WNK bodies are multikinase complexes that facilitate NCC activation during hypokalemia.

Kidney-Specific With-No-Lysine (KS-WNK1) is a distal convoluted tubule (DCT) scaffold protein that serves as a docking site for the WNK-SPAK/OSR1 kinase signaling pathway. During dietary K+ deprivation, KS-WNK1 condenses with the active WNK-SPAK/OSR1 cascade into large spherical membraneless structures in the DCT, termed WNK bodies. We propose that the assembly of these multikinase complexes promotes K+ conservation through the activation of the WNK-SPAK/OSR1 pathway and its downstream target, the sodium-chloride cotransporter (NCC). Mice lacking KS-WNK1 are unable to form WNK bodies and have diminished activation of WNK-SPAK/OSR1 pathway and NCC, resulting in a Gitelman-like phenotype. A critical question is whether the blunted NCC activation in KS-WNK1 KO mice is a direct consequence of the loss of WNK bodies, or a secondary effect from the generalized lack of KS-WNK1. Here we address the issue by using CRISPR-Cas technology to generate a mouse expressing a full-length mutant version of KS-WNK1 that does not form WNK bodies. The mutation was directed at a conserved N-terminal hydrophobic motif (VFVIV -> QQQQQ) within exon 4A, and thus referred to as the "5Q" mouse. We confirmed in HEK293 cells that this mutation to KS-WNK1 abolished WNK body formation. Next, we determiend the function of WNK bodies during 10 days of dietary K+ deprivation in the 5Q mouse versus littermates, assessing changes in WNK-SPAK/OSR1 & NCC activation by immunostaining and western blot, and on electrolyte handling. We found that K+ restricted 5Q mice were unable to form spherical WNK bodies, and instead formed irregularly shaped aggregates around the nucleus and basolateral membrane. These malformed aggregates were enriched in phosphorylated SPAK/OSR1, which was nearly absent from the apical membrane. Consistent with intracellular sequestration of pSPAK/pOSR1, the abundance of pSPAK/pOSR1 was increased in immunoblots of kidney homogenates by 25% (males; p = 0.0007) & 30% (females; p = 0.0012), but phosphorylated NCC abundance (a surrogate for NCC activation) was decreased by 25% (males; p = 0.06) & 70% (females; p = 0.0005). This reduction in phospho-NCC in the 5Q mutants resulted in a Gitelman-like phenotype that was more pronounced in females. Female 5Q mice had a trend towards decreased plasma [K+] (0.4 meq/L reduction; p = 0.1), and significantly decreased [Cl-] (4 meq/L reduction; p = 0.04) and increased [HCO3-] (4 meq/L increase; p = 0.05). In conclusion, the KS-WNK1 5Q mutation causes aberrant WNK body formation, resulting in mislocalization of the WNK-SPAK/OSR1 pathway in K+ deprived mice. While the 5Q mice can activate SPAK, SPAK is unable to leave the dysfunctional aggregates to activate NCC at the apical membrane. Similar to KS-WNK1 KO mice, this results in a Gitelman-like phenotype that predominates in females. These findings demonstrate that KS-WNK1 assembles WNK bodies to serve as functional protein scaffolds for multikinase complexes to enhance NCC activation during hypokalemia.