科研之友
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摘要
The present study aimed to determine the molecular mechanisms leading to the production of advanced glycation end-products (AGEs) and their effect on the morphology and function of rat glomerular capillary endothelial cells (GECs). Primary rat GECs were treated with AGE-modified human serum albumin (AGE-HSA) and divided into groups according to AGE concentration and treatment time. The structure and distribution of cytoskeletal protein F-actin and the cortical actin binding protein, cortactin, were analyzed using immunofluorescence and confocal microscopy. As the Ras-related C3 botulinum toxin substrate 1 (Racl) signaling pathway was previously identified to be involved in mediating the contraction of endothelial actin-myosin activity, Racl was examined subsequent to treatment of the cells with the Racl agonist 2'-O-methyladenosine-3',5'-cyclic monophosphate (O-Me-cAMP) for 1 h using a pull-down assay. Cell permeability was determined by the leakage rate of a fluorescein isothiocyanate fluorescent marker protein. AGE-HSA treatment resulted in alterations in the structure and distribution of F-actin and cortactin in a dose- and time-dependent manner, while no effect was observed with HSA alone. The effect of AGE on the cytoskeleton was inhibited by the addition of O-Me-cAMP. AGE-HSA significantly reduced the level of Racl activity (P<0.05); however, no effect was observed on total protein levels. Furthermore, AGE-HSA treatment led to a significant increase in the permeability of endothelial cells (P<0.01), which was inhibited by O-Me-cAMP (P<0.01). The Racl signaling pathway is thus suggested to serve an important function in mediating AGE-induced alterations in GEC morphology and function.
