| BackgroundDiabetic nephropathy (DN) is the most common complicationin of patients with diabetes. The main pathological changes of diabetic nephropathy is glomerular micro vascular disease. Diabetic nephropathy belongs to the microvascular complications of diabetes mellitus. The overall effect and the long-term prognosis of diabetic nephropathy is poor, is the leading cause of death in diabetic patients, Once diabetes mellitus developed to diabetes complicated with renal damage, the majority of patients will soon progressed to end-stage renal disease (ESRD). Once the development to ESRD, more than other kidney disease treatment more difficult, in addition to end-stage renal disease with higher medical costs, not only brings heavy economic burden to the families of the patients, but also bring heavy burden to society and the country, therefore actively to clarify its pathogenic mechanism, the search for new therapeutic targets, the development of new drugs, making better therapeutic effects prevention measures is particularly important and urgent.The pathogenesis of diabetic nephropathy is not clear, and its pathogenesis is complex. In recent years the domestic and foreign research that long-term formation in high glucose environment a lot of advanced glycosylation end products (advanced in glycationend products AGEs) play a key role in the progress of the whole course of disease. In a landmark event of diabetic nephropathy is the change of glomerular endothelial vascular permeability, the increasing permeability of renal capillary endothelial cells play a key role in the process of proteinuria, and increased vascular permeability mainly achieved by transcellular way, the intercellular damage, cell shrinkage, cell the gap enlarge, the formation of paracellular pathway, leading to vascular endothelial permeability increases which make fluid and macromolecules outflow. Further study found that endothelial junction destruction and the intercellular cleft formation is one of the important ways to increase vascular permeability and big molecular material outflow the vessel wall. Cytoskeletal proteins and connexin of endothelial cells is an important material foundation of the endothelial cell barrier. Endothelial cytoskeleton especially actin in various pathogenic factors stimulating the reorganization and redistribution is cause contraction of endothelial cells, intercellular crack formation, the permeability of the pathological basis. Assembly and remodeling of the actin cytoskeleton is regulated by multiple factors, of which the Rac signal is the key part of the control, while Rac is involved in the regulation of the specific mechanism is still not clear, so it is worth studying.Studies have shown that AGEs can increase the permeability of microvascular endothelial cells, the AGEs in a dose and time dependent manner cause increased permeability of vascular endothelial cells, Therefore, we hypothesized that AGEs should also can increase permeability of glomerular vascular endothelial cell, which further leads to the occurrence of proteinuria, but the domestic research in this area is less, so it is very worthy of further study. In addition, the more important thing to carry out the research is of great significant to clarify the role of AGEs in the pathogenesis and progression of diabetic nephropathy, is conducive to find the new therapeutic target for the treatment of diabetic nephropathy.we carry on the following research to clarify the following ideas:(1) whether AGEs can induce an increase in glomerular endothelial cell permeability?(2) Whether regulating the permeability of AGEs is varied by adjusting the change of glomerular endothelial cytoskeletal proteins F-actin and cortactin?(3) whether the AGEs through the Rac signaling pathway to regulate the permeability of glomerular endothelial cell?In order to illustrate the above problem,we carry on study as follows:1. To establish the isolation and culture of primary rat glomerular endothelial cell method.2. To change the situation changes and cytoskeletal proteins F-actin and cortactin morphology of the increasing permeability of glomerular endothelial cells after AGEs stimulation.3. To observe The Racl activity of glomerular endothelial cells after AGEs stimulation. To investigate the increasing permeability of glomerular endothelial cell after AGEs stimulation, is whether related with Racl signaling pathway.ObjectiveThe purpose of this study is to use the cell culture in vitro to clarify: The effect of AGEs on the permeability of rat glomerular endothelial cells (GECs) monolayer in vitro, change of morphology and function of structure and distribution of cytoskeletal proteins on the rat GECs Stimulated by AGEs. Whether the mechanism of AGEs mediated changes of rat GECs permeability is associated with Racl signal pathway? Further to provide a new target for clinical prevention and treatment of DN and thinking.MethodIn vitro cultured primary rat GECs, different concentrations of AGEs-HSA stimulating rat GECs with different time, and established the control group for comparison. To observe the structure and distribution of cytoskeleton F-actin, Cortical actin binding protein (Cortactin) through immunofluorescent staining and laser scanning confocal microscope technology.At the same time, Detecting the changes of Racl activity by the pull-down assay technology;With thransendothelial electrical resistance of TER and FITC fluorescent marker protein average permeability coefficient to detect the permeability of GECs monolayer cell.Result1. Rat primary GECs were isolated and cultured in vitro finely and identified by the inverted microscope, staining with FITC-BSI.2. AGEs stimulation after incubation for8hours, with the increase of AGEs concentration and the mean value of TER of GECs cell monolayers gradually decreased, but the average permeability coefficient of FITC-BSA gradually increased. The results suggest that the permeability of GECs cell monolayer increased with concentration of AGEs increased, showed the dose dependence.Concentrations of100μg/ml AGEs stimulated GECs cells monolayer after incubation, with stimulation time of AGEs prolonged the mean value of TER of GECs cells monolayer decreased gradually, the average permeability coefficient FITC-BSA increased, suggested that permeability of GECs cell monolayer increased gradually with thestimulation time of AGEs increases, showed the time dependence.3. Under normal conditions, endothelial cells are smooth and intact. F-actin is filamentous among the long axis and near cell junctions, and is distributed as reticular, intact, and continuous lines, mainly in cell edges and inner membranes. Some reticular nuclear matrix is also present in surrounding the nucleus. Cortactin is mainly distributed in the cytoplasm, and sometimes is also present in the cell membrane. With an increase in AGE-HAS treatment concentration or time, the F-actin peripheral dense band edge gradually changed to rough and irregular, with a jagged-like structure. F-actin was diffusely distributed in cells, and the stress fibers, composed of a single row of non-polar actin filaments, were gradually increased. The distribution of cytoplasmic cortactin gradually became disorganized, and became unclear in the cortical area and membrane. Cells became round and retracted when treated with AGE-HAS for8hours at a concentration of100μg/mL; however, HAS did not exhibit these effects.4. Reconstruction of the structure of F-actin, formation of central stress fibers, and retraction in GECs induced by AGE-HAS were dramatically inhibited by pre-treatment with O-Me-cAMP. In addition, similar inhibitory effects were noted in GE-HAS-induced cortactin disorganization and cell retraction when pre-treated with O-Me-cAMP; however, HAS did not exhibit an effect in this process. These findings further demonstrate that the Racl signaling pathway is involved in AGE-induced morphologic and structural changes in GECs.5. The level of Racl activity, but not total Racl level, in AGE-HSA (100μg/ml) treatment group was significantly decreased when compared to the control group; however, HAS did not exhibit this effect. These results suggest that the Racl signaling pathway plays an important role in mediating the function of AGE-induced functional changes in endothelial cells.6. The permeability coefficient Pa of FITC-BSA of Single layer GECs was significantly enhanced and the TER of GECs was significantly decreased when treated with AGE-HSA,. but This effect was inhibited by O-Me-cAMP.Conclusion 1. The results suggest that the permeability of GECs cell monolayer increased with concentration of AGEs increased, showed the dose dependence and the time dependence.2. AGEs can affected The morphological and functional changes of rat GECs by changing the cytoskeletal protein F-actin and cortactin structure and distribution. Which affected its permeability changes.3. Racl signaling pathway plays an important role in the morphological and functional changes of GECs mediated by AGEs. |
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