Physiological changes in plasma [K+] affect NCC function via Cl‐ inhibition of WNK4

Abstract

We have shown that dietary K+ modulates NCC via plasma K+ ([K+]p). [K+]p determines distal convoluted tubule (DCT) membrane potential and therefore, intracellular chloride ([Cl‐]i) (estimated to be near 20 mM). Extracellular [K+] modulates NCC in HEK cells via endogenous WNK1, but HEK [Cl‐]i is higher than DCT [Cl‐]i. WNK4 DCT abundance and redistribution in response to K+ intake suggests that it plays a dominant role in vivo, but this model requires that Cl‐ affects WNK4 at low, physiological [Cl‐]i.To test this, we compared WNK1 and WNK4 kinase activities on SPAK. Although Cl‐ inhibited both WNK1 and WNK4, the half maximal inhibiting [Cl‐] was much lower for WNK4 than WNK1.To confirm that WNK4 Cl‐ sensitivity affects SPAK and NCC activity in cells, we mutated the WNK4 Cl‐‐sensing domain (WNK4 LL‐FF) that is homologous to the WNK1 domain. Although WT WNK4 reduced pSPAK in HEK cells, WNK4 LL‐FF increased it. Kinase‐dead WNK4 LL‐FF returned pSPAK to WT WNK4 levels. Furthermore, WNK4 LL‐FF increased pNCC compared to WT WNK4.To confirm the physiological dominance of this pathway, we tested effects of multiple mouse models, including dietary manipulations (both Na+ and K+), drug treatments (amiloride), and genetic manipulations (MR knockout). A tight relationship between [K+]p and pNCC was observed (pNCC=42.95[K+]‐3.1, R2=0.77), which was linear in the physiological range of [K+]p. Between 5 mM and 3.5 mM, for every 0.1 mM decrease in [K+]p, pNCC increased by 20%.Lastly, dietary K+ titration (0% to 5%) produced a linear increase in [K+]p from 2.9 mM to 3.85 mM. [K+]p and pNCC were also highly correlated (pNCC=‐0.63[K+]+3.2, R2=0.63).WNK4 kinase activity is Cl‐‐sensitive in the range of DCT [Cl‐]i, while WNK1 inhibition requires higher [Cl‐]. pNCC tightly correlates with [K+]p throughout the physiological range in multiple models.