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The prevailing view is that ClC-Ka chloride channel (mouse Clc-k1) functions in thin ascending limb for urine concentration, whereas ClC-Kb (mouse Clc-k2) in thick ascending limb (TAL) for salt reabsorption, respectively. Mutations of ClC-Kb cause classic Bartter syndrome with renal salt wasting with onset from perinatal to adolescent.We study the roles of Clc-k channels in perinatal mouse kidneys using constitutive or inducible kidney-specific gene ablation and 2-D and advanced 3-D imaging of optically cleared kidneys. We show that Clc-k1 and -k2 are broadly expressed and colocalized in perinatal kidneys. Deletion of Clc-k1 and -k2 reveals that both participate in NKCC2-and NCC-mediated NaCl reabsorption in neonatal kidneys. Embryonic deletion of Clc-k2 causes tubular injury and impairs renal medulla and TAL development. Inducible deletion of Clc-k2 begins after medulla maturation produces mild salt wasting resulting from reduced NCC activity. Thus, both Clc-k1 and -k2 contribute to salt reabsorption in TAL and DCT in neonates, potentially explaining less severe phenotypes in classic Bartter. As opposed to the current understanding that salt wasting in adult Bartter patients is due to Clc-k2 deficiency in adult TAL, our results suggest that it is mainly originated from medulla and TAL defects during development. Table 2. Plasma and urine biochemistries of 8-week-old Clc-k1-null and Clc-k2-null mice WT Clc-k1 -/-WT Clc-k2 -/-Plasma BUN (mg/dL) 29.7±1.6 26.6±2.0 30.4±1.7 63.6±3.0** Creatinine (mg/dL) 0.25±0.03 0.29±0.03 0.26±0.01 0.39±0.03** Table 3 Plasma and urine biochemistries in 10-week-old inducible Clc-k2 deficient mice.

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.

Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) responding to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs of the brain (CVOs) focus on mechanosensitive membrane proteins. The present study demonstrated that an 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. Neuronal-specific knockout (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-indued 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.