, electrophysiology of channels expressed in 293T cells demonstrated that KCNJ5(G151E) was the more extreme mutation, producing a much larger Na(+) conductance than KCNJ5(G151R), resulting in rapid Na(+)-dependent cell lethality. We infer that this increased lethality limits adrenocortical cell mass and the severity of aldosteronism in vivo, accounting for the milder phenotype among these patients. These findings demonstrate striking variations in phenotypes and clinical outcome resulting from different mutations of the same amino acid in KCNJ5 and have implications for the diagnosis and pathogenesis of primary aldosteronism with and without adrenal hyperplasia.

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K(+) and H(+) excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin-RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K(+) and pH homeostasis.

Endocrine tumors such as aldosterone-producing adrenal adenomas (APAs), a cause of severe hypertension, feature constitutive hormone production and unrestrained cell proliferation; the mechanisms linking these events are unknown. We identify two recurrent somatic mutations in and near the selectivity filter of the potassium (K(+)) channel KCNJ5 that are present in 8 of 22 human APAs studied. Both produce increased sodium (Na(+)) conductance and cell depolarization, which in adrenal glomerulosa cells produces calcium (Ca(2+)) entry, the signal for aldosterone production and cell proliferation. Similarly, we identify an inherited KCNJ5 mutation that produces increased Na(+) conductance in a Mendelian form of severe aldosteronism and massive bilateral adrenal hyperplasia. These findings explain pathogenesis in a subset of patients with severe hypertension and implicate loss of K(+) channel selectivity in constitutive cell proliferation and hormone production.

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.

Context: Primary aldosteronism is a leading cause of secondary hypertension (HTN), but the mechanisms underlying the characteristic renin-independent secretion of aldosterone remain unknown in most patients., Objectives: We report a new familial form of aldosteronism in a father and two daughters. All were diagnosed with severe HTN refractory to medical treatment by age 7 yr. We performed a variety of clinical, biochemical, and genetic studies to attempt to clarify the underlying molecular defect., Results: Biochemical studies revealed hyporeninemia, hyperaldosteronism, and very high levels of 18-oxocortisol and 18-hydroxycortisol, steroids that reflect oxidation by both steroid 17-alpha hydroxylase and aldosterone synthase. These enzymes are normally compartmentalized in the adrenal fasciculata and glomerulosa, respectively. Administration of dexamethasone failed to suppress either aldosterone or cortisol secretion; these findings distinguish this clinical syndrome from glucocorticoid-remediable aldosteronism, another autosomal dominant form of HTN, and suggest a global defect in the regulation of adrenal steroid production. Genetic studies excluded mutation at the aldosterone synthase locus, further distinguishing this disorder from glucocorticoid-remediable aldosteronism. Because of unrelenting HTN, all three subjects underwent bilateral adrenalectomy, which in each case corrected the HTN. Adrenal glands showed dramatic enlargement, with paired adrenal weights as high as 82 g. Histology revealed massive hyperplasia and cellular hypertrophy of a single cortical compartment that had features of adrenal fasciculata or a transitional zone, with an atrophic glomerulosa., Conclusion: These findings define a new inherited form of aldosteronism and suggest that identification of the underlying defect will provide insight into normal mechanisms regulating adrenal steroid biosynthesis.

The effects of alleles in many genes are believed to contribute to common complex diseases such as hypertension. Whether risk alleles comprise a small number of common variants or many rare independent mutations at trait loci is largely unknown. We screened members of the Framingham Heart Study (FHS) for variation in three genes-SLC12A3 (NCCT), SLC12A1 (NKCC2) and KCNJ1 (ROMK)-causing rare recessive diseases featuring large reductions in blood pressure. Using comparative genomics, genetics and biochemistry, we identified subjects with mutations proven or inferred to be functional. These mutations, all heterozygous and rare, produce clinically significant blood pressure reduction and protect from development of hypertension. Our findings implicate many rare alleles that alter renal salt handling in blood pressure variation in the general population, and identify alleles with health benefit that are nonetheless under purifying selection. These findings have implications for the genetic architecture of hypertension and other common complex traits.

