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Abstract: FR-OR27

Calcium-Binding Protein 39 (Cab39) Is Required for Na+-Cl- Cotransporter (NCC) Phosphorylation in Mice

Session Information

Category: Fluid, Electrolytes, and Acid-Base Disorders

  • 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Ferdaus, Mohammed Zubaerul, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Welling, Paul A., Johns Hopkins Medicine, Baltimore, Maryland, United States
  • Delpire, Eric J., Vanderbilt University Medical Center, Nashville, Tennessee, United States
Background

The Na+-Cl- cotransporter (NCC), expressed along the distal convoluted tubule (DCT) in the kidney, is essential for sodium and potassium homeostasis and blood pressure (BP) regulation. We showed previously that WNK4 (with-no-lysine kinase 4) phosphorylates and activates SPAK (STE20/SPS1-related proline/alanine-rich kinase), which in turn phosphorylates and activates NCC. According to in vitro studies, calcium-binding protein 39 (Cab39) is a scaffold that facilitates SPAK activation by stabilizing a fully active kinase conformation. Cab39 and its homolog, Cab39-like (Cab39l), are both are expressed in the DCT. A previous study published in abstract form showed that Cab39l-knockout (Cab39l-KO) mice did not exhibit differences in electrolyte homeostasis or BP compared with wild-type mice. Here we created a Cab39/Cab39l double knockout mouse to test if the adaptor proteins are necessary for WNK-SPAK-NCC signaling in vivo.

Methods

Inducible NCC-Cre was used to remove Cab39 along the DCT, creating DCT-specific double knockout (DKO) mice against a background of global Cab39l-KO mice. With blood electrolyte analysis, protein quantification and localization analyses were carried out using Western blotting and immunofluorescence, respectively in wild-type or single Cab39l-KO and Cab39/Cab39l double knockout (DKO) mice.

Results

We showed that phosphorylated NCC abundance, a proxy for NCC function, and total NCC abundance were significantly lower in DKO mice. Despite having higher levels of WNK4 and equal levels of total and phosphorylated (active) SPAK, DKO mice on a low K+ diet, a known activator of NCC function, had lower blood [K+] and significantly less phosphorylated and total NCC abundance. Importantly, SPAK was distributed in puncta in the DKO mice, compared to the apical membrane of wild-type mice.

Conclusion

Our study demonstrates that Cab39 is critical for SPAK phosphorylation and localization and NCC function. Deletion of Cab39/Cab39l along the DCT prevents SPAK activation and causes SPAK to be confined to cytosolic puncta. Mice lacking Cab39 have compromised NCC function, reminiscent of Gitelman syndrome.

Funding

  • NIDDK Support