The E3 ubiquitin-protein ligase Nedd4-2 regulates the sodium chloride cotransporter (NCC) but is not required for a potassium-induced reduction of NCC expression

STUDY OBJECTIVE: The activity of the sodium chloride cotransporter (NCC) in the kidney distal convoluted is important for kidney sodium (Na+ ) and potassium (K+ ) handling. NCC abundance is reduced by high plasms K+ levels. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) is involved in regulation of NCC, but its precise role is unclear. The aim of this study was to examine the functional role of Nedd4-2 on NCC regulation and whether Nedd4-2 is involved in the high K+ effects on NCC. HYPOTHESIS: Nedd4-2 regulates NCC membrane abundance by influencing NCC trafficking or degradation and plays an important role in the high K+ induced reduction in NCC. METHODS: In vitro studies were performed using tetracycline inducible MDCKI cell lines stably expressing human NCC combined with Nedd4-2 knockdown using shRNA. The cell lines were characterized using immunoprecipitation coupled to biotin-based membrane abundance assays. The effects of normal or high extracellular K+ on NCC in ex vivo isolated mouse kidney tubules from wildtype or Nedd4-2 knockout (KO) mice were assessed. RESULTS: In MDCK cells the levels of NCC and phosphorylated NCC (pNCC) were increased with Nedd4-2 deletion, and NCC levels on the membrane were elevated. No significant changes were seen for the SPS1-related proline/alanine-rich serine-threonine kinase (SPAK) and phosphorylated SPAK (pSPAK) after Nedd4-2 knockdown. Deletion of Nedd4-2 had no effect on the endocytosis rate of NCC, but NCC half-life was increased. In tubules high K+ reduced total and pNCC regardless of genotype. The levels of the inwardly rectifying potassium channel subunits Kir4.1 and Kir5.1 were increased in Nedd4-2 KO tubules. High K+ treatment decreased the level of Kir4.1, but not Kir5.1, only when Nedd4-2 was eliminated. pSPAK was decreased with high K+ treatment regardless of the genotype. CONCLUSION: Nedd4-2 is involved in ubiquitylation of NCC, regulation of NCC membrane abundance and degradation, but it does not play role in the K+ induced inhibition of NCC. © FASEB.