The serine-threonine kinase WNK3 modulates Cl- transport into and out of cells through its regulation of SLC12A cation/Cl- cotransporters, implicating it as (one of) the long-sought Cl-/volume-sensitive kinase(s). Integrators in homeostatic systems regulate structurally diverse but functionally coupled elements. For example, the related kinase WNK4 regulates the Na-Cl co-transporter (NCC), paracellular Cl- flux, and the K+ channel ROMK1 (Kir1.1) to maintain renal NaCl and K+ homeostasis; mutations in PRKWNK4, encoding WNK4, cause a Mendelian disease featuring hypertension and hyperkalemia. It is known that WNK3 is expressed in the nephron's distal convoluted tubule (DCT) and stimulates NCC activity. Here, we show that WNK3 is also expressed in cortical and outer medullary collecting duct principal cells. Accordingly, we tested WNK3's effect on the mediators of NaCl and K+ handling in these nephron segments--the epithelial sodium channel (ENaC), paracellular Cl- flux, and ROMK1--using established model systems. WNK3 did not alter paracellular Cl- flux in tetracycline-responsive MDCK II cells, nor affect amiloride-sensitive currents when co-expressed with ENaC in Xenopus laevis oocytes. However, additional co-expression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (p < .0001) by altering its plasmalemmal surface expression; WNK3 did not affect ROMK1's conductance or open/closed probability. In contrast, WNK3 had no effect on the activity of the cardiac long-QT syndrome K+ channel KCNQ1/KCNE1 when co-expressed in oocytes. Inhibition of ROMK1 is independent of WNK3's catalytic activity and is mediated by WNK3's carboxyl terminus--a mechanism distinct from its known kinase-dependent activation of NCC. A kinase-inactivating point mutation, or a missense mutation homologous to one in WNK4 that causes disease produced a gain-of-function effect, enhancing WNK3's inhibition of ROMK1 greater than 2.5-fold relative to wild type kinase (p < .0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its co-expression with its targets in the distal nephron, and the established in vivo effect of WNK4 at these same targets provide evidence that WNK3's action is physiologically relevant. WNK3 is likely a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.

Hypertension and dyslipidemia are risk factors for atherosclerosis and occur together more often than expected by chance. Although this clustering suggests shared causation, unifying factors remain unknown. We describe a large kindred with a syndrome including hypertension, hypercholesterolemia, and hypomagnesemia. Each phenotype is transmitted on the maternal lineage with a pattern indicating mitochondrial inheritance. Analysis of the mitochondrial genome of the maternal lineage identified a homoplasmic mutation substituting cytidine for uridine immediately 5' to the mitochondrial transfer RNA(Ile) anticodon. Uridine at this position is nearly invariate among transfer RNAs because of its role in stabilizing the anticodon loop. Given the known loss of mitochondrial function with aging, these findings may have implications for the common clustering of these metabolic disorders.

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.

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.

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.

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain. [ABSTRACT FROM AUTHOR]

Aldosterone-producing adenomas (APAs) and bilateral adrenal hyperplasia are important causes of secondary hypertension. Somatic mutations in KCNJ5, CACNA1D, ATP1A1, ATP2B3 and CTNNB1 have been described in APAs. Objective To characterize clinical--pathological features in APAs and unilateral adrenal hyperplasia, and correlate them with genotypes. Design Retrospective study. Subjects and Measurements Clinical and pathological characteristics of 90 APAs and seven diffusely or focally hyperplastic adrenal glands were reviewed, and samples were examined for mutations in known disease genes by Sanger or exome sequencing. Results Mutation frequencies were as follows: KCNJ5, 37.1%; CACNA1D, 10.3%; ATP1A1, 8.2%; ATP2B3, 3.1%; and CTNNB1, 2.1%. Previously unidentified mutations included I157K, F154C and two insertions (I150_G151insM and I144_E145insAI) in KCNJ5, all close to the selectivity filter, V426G_V427Q_A428_L433del in ATP2B3 and A39Efs*3 in CTNNB1. Mutations in KCNJ5 were associated with female and other mutations with male gender (P = 0.007). On computed tomography, JCCiV/5-mutant tumours displayed significantly greater diameter (P = 0.023), calculated area (P = 0.002) and lower precontrast Hounsfleld units (P = 0.0002) vs tumours with mutations in other genes. Accordingly, JCCiV/5-mutant tumours were predominantly comprised of lipid-rich fasciculata-like clear cells, whereas other tumours were heterogeneous (P = 5 x 10 vs TIOTI-KCNJ5 mutant and P = 0.0003 vs wild-type tumours, respectively). CACNA1D mutations were present in two samples with hyperplasia without adenoma. Conclusions JCCiV/5-mutant tumours appear to be associated with fasciculata-like clear cell predominant histology and tend to be larger with a characteristic imaging phenotype. Novel somatic KCNJ5 variants likely cause adenomas by loss of potassium selectivity, similar to previously described mutations. [ABSTRACT FROM AUTHOR]

Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum K[sup+] levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and K[sup+] secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the K[sup+] chan- nel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (Angli) levels. AnglI signaling might modulate WNK4 activity. We show that AnglI 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 AnglI, 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 Angil signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. Angil'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 Angll signaling. These effects extend to mammalian cells. AnglI 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 AnglI and NCC. and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing K secretion. [ABSTRACT FROM AUTHOR]

The effects of alleles in many genes are believed to contribute to common complex diseases such as hypertension. Whether risk alleles comprise a small number of common variants or many rare independent mutations at trait loci is largely unknown. We screened members of the Framingham Heart Study (FHS) for variation in three genes—SLC12A3 (NCCT), SLC12A1 (NKCC2) and KCNJ1 (ROMK)—causing rare recessive diseases featuring large reductions in blood pressure. Using comparative genomics, genetics and biochemistry, we identified subjects with mutations proven or inferred to be functional. These mutations, all heterozygous and rare, produce clinically significant blood pressure reduction and protect from development of hypertension. Our findings implicate many rare alleles that alter renal salt handling in blood pressure variation in the general population, and identify alleles with health benefit that are nonetheless under purifying selection. These findings have implications for the genetic architecture of hypertension and other common complex traits. [ABSTRACT FROM AUTHOR]

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 (WNK4S1169D) 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. [ABSTRACT FROM AUTHOR]

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 reninangiotensin-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 β- and γ-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. [ABSTRACT FROM AUTHOR]

The serine–threonine kinase WNK3 modulates Cl− transport into and out of cells through its regulation of SLC12A cation–Cl− cotransporters, implicating it as (one of) the long-sought Cl−/volume-sensitive kinase(s). Integrators in homeostatic systems regulate structurally diverse but functionally coupled elements. For example, the related kinase WNK4 regulates the Na+–Cl− cotransporter (NCC), paracellular Cl− flux, and the K+ channel ROMK1 (Kir1.1) to maintain renal NaCl and K+ homeostasis; mutations in PRKWNK4, encoding WNK4, cause a Mendelian disease featuring hypertension and hyperkalaemia. It is known that WNK3 is expressed in the nephron's distal convoluted tubule (DCT) and stimulates NCC activity. Here, we show that WNK3 is also expressed in cortical and outer medullary collecting duct principal cells. Accordingly, we tested WNK3's effect on the mediators of NaCl and K+ handling in these nephron segments – the epithelial sodium channel (ENaC), paracellular Cl− flux, and ROMK1 – using established model systems. WNK3 did not alter paracellular Cl− flux in tetracycline-responsive MDCK II cells, nor affect amiloride-sensitive currents when coexpressed with ENaC in Xenopus laevis oocytes. However, additional coexpression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold ( P < 0.0001) by altering its plasmalemmal surface expression; WNK3 did not affect ROMK1's conductance or open/closed probability. In contrast, WNK3 had no effect on the activity of the cardiac long-QT syndrome K+ channel KCNQ1/KCNE1 when coexpressed in oocytes. Inhibition of ROMK1 is independent of WNK3's catalytic activity and is mediated by WNK3's carboxyl terminus – a mechanism distinct from its known kinase-dependent activation of NCC. A kinase-inactivating point mutation or a missense mutation homologous to one in WNK4 that causes disease produced a gain-of-function effect, enhancing WNK3's inhibition of ROMK1 greater than 2.5-fold relative to wild-type kinase ( P < 0.0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its coexpression with its targets in the distal nephron, and the established in vivo effect of WNK4 at these same targets provide evidence that WNK3's action is physiologically relevant. WNK3 is probably a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion. [ABSTRACT FROM AUTHOR]

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 CI- efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular CI- 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 shrink-age-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 CI- influx and efflux branches of the SLC12A cotransporter family. [ABSTRACT FROM AUTHOR]

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 NaCI 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 NaCI 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 NaCI, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin. [ABSTRACT FROM AUTHOR]

Investigates the molecular mechanisms of human hypertension. Factors influencing the epidemiology of the condition; Identification of genes underlying blood pressure variation; Forms of Mendelian hypertensive syndromes; Role of net salt balance; Implications for the development and use of antihypertensive treatments.

