EnaC Knockdown in the Mouse Lung using RNAI.

Hyde SC, Painter H, Gill DR

Pediatric Pulmonology, 42 S30 306

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The North American Cystic Fibrosis Conference, Anaheim, 2007

Lung pathology in individuals with Cystic Fibrosis (CF) is linked to sodium hyper-absorption. The defective regulation of the epithelial sodium channel ENaC is thought to be a major contributor to reduced Airway Surface Liquid (ASL) volume and impaired mucociliary clearance of the airways. Thus, strategies designed to inhibit ENaC function may result in clinical benefit. RNA interference (RNAi), mediated by short, double-stranded RNA molecules, can be used to target complementary RNA sequences for degradation via the RNA Induced Silencing Complex (RISC). We investigated the possibility of using RNAi to reduce expression of ENaC in the mouse lung, as proof of principle for a strategy to reduce sodium hyper-absorption in the CF lung. Potent siRNA molecules capable of efficient knockdown of ENaC alpha, beta and gamma subunits were identified in an optimised cell culture system utilising mouse kidney M1 cells (50,000 cells per 24-well; 20pmol siRNA complexed with Lipofectamine 2000; 48 hour harvest) in which ENaC expression was quantified using real-time RT-PCR. Approximately 80% knockdown of each ENaC subunit was observed with the most potent siRNA molecules in this system. Subsequently, siRNA molecules were delivered to the lungs of female BALB/c mice via hydrodynamic tail vein injection (40 µg naked siRNA, n=5-6). After 24 hours, lungs were harvested, RNA extracted and ENaC mRNA measured using real-time TaqMan PCR. Whereas, ENaC subunit mRNA levels in mice treated with a negative control siRNA were not different from untreated mice (p < 0.05), delivery of ENaC alpha and beta-specific siRNA molecules resulted in a reduction to 56.5 ± 12.3 % or 33.3 ± 4.7 % of the expression levels observed in untreated mice, respectively (p < 0.05, Mann-Whitney U). Interestingly, despite efficient knockdown in cell culture, in vivo treatment with the most potent ENaC gamma-specific siRNA molecule led to no reduction in ENaC gamma expression compared with untreated controls (p > 0.05). These data show proof of principle that ENaC expression can be reduced in the lung using siRNA. Further work is needed to assess the functional consequences of inhibiting ENaC in the lung.