Kinase Scaffold Cab39 Is Necessary for Phospho-Activation of the Thiazide-Sensitive NCC

Potassium regulates the WNK (with no lysine kinase)-SPAK (STE20/SPS1-related proline/alanine-rich kinase) signaling axis, which in turn controls the phosphorylation and activation of the distal convoluted tubule thiazide-sensitive NCC (sodium-chloride cotransporter) for sodium-potassium balance. Although their roles in the kidney have not been investigated, it has been postulated that Cab39 (calcium-binding protein 39) or Cab39l (Cab39-like) is required for SPAK/OSR1 (oxidative stress response 1) activation. This study demonstrates how they control the WNK-SPAK/OSR1-NCC pathway.

We created a global knockout of Cab39l and a tamoxifen-inducible, NCC-driven, Cab39 knockout. The 2 lines were crossed to generate Cab39-DKO (Cab39 double knockout) animals. Mice were studied under control and low-potassium diet, which activates WNK-SPAK/OSR1-NCC phosphorylation. Western blots were used to assess the expression and phosphorylation of proteins. Blood and urine electrolytes were measured to test for compromised NCC function. Immunofluorescence studies were conducted to localize SPAK and OSR1.

Both Cab39l and Cab39 are expressed in distal convoluted tubule, and only the elimination of both leads to a striking absence of NCC phosphorylation. Cab39-DKO mice exhibited a loss-of-NCC function, like in Gitelman syndrome. In contrast to the apical membrane colocalization of SPAK with NCC in wild-type mice, SPAK and OSR1 become confined to intracellular puncta in the Cab39-DKO mice.

In the absence of Cab39 proteins, NCC cannot be phosphorylated, resulting in a Gitelman-like phenotype. Cab39 proteins function to localize SPAK at the apical membrane with NCC, reminiscent of the Cab39 yeast homolog function, translocating kinases during cytokinesis.