Control of the Renal WNK Signaling Pathway by Phase Transitions

With-No-Lysine (WNK) kinases are ubiquitous regulators of cation chloride cotransport. Disease-causing mutations of these kinases cause salt-sensitive hypertension, hyperkalemia, and type IV renal tubular acidosis, indicating their importance in kidney tubular function. Despite advances in our understanding of the WNK signaling pathway, fundamental questions remain. The most significant knowledge gap concerns the mechanism by which WNK kinases are activated by cell volume changes. Recently, we reported that the “Long” kinase- active form of WNK1 (L-WNK1) activates within biomolecular condensates during hypertonic cell shrinkage. These membraneless liquid-like assemblies form via phase separation, induced by macromolecular crowding. This process bypasses the inhibitory effects of increased intracellular chloride caused by exosmosis, rapidly triggering net ion influx and volume recovery. The importance of protein phase behavior in WNK signaling is further supported by our work in distal convoluted tubule (DCT). In this nephron segment, KS-WNK1, a truncated WNK1 isoform that likely functions as a scaffold for phase transitions, drives the formation of specialized biomolecular condensates termed WNK bodies. These structures form in the DCT during hypokalemia, and their presence correlates with the phosphorylation-dependent activation of the thiazide-sensitive NaCl cotransporter NCC. This suggests a relationship between extracellular K+ sensing, WNK phase behavior, and distal nephron salt handling. Here, we will test the hypothesis that WNK kinases undergo phase transitions to respond to their environment and amplify ion transport. Aim 1 will determine how phosphorylation-dependent charge switching of the L-WNK1 C-terminus activates ion flux and volume recovery. Aim 2 will investigate how intracellular chloride and crowding-induced phase separation interact to control WNK activity. Aim 3 will determine if DCT WNK body condensates are necessary for NCC activation, and whether disrupting WNK bodies reverses Familial Hyperkalemic Hypertension. This proposal combines the complementary expertise of two leaders in the WNK signaling field. The MPIs will use a multifaceted and innovative approach that includes mouse models of altered WNK body function, newly developed live cell imaging methods, and Drosophila Malpighian tubule, a tractable and genetically malleable model of the nephron. The discovery that WNK kinases function as physiological crowding sensors marks a conceptual advance that has enabled fresh thinking and logical testable hypotheses, viewed through the lens of condensate biology. Thus, we expect that the knowledge gained from these studies will transform our understanding of epithelial cell volume regulation and ion transport, while testing the functional contributions of biomolecular condensates to kidney physiology and disease.