RNA interference for -ENaC inhibits rat lung fluid absorption in vivo

Li, Tianbo, and Hans G. Folkesson. RNA interference for -ENaC inhibits rat lung fluid absorption in vivo. Am J Physiol Lung Cell Mol Physiol 290: L649–L660, 2006. First published October 28, 2005; doi:10.1152/ajplung.00205.2005.—We used siRNA against the -ENaC (epithelial Na channel) subunit to investigate ENaC involvement in lung fluid absorption in rats by the impermeable tracer technique during baseline and after -adrenoceptor stimulation by terbutaline. Terbutaline stimulation of lung fluid absorption increased fluid absorption by 165% in pSi-0-pretreated rat lungs (irrelevant siRNA-generating plasmid). Terbutaline failed to increase lung fluid absorption in rats given the specific -ENaC siRNA-generating plasmid (pSi-4). pSi-4 pretreatment reduced baseline lung fluid absorption by 30%. -ENaC was undetectable in pSi-4-pretreated lungs, regardless of condition but was normal in pSi-0-pretreated lungs. We carried out a dose-response analysis where rats were given 0–200 g/kg body wt pSi-4, and -ENaC mRNA and protein expressions were analyzed. To reach IC50 for -ENaC mRNA expression, 32 g/kg body wt pSi-4 was needed, and to reach IC50 for -ENaC protein expression, 59 g/kg body wt pSi-4 was needed. We tested for lung tissue specificity and found no changes in -ENaC expression, at either mRNA or protein level, as well as no changes in 1-Na-KATPase protein expression. We isolated alveolar epithelial type II cells 24 h after in vivo pSi-4 pretreatment. In these cells, -ENaC mRNA was undetectable, demonstrating that alveolar epithelial ENaC expression was attenuated after intratracheal -ENaC siRNA-generating plasmid DNA instillation. We tested for organ specificity and found no changes in kidney and -ENaC mRNA and protein expression. Thus we provide conclusive evidence that -adrenoceptor stimulation of lung fluid absorption is critically ENaC dependent, whereas baseline lung fluid absorption seemed less ENaC dependent.