Hypoxia Affects Alveolar Epithelial Na~+ Transport Through ERK/NF-κB Signaling Pathway

Objective:Hypoxia is associated with many respiratory diseases,including acute lung injury and acute respiratory distress syndrome,which are characterized by dyspnea,destruction of the alveolar capillary barrier,pulmonary edema,and decreased lung volume.The formation of hypoxic environment is partly attributed to the accumulation of edema fluid and mucus on the surface of alveolar epithelial cell(AEC),which forms the oxygen delivery barriers and disrupts the balance of ion transport.Epithelial sodium channel(ENaC)on the apical side of AEC plays a crucial role to maintain the electrochemical gradient of Na~+and water reabsorption,thus becomes the key point for edema fluid removal under hypoxia.Transepithelial clearance of edema fluid driven by epithelial ion transport in turn alleviates the hypoxic state of AEC.The aim of this study was to explore the imbalance of ion transport and its possible mechanisms under hypoxia in vivo and in vitro.Methods:1.Excess volume of culture medium was added on the surface of AEC to simulate the hypoxic environment of alveoli in the state of pulmonary edema by limiting the oxygen delivery to cells,supported by the evidence of increased hypoxia-inducible factor-1 expression,which is an indicator of hypoxia.2.After the hypoxic model was established successfully,the changes of ENaC protein and m RNA expression under different volume of culture medium in different time were detected to explore the effect of hypoxia.3.Human A549 cells and primary mouse alveolar type 2 epithelial cells were used in parallel experiments to explore the expression of AEC-related pathway under hypoxia by Western blot,such as extracellular signal-regulated kinase(ERK)and nuclear factorκB(NF-κB).4.To investigate the relationship between ERK and NF-κB signaling pathway,the phosphorylation of NF-κB inhibitorαand p65 was detected by Western blot.Moreover,the related mechanism of hypoxia on AEC ion transport was detected by qRT-PCR and immunofluorescence assay.5.The hypoxic animal model was established by hypoxia incubator,and the alveolar fluid clearance and lung wet/dry weight ratio were used to detected whether hypoxia affected the alveolar epithelial ion transport through NF-κB signaling pathway in vivo.6.HE staining was applied to study the effect of NF-κB on histopathology changes in the hypoxic mouse lung tissue.7.The effect of hypoxia and NF-κB inhibitor on amiloride-sensitive short-circuit current of H441 cell monolayers and tracheal epithelium was detected by Ussing chamber experiment.Results:1.The expression of hypoxia-inducible factor-1 was upregulated,which proved the feasibility of hypoxia mode establishment.2.The expression levels of ENaC protein and m RNA in A549 cells were decreased under hypoxia.3.In the parallel experiment of A549 cells and mouse alveolar type 2 epithelial cells,hypoxia induced the decrease of ENaC protein/m RNA expression,while ERK and NF-κB signaling pathways were proved to be activated under hypoxia.4.The phosphorylation of NF-κB inhibitorαand p65 decreased after ERK inhibition,suggesting that NF-κB as a downstream pathway of ERK signaling pathway.Moreover,both ERK and NF-κB inhibitors could inhibit the decreased expression ofα-ENaC under hypoxia.5.The intervention of NF-κB signaling pathway could alleviate the symptoms of pulmonary edema in mice under hypoxia.6.NF-κB signaling pathway might mediate hypoxia-induced lung injury in mice.7.When permeabilized H441 cell monolayers and the mice were exposed to hypoxia,the amiloride sensitive short-circuit current of tracheal epithelium decreased significantly,which could be alleviated after the inhibition of NF-κB.Conclusion:Under hypoxic conditions induced by excessive culture medium,ENaC expression levels were reduced in AEC,which might be mediated by ERK/NF-κB signaling pathway,thereby inhibiting alveolar epithelial ion transport and accelerating the formation of edema fluid.