The Effect of WNK4 on the Na+:Cl‐ Cotransporter is modulated by Intracellular Chloride

Abstract

IntroductionThe effect of WNK4 on the renal Na+:Cl‐ cotransporter, NCC is controversial since several studies in vitro and in vivo have shown negative or positive effects. We hypothesize that both effects coexist and are modulated by the intracellular chloride concentration ([Cl‐]i). Piala et al. (2014) showed that L‐WNK1 is a Cl‐‐sensitive kinase and identified two leucine residues forming the Cl‐‐binding site. These leucines are conserved in WNK isoforms.Materials and MethodsWe used the functional expression system of Xenopus oocytes and HEK293 cells. NCC activity was measured by 22Na+ uptake and/or amino acid threonine phosphorylation in the absence or presence of wild type or mutant WNK4, in control conditions, or after [Cl‐]i depleting maneuvers. WNK4 autophosphorylation was also assessed in these conditions.ResultsAt baseline [Cl‐]i ∼ 45 mM, WNK4 autophosphorylation was undetectable and NCC activity was either inhibited or unaffected. [Cl‐]i reduction (25‐30 mM) promoted autophosphorylation transforming WNK4 to an activator of NCC in a kinase‐dependent manner. The L322F substitution transformed WNK4 into a constitutively autophosphorylated kinase that activated NCC without chloride depletion.ConclusionWNK4 is Cl‐ sensitive kinase exerting differential effects on NCC depending on the [Cl‐]i. It is possible that small changes in [Cl‐]i in distal convoluted tubule cells may have a crucial role determining WNK4 effect on NCC, suggesting that diverse physiological processes could converge in the Cl‐modulation of WNK's.