Abstract
Most patients with acute lung injury (ALI) have reduced alveolar fluid clearance that has been associated with higher mortality. Several mechanisms may contribute to the decrease in alveolar fluid clearance. In this study, we tested the hypothesis that pulmonary edema fluid from patients with ALI might reduce the expression of ion transport genes responsible for vectorial fluid transport in primary cultures of human alveolar epithelial type II cells. Following exposure to ALI pulmonary edema fluid, the gene copy number for the major sodium and chloride transport genes decreased. By Western blot analyses, protein levels of alphaENaC, alpha1Na,K-ATPase, and cystic fibrosis transmembrane conductance regulator decreased as well. In contrast, the gene copy number for several inflammatory cytokines increased markedly. Functional studies demonstrated that net vectorial fluid transport was reduced for human alveolar type II cells exposed to ALI pulmonary edema fluid compared with plasma (0.02 +/- 0.05 versus 1.31 +/- 0.56 microl/cm2/h, p < 0.02). An inhibitor of p38 MAPK phosphorylation (SB202190) partially reversed the effects of the edema fluid on net fluid transport as well as gene and protein expression of the main ion transporters. In summary, alveolar edema fluid from patients with ALI induced a significant reduction in sodium and chloride transport genes and proteins in human alveolar epithelial type II cells, effects that were associated with a decrease in net vectorial fluid transport across human alveolar type II cell monolayers.
Highlights
Our results demonstrate that acute lung injury (ALI) pulmonary edema fluid contains soluble factors that are capable of adversely affecting the resolution of pulmonary edema in patients who developed ALI from sepsis
Transmission Electron Microscopy—To examine the ultrastructure of the human alveolar epithelial type II cells exposed to ALI pulmonary edema fluid or plasma, treated human type II cell monolayers grown on Transwell membranes were fixed with 3% (w/v) Karnovsky fixative for 1 h at 0 °C, and the membranes were removed
Effect of ALI Pulmonary Edema Fluid on Net Fluid Transport— Net fluid transport was essentially zero for human alveolar type II cells exposed to ALI pulmonary edema fluid compared with simultaneously collected plasma (0.02 Ϯ 0.05 versus 1.31 Ϯ 0.56 l/cm2/h, mean Ϯ S.D., p Ͻ 0.02)
Summary
Isolation of Human Alveolar Type II Pneumocytes—Type II epithelial cells were isolated from human donor lungs (preserved at 4 °C for 4 – 8 h) that were declined for transplantation by the California Transplant Donor Network using established methods (9). 72 h after the isolation, the type II cells were washed once with PBS and exposed to 1 ml of ALI pulmonary edema fluid with and without inhibitors or the corresponding plasma for 24 h. Human type II cells, seeded on Transwell plates for 5 days, were exposed to plasma or pulmonary edema fluid with and without SB202190 from patients with ALI for 24 h and fixed in 4% formaldehyde for 30 min. Transmission Electron Microscopy—To examine the ultrastructure of the human alveolar epithelial type II cells exposed to ALI pulmonary edema fluid or plasma, treated human type II cell monolayers grown on Transwell membranes were fixed with 3% (w/v) Karnovsky fixative for 1 h at 0 °C, and the membranes were removed. Inhibitor Studies—To test the mechanisms through which ALI edema fluid decreased gene and protein expression, human type II cells were exposed to five inhibitors individually 30 min prior to exposure to ALI pulmonary edema fluid or correspond-
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