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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 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.

Mutations in the serine-threonine kinases WNK1 and WNK4 cause a Mendelian disease featuring hypertension and hyperkalemia. In vitro and in vivo studies have revealed that these proteins are molecular switches that have discrete functional states that impart different effects on downstream ion channels, transporters, and the paracellular pathway. These effects enable the distal nephron to allow either maximal NaCl reabsorption or maximal K+ secretion in response to hypovolemia or hyperkalemia, respectively. The related kinase WNK3 has reciprocal actions on the primary mediators of cellular Cl(-) influx and efflux, effects that can serve to regulate cell volume during growth and in response to osmotic stress as well as to modulate neuronal responses to GABA. These findings define a versatile new family of kinases that coordinate the activities of diverse ion transport pathways to achieve and maintain fluid and electrolyte homeostasis.

The mechanisms that govern homeostasis of complex systems have been elusive but can be illuminated by mutations that disrupt system behavior. Mutations in the gene encoding the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and hyperkalemia. We show that physiology in mice transgenic for genomic segments harboring wild-type (TgWnk4(WT)) or PHAII mutant (TgWnk4(PHAII)) Wnk4 is changed in opposite directions: TgWnk4(PHAII) mice have higher blood pressure, hyperkalemia, hypercalciuria and marked hyperplasia of the distal convoluted tubule (DCT), whereas the opposite is true in TgWnk4(WT) mice. Genetic deficiency for the Na-Cl cotransporter of the DCT (NCC) reverses phenotypes seen in TgWnk4(PHAII) mice, demonstrating that the effects of the PHAII mutation are due to altered NCC activity. These findings establish that Wnk4 is a molecular switch that regulates the balance between NaCl reabsorption and K+ secretion by altering the mass and function of the DCT through its effect on NCC.

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.

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 (KLHL3S433-P) via protein kinase C, resulting in increased WNK4 levels and increased NCC activity. Mechanisms mediating KLHL3S433-P dephosphorylation have heretofore been unknown. We now demonstrate that calcineurin expressed in DCT is a potent KLHL3S433-P phosphatase. In mammalian cells, the calcium ionophore ionomycin, a calcineurin activator, reduces KLHL3S433-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 KLHL3S433-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 KLHL3S433-P. These findings demonstrate that KLHL3S433-P is a calcineurin substrate and implicate increased KLHL3 phosphorylation in tacrolimus-induced pathologies. [ABSTRACT FROM AUTHOR]

The Na-K-Cl cotransporter (NKCC2) is the major salt transport pathway in the thick ascending limb of Henle's loop and is part of the molecular mechanism for blood pressure regulation. Recent screening of ~3,000 members of the Framingham Heart Study identified nine rare independent mutations in the gene encoding NKCC2 (SLC12A1) associated with clinically reduced blood pressure and protection from hypertension (Ji WZ, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State M, Levy D, Lifton RP. Nat Genet 40: 592-599, 2008). To investigate their functional consequences, we introduced the nine mutations in human NKCC2A and examined protein function, expression, localization, regulation, and ion transport kinetics using heterologous expression in Xenopus laevis oocytes and HEK-293 cells. When expressed in oocytes, four of the mutants (T235M, R302W, L505V, and P569H) exhibited reduced transport function compared with wild-type. In HEK-293 cells, the same four mutants exhibited reduced function, and in addition N399S and P1083A had significantly lower activity than wild-type. The two most functionally impaired mutants (R302W and L505V) exhibited dramatically diminished production of complex-glycosylated protein and a decrease in or absence of plasma membrane localization, indicative of a processing defect. All of the functional human (h) NKCC2A variants were regulated by changes in oocyte volume and intracellular chloride in HEK cells, but P254A and N399S exhibited a lower constitutive activity in HEK cells. The P569H mutant exhibited a 50% reduction in sodium affinity compared with wild-type, predicting lower transport activity at lower intratubular salt concentrations, while the P254A mutant exhibited a 35% increase in rubidium affinity. We conclude that defects in NKCC2 processing, transport turnover rate, regulation, and ion affinity contribute to impaired transport function in six of the nine identified mutants, providing support for the predictive approach of Ji et al. to identify functionally important residues by sequence conservation. Such mutations in hNKCC2A are likely to reduce renal salt reabsorption, providing a mechanism for lower blood pressure. [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]

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]

A key question in systems biology is how diverse physiologic processes are integrated to produce global homeostasis. Genetic analysis can contribute by identifying genes that perturb this integration. One system orchestrates renal NaCl and K+ flux to achieve homeostasis of blood pressure and serum K+ concentration (refs. 2,3). Positional cloning implicated the serine-threonine kinase WNK4 in this process; clustered mutations in PRKWNK4, encoding WNK4, cause hypertension and hyperkalemia (pseudohypoaldosteronism type II, PHAII) by altering renal NaCl and K+ handling. Wild-type WNK4 inhibits the renal Na-Cl cotransporter (NCCT); mutations that cause PHAII relieve this inhibition. This explains the hypertension of PHAII but does not account for the hyperkalemia. By expression in Xenopus laevis oocytes, we show that WNK4 also inhibits the renal K+ channel ROMK. This inhibition is independent of WNK4 kinase activity and is mediated by clathrin-dependent endocytosis of ROMK, mechanisms distinct from those that characterize WNK4 inhibition of NCCT. Most notably, the same mutations in PRKWNK4 that relieve NCCT inhibition markedly increase inhibition of ROMK. These findings establish WNK4 as a multifunctional regulator of diverse ion transporters; moreover, they explain the pathophysiology of PHAII. They also identify WNK4 as a molecular switch that can vary the balance between NaCl reabsorption and K+ secretion to maintain integrated homeostasis. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]