Cab39 is the major scaffolding protein for SPAK-mediated NCC activation in the mouse kidney

STE20/SPS1-related proline/alanine-rich kinase (SPAK) phosphorylates the sodium-chloride cotransporter (NCC) to stimulate sodium reabsorption in the Distal Convoluted Tubule (DCT) for K + homeostasis and blood pressure. The scaffold proteins, Calcium-binding protein 39 (Cab39) and Cab39-like (Cab39l) activate SPAK in a Xenopus oocyte expression system. Still, it has not been determined whether Cab39 or Cab39-like are required for NCC phosphorylation in the DCT. Here, we developed DCT-specific Cab39 knockout (KO), global Cab39l-KO, and Cab39/Cab39l double knockout (DKO) mice to assess the role of the two adaptor proteins in the Kidney. The SPAK-NCC signaling axis was evaluated by Western Blot and immunofluorescence microscopy. Physiological consequences of altered signaling were evaluated with blood and urine electrolyte measurements. Cab39-KO mice had significantly lower total NCC (tNCC) and phosphorylated NCC (pNCC) abundance than wild-type mice. Phospho-activation of NCC with low K + diet was also blunted in the Cab39 KO mice. By contrast, the Cab39l-KO mice did not show differences in total or phosphorylated NCC signals. However, when Cab39 was eliminated on the Cab39l KO background (DKO), pNCC abundance further decreased to almost undetectable levels. Remarkably, the DKO did not affect the phospho-activation of SPAK. Instead, SPAK failed to move to the apical membrane and accumulated into cytoplasmic puncta in the DKO mice. In summary, these data indicate that Cab39 and Cab39l act as major and minor regulators of SPAK-NCC signaling, providing an adaptor for translocating SPAK to the apical membrane for NCC interaction and phosphorylation. NIH grant R01DK093501 to Professor Eric Delpire and Professor Paul Welling, The Leducq Foundation grant 17CVD05 to Professor Eric Delpire and Professor Paul Welling, and a Crawford Long Innovation grant to Professor Eric Delpire. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.