LSC Abstract – Epithelial sodium channel (ENaC) currents and superoxide and hydroxyl formation in influenza H1N1 infected rat primary alveolar type II cells

Rationale: Alveolar type II (ATII) cells actively transport sodium from the alveolar to the interstitial side primarily through the amiloride sensitive sodium channel (ENaC). This property is crucial in acute lung injury by limiting the built up of edematous fluid. Binding of Influenza virus to ATII cells reduces ENaC activity in vitro and alveolar fluid clearance in vivo. Aim: To investigate the effects of Influenza H1N1 virus infection of rat primary ATII cells with respect to ENaC function and ROS formation. Methods: Rat ATII cells were isolated and grown in air-liquid interface. They were then exposed to influenza for 15 min. At 1, 3 and 6 h post-infection, the ENaC current was measured in Ussing chambers. In a different set, cells were treated with a superoxide and hydroxyl radicals spin trap. Supernatant measurements were done by Electron Paramagnetic Resonance (EPR) to assess extracellular oxidative stress. UV-inactivated virus served as control in all cases. RT-PCR verified that it was not replicating and flow cytometry verified that it did not increase apoptosis compared to wild type virus. Results: Infection of rat ATII cells with H1N1 virus resulted in a significant decrease in the ENaC current at 3 and 6 hours post-infection. Interestingly, infection of cells with the UV inactivated virus also resulted in a significant reduction at 6 hours post-infection. EPR measurements showed significantly increased extracellular reactive species at 1,3 and 6 h post-infection. Conclusion: Infection of ATII cells with H1N1 leads to prolonged decline of ENaC function that is accompanied by a sustained extracellular oxidative stress.