Involvement of αENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysis

To explore interactions between the epithelial Na channel (ENaC) and neural precursor expressed, developmentally downregulated protein 4-2 (Nedd4-2) at the conversion of the rat lung from fluid secretion to absorption at birth, we used small-interfering RNA (siRNA) against alpha ENaC and Nedd4-2. siRNA-generating plasmid DNA (pDNA) was administered via trans-thoracic intrapulmonary (ttip) injection 24 h before ENaC and Nedd4-2 expression, extravascular lung water, and mortality were measured. alpha ENaC mRNA and protein were specifically reduced by similar to 65% after pSi-4 injection. Nedd4-2 mRNA and protein were reduced by similar to 60% after pSi-N1 injection. Interestingly, alpha ENaC and alpha ENaC mRNA and protein expression were increased after Nedd4-2 silencing. Extravascular lung water was significantly increased after alpha ENaC silencing and reduced after Nedd4-2 silencing. alpha ENaC silencing resulted in a fourfold increase in newborn mortality, whereas silencing Nedd4-2 did not affect mortality. We also isolated distal lung epithelial (DLE) cells after in vivo alpha ENaC or Nedd4-2 silencing and measured alpha ENaC or Nedd4-2 expression in freshly isolated DLE cells. In these DLE cells, there were attenuated alpha ENaC or Nedd4-2 mRNA and protein, thus demonstrating that alpha ENaC and Nedd4-2 silencing occurred in alveolar epithelial cells after ttip injection. We also looked for pDNA by PCR to determine pDNA presence in the lungs and found strong evidence for pDNA presence in both lungs. Thus we provide evidence that ENaC and Nedd4-2 are involved in the transition from lung fluid secretion to fluid absorption near term and at birth.