Objective: To define the clinical spectrum of glucocorticoid-remediable aldosteronism (GRA) in a large kindred.Design: Screening all at-risk relatives of a proband for GRA using a specific biochemical phenotype and collecting of medical histories of kindred members from five generations.Setting: Outpatient General Clinical Research Centers and patients' homes.Measurements: Screening was done while patients were on a self-selected diet and included blood pressure determinations; serum potassium and plasma renin activity and aldosterone measurements; and 24-hour urinary tetrahydroaldosterone, 18-oxotetrahydrocortisol, and 18-hydroxycortisol measurements.Results: Diagnosis of GRA was established on the basis of a previously described specific biochemical abnormality, overproduction of the cortisol C-18 oxidation products (18-oxotetrahydrocortisol and 18-hydroxycortisol) in urine and their ratio relative to tetrahydroaldosterone. Glucocorticoid-remediable aldosteronism was diagnosed in 11 additional patients spanning three generations; this group included the youngest patient (3 months old) ever diagnosed with GRA. Complete penetrance of the biochemical abnormality is likely, with 11 of 18 at-risk patients displaying the phenotype. All patients with GRA had elevated blood pressure. Affected adult patients had been diagnosed as hypertensive before reaching 21 years of age (n = 7 mean, 16.1 +/- 3.4 years). All affected patients were normokalemic (4.3 +/- 0.3 mmol/L).Conclusion: Hypertension is a characteristic feature of GRA. Elevated blood pressure in this kindred developed at an early age and often was severe. Because a normal potassium level does not exclude the diagnosis of GRA, the disorder may be underdiagnosed. The value of a specific cortisol C-18 oxidation phenotype in the diagnosis of GRA has been confirmed. [ABSTRACT FROM AUTHOR]

Mutations in the kinase WNK4 cause an inherited form of human hypertension, pseudohypoaldosteronism type II (PHAII). WNK4 has been shown to regulate the activity of several mediators of renal NaCl and K+ flux. We now show by experiments in Xenopus oocytes and in mice with PHAII-mutant WNK4 that WNK4 regulates the epithelial Na+ channel (ENaC), a major effector of aldosterone signaling. In oocytes, WNK4 inhibits ENaC activity, as measured by amiloride-sensitive whole cell currents (>50%, p< 2 x 10-7), via a kinase-independent mechanism that requires C-termini of the channel's β and γ subunits. A PHAII-causing mutation in WNK4 abolishes this inhibition. SGK1, a known aldosterone-stimulated activator of ENaC, binds WNK4 and specifically phosphorylates it at serine 1169. Mutations that mimic this phosphorylation abrogate WNK4's inhibition of ENaC. We also measured amiloride-sensitive short circuit currents in distal colonic epithelial explants and showed that PHAII mice exhibit 3.5-fold increased ENaC activity (p<0.02) compared to wild type littermates. These studies identify a novel downstream target of WNK4 and show that WNK4 itself is regulated by components of the aldosterone signaling pathway. Moreover, they provide the first functional demonstration of a WNK kinase regulating an ion channel in vivo and reveal a novel role for WNK kinases in regulating salt absorption outside the kidney. [ABSTRACT FROM AUTHOR]

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT. [ABSTRACT FROM AUTHOR]

Endocrine tumors such as aldosterone-producing adrenal adenomas (APAs), a cause of severe hypertension, feature constitutive hormone production and unrestrained cell proliferation; the mechanisms linking these events are unknown. We identify two recurrent somatic mutations in and near the selectivity filter of the potassium (K+) channel KCNJ5 that are present in 8 of 22 human APAs studied. Both produce increased sodium (Na+) conductance and cell depolarization, which in adrenal glomerulosa cells produces calcium (Ca2+) entry, the signal for aldosterone production and cell proliferation. Similarly, we identify an inherited KCNJ5 mutation that produces increased Na+ conductance in a Mendelian form of severe aldosteronism and massive bilateral adrenal hyperplasia. These findings explain pathogenesis in a subset of patients with severe hypertension and implicate loss of K+ channel selectivity in constitutive cell proliferation and hormone production. [ABSTRACT FROM AUTHOR]