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

Abstract

Study objectiveThe 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.HypothesisNedd4‐2 regulates NCC membrane abundance by influencing NCC trafficking or degradation and plays an important role in the high K+ induced reduction in NCC.MethodsIn 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.ResultsIn 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.ConclusionNedd4‐2 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.