title: Regulation of ENaC-mediated alveolar fluid clearance by insulin via PI3K/Akt pathway in LPS-induced acute lung injury.
abstract: BACKGROUND
  Stimulation of epithelial sodium channel (ENaC) increases Na(+) transport, a driving force of alveolar fluid clearance (AFC) to keep alveolar spaces free of edema fluid that is beneficial for acute lung injury (ALI). It is well recognized that regulation of ENaC by insulin via PI3K pathway, but the mechanism of this signaling pathway to regulate AFC and ENaC in ALI remains unclear. The aim of this study was to investigate the effect of insulin on AFC in ALI and clarify the pathway in which insulin regulates the expression of ENaC in vitro and in vivo.
  
  
  METHODS
  A model of ALI (LPS at a dose of 5.0 mg/kg) with non-hyperglycemia was established in Sprague-Dawley rats receiving continuous exogenous insulin by micro-osmotic pumps and wortmannin. The lungs were isolated for measurement of bronchoalveolar lavage fluid(BALF), total lung water content(TLW), and AFC after ALI for 8 hours. Alveolar epithelial type II cells were pre-incubated with LY294002, Akt inhibitor and SGK1 inhibitor 30 minutes before insulin treatment for 2 hours. The expressions of α-,β-, and γ-ENaC were detected by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting.
  
  
  RESULTS
  In vivo, insulin decreased TLW, enchanced AFC, increased the expressions of α-,β-, and γ-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI, the effects of which were blocked by wortmannin. Amiloride, a sodium channel inhibitor, significantly reduced insulin-induced increase in AFC. In vitro, insulin increased the expressions of α-,β-, and γ-ENaC as well as the level of phosphorylated Akt but LY294002 and Akt inhibitor significantly prevented insulin-induced increase in the expression of ENaC and the level of phosphorylated Akt respectively. Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway.
  
  
  CONCLUSIONS
  Our study demonstrated that insulin alleviated pulmonary edema and enhanced AFC by increasing the expression of ENaC that dependent upon PI3K/Akt pathway by inhibition of Nedd4-2.
authors.affiliation: Department of Respiratory Medicine, Second Affiliated Hospital of Chongqing Medical University, Yuzhong District, Chongqing, China.
authors.forename: Wang
authors.initials: W
authors.lastname: Deng
authors.forename: Chang-Yi
authors.initials: CY
authors.lastname: Li
authors.forename: Jin
authors.initials: J
authors.lastname: Tong
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Dao-Xin
authors.initials: DX
authors.lastname: Wang
mesh_terms: D055371:Acute Lung Injury / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D002478:Cells, Cultured; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D066298:In Vitro Techniques; D007328:Insulin / Q000097:blood / Q000494:pharmacology*; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000009:adverse effects; D008297:Male; D023421:Models, Animal; D000075702:Nedd4 Ubiquitin Protein Ligases; D009195:Peroxidase / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D001786:Blood Glucose; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D007328:Insulin; D015850:Interleukin-6; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009195:Peroxidase; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618484:Nedd4 protein, rat; D044767:Ubiquitin-Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt