PULMONARY ENDOTHELIAL CELL ACTIVATION DURING EXPERIMENTAL ACUTE KIDNEY INJURY
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AbstractAcute kidney injury (AKI) leads to increased lung microvascular permeability, leukocyte infiltration and upregulation of soluble inflammatory proteins in rodents. Most work investigating connections between AKI and pulmonary dysfunction, however, has focused on characterizing whole lung tissue changes associated with AKI. Studies at the cellular level are essential to understanding the molecular basis of lung changes during AKI. Given that the pulmonary microvascular barrier is functionally abnormal during AKI, we hypothesized that AKI induces a specific pro-inflammatory and pro-apoptotic lung endothelial cell (EC) response. Four and 24 hours after kidney ischemia/reperfusion injury (IRI) or bilateral nephrectomy (BNx), murine pulmonary endothelial cells were isolated via tissue digestion followed by magnetic bead sorting. Purified lung ECs were analyzed for changes in mRNA expression using real time “Superarray” PCR analysis of genes related to endothelial cell function. In parallel experiments, confluent rat pulmonary microvascular ECs were treated with AKI or control serum to evaluate functional cellular alterations. Immunocytochemistry and FACS analysis of Annexin V/PI staining were employed to evaluate cytoskeletal changes and promotion of apoptosis. Isolated murine pulmonary endothelial cells exhibited significant changes in expression of gene products related to inflammation, vascular reactivity and programmed cell death. Further experiments using an in vitro rat pulmonary microvascular EC system revealed that AKI serum induced functional cellular changes related to apoptosis, including structural actin alterations and phosphatidylserine translocation. Analysis and segregation of both upregulated and downregulated genes into functional roles suggests that these transcriptional events likely participate in the transition to an activated pro-inflammatory and pro-apoptotic endothelial cell phenotype during AKI. Further mechanistic analysis of EC-specific events in the lung during AKI might reveal potential novel therapeutic targets for the deleterious kidney-lung crosstalk in the critically ill patient.