Nedd4‐2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia

Nedd4‐2, a E3 ubiquitin ligase, regulates epithelial sodium channel (ENaC) mediated transcellular Na+ transport in the collecting duct. We investigated the effect of Nedd4‐2 on the junctional complex and paracellular conductance in mpkCCDc14, a collecting duct cell line. We demonstrate that Nedd4‐2 co‐immunoprecipitated with and reduced the expression of occludin. This interaction was mediated via a conserved PY motif in the C‐terminus of occludin and mutation of this PY motif increased the half life of transfected occludin in HEK293 cells. We demonstrate that Nedd4‐2 ubiquitinates occludin which was not seen with a catalytically inactive form of Nedd4‐2. Overexpression of Nedd4‐2 in mpkCCDc14, reduced occludin expression at the tight junctions (TJs) and increased paracellular conductance consistent with a delay in the formation of TJs. Conversely, siRNA‐mediated knockdown of Nedd4‐2 increased occludin levels and reduced paracellular conductance. In conclusion, our data suggest that Nedd4‐2 interacts with occludin leading to its ubiquitination thereby modulating the assembly of TJs and the regulation of paracellular conductance in the collecting duct. NHLBI and VA Merit