Role of megalin in albumin transport across the alveolar epithelium and its dysregulation by transforming growth factor beta
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The acute respiratory distress syndrome (ARDS) displays mortality rates of up to 40 % in approximately 200,000 critically ill patients in the United States annually. The disease is characterized by accumulation of protein-rich edema in the alveolar space due to impaired endothelial and epithelial barrier function in the lung. Of great importance is the clearance of plasma proteins from the alveolar space. Sodium and fluid is cleared from the lung quite rapidly via active transport processes, leading to increased protein concentration in the alveolar compartment. This increased concentration of protein creates an oncotic gradient which further promotes alveolar edema formation, thereby preventing recovery. Albumin clearance and edema resolution are crucial for the patient s survival. However, the mechanisms by which albumin transport is facilitated have not been fully elucidated. In the present study we used primary alveolar epithelial cells and an intact organ model to investigate the mechanisms of protein clearance from the alveolar space. In tight monolayers of alveolar epithelial type II cells and type I-like cells, transport of 125I-albumin was almost completely blocked by competition with unlabeled albumin. Similar results were obtained by assessing the uptake of FITC-albumin by fluorescence microscopy, indicating that albumin transport is facilitated by an active and receptor-mediated transport process. A number of previous studies have reported that clathrin-dependent endocytosis is the main pathway for protein uptake along epithelia, and the multi-ligand receptors megalin and cubilin have been established to facilitate clathrin-dependent endocytosis of proteins in the kidney. To investigate the role of clathrin-dependent endocytosis and megalin in the physiological significance of albumin transport in the lung, we applied receptor associated protein (RAP), an inhibitor of megalin-mediated endocytosis, to our cell monolayers and detected significantly decreased albumin transport rates. Since pharmacological inhibitors may have non-specific effects on unrelated proteins, we employed a genetic approach to further elucidate the role of megalin in albumin uptake by alveolar epithelial cells. We sought to specifically silence the megalin gene by RNA interference. Since primary ATII cells display very low transfection efficiency, we employed cultured RLE-6TN cells, an established in vitro model system for ATII cells. In these cells, gene silencing of megalin resulted in an almost complete block of albumin uptake. Collectively, our findings suggested that under physiological conditions, albumin endocytosis in the alveolar epithelium was mediated by megalin. Importantly, we observed similar protein transport rates in both alveolar epithelial cell types. These findings are of high significance, since type I pneumocytes represent more than 95% of the alveolar surface area. To determine the mechanisms of albumin transport in the pathophysiology of acute lung injury we applied TGF-beta1, a key mediator of ALI/ARDS, to alveolar epithelial cells and to the isolated, ventilated and perfused rabbit lung and detected significantly impaired albumin transport rates (within 30 minutes in cell monolayers and within 120 minutes in the isolated rabbit lung). Furthermore, we observed markedly decreased megalin membrane abundance in response to a pathologically relevant concentration of TGF-beta1 via immunofluorescence. We found evidence that TGF-beta1 activates both subunits of glycogen synthase kinase 3 (GSK3), a kinase that phosphorylates megalin and thereby induces endocytosis of the receptor. Importantly, maximal activation of GSK3 under TGF-beta1 stimulation was observed within minutes, which was in line with the rapid impairment of albumin transport we detected under TGF-beta1 challenge in primary alveolar epithelial cells and in the isolated rabbit lung. When GSK3 activity was inhibited by administration of the specific inhibitor SB 216763, TGF-beta1-mediated downregulation of megalin membrane expression was prevented and albumin transport remained unaffected. These data implied that TGF-beta1-mediated activation of GSK3 induced phosphorylation and subsequent endocytosis of megalin, thereby disrupting albumin transport in the alveolar epithelium. Since inhibition of GSK3 as a therapeutic strategy has been presumed to be beneficial for several diseases, we set out to investigate a potential role for SB 216763 in the therapy of acute lung injury. Treatment of alveolar epithelial cells with the specific GSK3 inhibitor after TGF-beta1 challenge restored normal megalin surface distribution and albumin transport. These findings may suggest a potential role for GSK3 inhibition as a novel therapeutic approach in ALI/ARDS.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler