Influenza A H1N1 Inhibits The Ion Transport Of Epithelial Sodium Channels Via With-no-lysine-kinase-4 In Mouse Tracheal Epithelial Cells

Objective:The influenza is a seriously respiratory infection disease.There have been many outbreaks of influenza in human history,including the outbreak in Hong Kong in1968.However,the pathogenesis of influenza virus is not fully understood.The airway surface is the first line of defense barrier against the pathogen from the external environment.Under normal physiological conditions,the airway surface liquid contains highly glycosylated mucins rich in terminal sialic acid,and it could trap pathogens from the environment and eliminate them from respiration system by cilia beating in a mechanical manner.The epithelial sodium channels(ENaC)located on the apical side of airway epithelia are responsible for the trans-epithelial transport of Na~+,which play an important role in maintaining the balance of salt-water reabsorption in airway epithelia.And studies have shown that pulmonary edema is one of the characteristics of acute lung injury caused by influenza virus.We speculate that the influenza virus may destroy the balance of salt-water transport by changing the function of ENaC.The aim of this study was to investigate the effects and mechanism of influenza virus on ENaC.Methods:1.Protein kinase and DNA enzyme were applied to isolate mouse tracheal epithelial cells(MTECs).MTECs were cultured in Transwell until forming a monolayer.2.Proliferation and tittering of influenza virus were performed in chicken embryo and Madin-Darby canine kidney cells.3.Observe the submicroscopic structure of MTECs by transmission electron microscopy and conform whether influenza virus disrupts the tight junctions of MTECs.4.We applied Ussing chamber system to detect the effects of influenza virus on ENaC.5.Real-time polymerase chain reaction and Western blot method were used respectively to detect mRNA levels and the protein expression of ENaC subunits.6.The height and viscosity of the airway surface liquid were measured by confocal mircroscope.7.Effect of influenza virus on with-no-lysine-kinase 4(WNK4)was verified by ELISA and western blotting.8.siRNA approach was applied for with-no-lysine-kinase-4 knockdown.Results:1.Amiloride-sensitive short-circuit-current was significantly reduced by influenza virus.2.Influenza virus inhibited ENaC activity by suppressing the ENaC protein and mRNA expression level.3.Influenza virus could increase the expression level of WNK4 in a time dependent manner.4.With-no-lysine-kinase-4 knockdown alleviate the suppression of ENaC by influenza virus.Conclusions:Influenza virus impairs the fluid transport in airway by disturbing ENaC function via WNK4 pathway.