Deletion of Cab39 adaptors results in KS-WNK1 independent accumulation of SPAK in biomolecular condensates

The sodium-chloride cotransporter (NCC) in the distal convoluted tubule regulates renal sodium reabsorption, potassium homeostasis, and blood pressure. NCC phosphorylation is controlled by extracellular potassium via the WNK-SPAK kinase cascade, proposed to initiate within biomolecular condensates termed WNK bodies. KS-WNK1 and Cab39 play different roles in WNK body formation and dissolution. While KS-WNK1 is required for organizing WNK bodies, previous studies suggest Cab39 promotes SPAK translocation from the WNK bodies to the apical membrane; Cab39 deletion traps phosphorylated SPAK in intracellular puncta and suppresses NCC phosphorylation. Whether these puncta represent bona fide WNK bodies or distinct condensates is unknown. To test whether SPAK puncta require KS-WNK1, we generated mice with DCT-specific deletion of both Cab39 isoforms and KS-WNK1 (triple knockout). NCC, phosphorylated SPAK, and WNK4 expression and localization were assessed by immunoblotting, immunofluorescence, and qPCR under low- and high-potassium diets that activate or inhibit the WNK-SPAK-NCC pathway. Despite elevated WNK4, triple-knockout mice exhibited marked NCC hypo-phosphorylation. Phosphorylated SPAK accumulated in cytoplasmic puncta resembling WNK bodies even without KS-WNK1, indicating they are distinct from canonical WNK bodies. Under high-potassium conditions when WNK4 and SPAK are dephosphorylated, these puncta were absent, suggesting dependence on upstream kinase activity. Thus, SPAK puncta form independently of KS-WNK1, previously considered necessary for WNK body formation, revealing distinct signaling condensates.