WNK4 kinase is a physiological intracellular chloride sensor.

With-no-lysine (WNK) kinases regulate renal sodium-chloride cotransporter (NCC) to maintain body sodium and potassium homeostasis. Gain-of-function mutations of WNK1 and WNK4 in humans lead to a Mendelian hypertensive and hyperkalemic disease pseudohypoaldosteronism type II (PHAII). X-ray crystal structure and in vitro studies reveal chloride ion (Cl-) binds to a hydrophobic pocket within the kinase domain of WNKs to inhibit its activity. The mechanism is thought to be important for physiological regulation of NCC by extracellular potassium. To test the hypothesis that WNK4 senses the intracellular concentration of Cl- physiologically, we generated knockin mice carrying Cl--insensitive mutant WNK4. These mice displayed hypertension, hyperkalemia, hyperactive NCC, and other features fully recapitulating human and mouse models of PHAII caused by gain-of-function WNK4. Lowering plasma potassium levels by dietary potassium restriction increased NCC activity in wild-type, but not in knockin, mice. NCC activity in knockin mice can be further enhanced by the administration of norepinephrine, a known activator of NCC. Raising plasma potassium by oral gavage of potassium inactivated NCC within 1 hour in wild-type mice, but had no effect in knockin mice. The results provide compelling support for the notion that WNK4 is a bona fide physiological intracellular Cl- sensor and that Cl- regulation of WNK4 underlies the mechanism of regulation of NCC by extracellular potassium.

WNK4 kinase: role of chloride sensing in the distal convoluted tubule.

PURPOSE OF REVIEW: This review focuses on recent efforts in identifying with-no-lysine kinase 4 (WNK4) as a physiological intracellular chloride sensor and exploring regulators of intracellular chloride concentration ([Cl-]i) in the distal convoluted tubule (DCT). RECENT FINDINGS: The discovery of WNK1's chloride-binding site provides the mechanistic details of the chloride-sensing regulation of WNK kinases. The subsequent in-vitro studies reveal that the chloride sensitivities of WNK kinases were variable. Because of its highest chloride sensitivity and dominant expression, WNK4 emerges as the leading candidate of the chloride sensor in DCT. The presentation of hypertension and increased sodium-chloride cotransporter (NCC) activity in chloride-insensitive WNK4 mice proved that WNK4 is inhibitable by physiological [Cl-]i in DCT. The chloride-mediated WNK4 regulation is responsible for hypokalemia-induced NCC activation but unnecessary for hyperkalemia-induced NCC deactivation. This chloride-sensing mechanism requires basolateral potassium and chloride channels or cotransporters, including Kir4.1/5.1, ClC-Kb, and possibly KCCs, to modulate [Cl-]i in response to the changes of plasma potassium. SUMMARY: WNK4 is both a master NCC stimulator and an in-vivo chloride sensor in DCT. The understanding of chloride-mediated regulation of WNK4 explains the inverse relationship between dietary potassium intake and NCC activity.

Functional severity of CLCNKB mutations correlates with phenotypes in patients with classic Bartter's syndrome.

KEY POINTS: The highly variable phenotypes observed in patients with classic Bartter's syndrome (BS) remain unsatisfactorily explained. The wide spectrum of functional severity of CLCNKB mutations may contribute to the phenotypic variability, and the genotype-phenotype association has not been established. Low-level expression of the human ClC-Kb channel in mammalian cells impedes the functional study of CLCNKB mutations, and the underlying cause is still unclear. The human ClC-Kb channel is highly degraded by proteasome in human embryonic kidney cells. The C-terminal in-frame green fluorescent protein fusion may slow down the proteasome-mediated proteolysis. Barttin co-expression necessarily improves the stability, membrane trafficking and gating of ClC-Kb. CLCNKB mutations in barttin-binding sites, dimer interface or selectivity filter often have severe functional consequences. The remaining chloride conductance of the ClC-Kb mutant channel significantly correlates with the phenotypes, such as age at diagnosis, plasma chloride concentration, and the degree of calciuria in patients with classic BS. ABSTRACT: Mutations in the CLCNKB gene encoding the human voltage-gated chloride ClC-Kb (hClC-Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests with highly variable phenotypes. The functional severity of the mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC-Kb in heterologous expression systems, the functional consequences of mutant channels have not been thoroughly examined, and the genotype-phenotype association has not been established. In this study, we found that hClC-Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In-frame fusion of green fluorescent protein (GFP) to the C-terminus of the channel may ameliorate proteasome degradation. Co-expression of barttin increased protein abundance and membrane trafficking of hClC-Kb and markedly increased functional chloride current. We then functionally characterized 18 missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC-Kb-GFP. Most CLCNKB mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin binding sites, dimer interface and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well-described functional consequences and clinical presentations for genotype-phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate. In conclusion, hClC-Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C-terminus of hClC-Kb improves protein expression. The functional severity of the CLCNKB mutation is an important determinant of the phenotype in classic BS.

WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release.

Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) in response to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs (CVOs) of the brain focus on mechanosensitive membrane proteins. The present study demonstrated that intracellular protein kinase WNK1 was involved. Focusing on vascular-organ-of-lamina-terminalis (OVLT) nuclei, we showed that WNK1 kinase was activated by water restriction. Neuron-specific conditional KO (cKO) of Wnk1 caused polyuria with decreased urine osmolality that persisted in water restriction and blunted water restriction-induced AVP release. Wnk1 cKO also blunted mannitol-induced AVP release but had no effect on osmotic thirst response. The role of WNK1 in the osmosensory neurons in CVOs was supported by neuronal pathway tracing. Hyperosmolality-induced increases in action potential firing in OVLT neurons was blunted by Wnk1 deletion or pharmacological WNK inhibitors. Knockdown of Kv3.1 channel in OVLT by shRNA reproduced the phenotypes. Thus, WNK1 in osmosensory neurons in CVOs detects extracellular hypertonicity and mediates the increase in AVP release by activating Kv3.1 and increasing action potential firing from osmosensory neurons.

Impairment in renal medulla development underlies salt wasting in Clc-k2 channel deficiency.

The prevailing view is that the ClC-Ka chloride channel (mouse Clc-k1) functions in the thin ascending limb to control urine concentration, whereas the ClC-Kb channel (mouse Clc-k2) functions in the thick ascending limb (TAL) to control salt reabsorption. Mutations of ClC-Kb cause classic Bartter syndrome, characterized by renal salt wasting, with perinatal to adolescent onset. We studied the roles of Clc-k channels in perinatal mouse kidneys using constitutive or inducible kidney-specific gene ablation and 2D and advanced 3D imaging of optically cleared kidneys. We show that Clc-k1 and Clc-k2 were broadly expressed and colocalized in perinatal kidneys. Deletion of Clc-k1 and Clc-k2 revealed that both participated in NKCC2- and NCC-mediated NaCl reabsorption in neonatal kidneys. Embryonic deletion of Clc-k2 caused tubular injury and impaired renal medulla and TAL development. Inducible deletion of Clc-k2 beginning after medulla maturation produced mild salt wasting resulting from reduced NCC activity. Thus, both Clc-k1 and Clc-k2 contributed to salt reabsorption in TAL and distal convoluted tubule (DCT) in neonates, potentially explaining the less-severe phenotypes in classic Bartter syndrome. As opposed to the current understanding that salt wasting in adult patients with Bartter syndrome is due to Clc-k2 deficiency in adult TAL, our results suggest that it originates mainly from defects occurring in the medulla and TAL during development.

An epidermal growth factor inhibitor, Gefitinib, induces apoptosis through a p53-dependent upregulation of pro-apoptotic molecules and downregulation of anti-apoptotic molecules in human lung adenocarcinoma A549 cells.

A selective epidermal growth factor receptor inhibitor, Gefitinib, has been clinically demonstrated to be effective for certain cancer cell types including lung cancer. Our previous study indicated that Gefitinib induced Fas/caspase-dependent apoptosis in human lung adenocarcinoma A549 cells. However, the pathway relaying the signals of Gefitinib-induced cell death has not been fully elucidated. Loss of normal function of p53 facilitates the development of neoplastic lesions and possibly contributes to the development of resistance to chemotherapy. Thus, the current study was designed to examine the role of p53 in Gefitinib-induced apoptosis. Incubation of human lung adenocarcinoma A549 cells with 25 microM Gefitinib resulted in phosphorylation and activation of p53 such as enhanced DNA binding activity, which was accompanied by the upregulation of PUMA (p53 upregulated modulator of apoptosis) and Fas, and downregulation of survivin and XIAP (X-linked inhibitor of apoptosis protein). The Gefitinib-mediated Fas, PUMA, survivin, XIAP regulation and subsequent apoptosis were significantly inhibited in stable p53-shRNA transfectants. Similarly, H1299/p53 cells were more sensitive to Gefitinib compared to H1299 cells in clonogenic survival assay. This event was accompanied by p53 phosphorylation, as well as Fas, PUMA, survivin, and XIAP modulation. Collectively, the results support an important role of p53 in Gefitinib-induced apoptosis in human lung cancer cells. p53 may induce apoptosis through the regulation of apoptotic (Fas and PUMA) and anti-apoptotic (XIAP and survivin) genes. Our studies not only pave a way to the understanding of pharmacological mechanisms of Gefitinib, but also implicate for the necessity to prescreen p53 expression level before clinical application of Gefitinib in human cancer therapy.