Hypoxia-Induced Inhibition of Epithelial Na+ Channels in the Lung Role of Nedd4-2 and the Ubiquitin-Proteasome Pathway

Transepithelial sodium transport via alveolar epithelialNa(+)channels (ENaC) and Na+, K+-ATPase constitutes the driving force for removal of alveolar edema fluid. Alveolar hypoxia associated with pulmonary edema may impair ENaC activity and alveolar Na+ absorption through a decrease of ENaC subunit expression at the apical membrane of alveolar epithelial cells (AECs). Here, we investigated the mechanism(s) involved in this process in vivo in the beta-Liddle mouse strain mice carrying a truncation of beta-ENaC Cterminus abolishing the interaction between beta-ENaC and the ubiquitin protein-ligase Nedd 4-2 that targets the channel for endocytosis and degradation and in vitro in rat AECs. Hypoxia (8% O-2 for 24 h) reduced amiloride-sensitive alveolar fluid clearance by 69% in wild-type mice but had no effect in homozygous mutated beta-Liddle littermates. In vitro, acute exposure of AECs to hypoxia (0.5-3% O-2 for 1-6 h) rapidly decreased transepithelial Na+ transport as assessed by equivalent short-circuit current Ieq and the amiloride-sensitive component of Na+ current across the apical membrane, reflecting ENaC activity. Hypoxia induced a decrease of ENaC subunit expression in the apical membrane of AECs with no change in intracellular expression and induced a 2-fold increase in alpha-ENaC polyubiquitination. Hypoxic inhibition of amiloridesensitive I-eq was fully prevented by preincubation with the proteasome inhibitorsMG132and lactacystin or with the antioxidant N-acetyl-cysteine. Our data strongly suggest that Nedd4-2-mediated ubiquitination of ENaC leading to endocytosis and degradation of apical Na+ channels is a key feature of hypoxia-induced inhibition of transepithelial alveolar Na+ transport.