The Preliminary Study Of High Glucose And Glycation End Products Stimulate Podocytes To Produce Microvesicles

Objective:In this study, we use MPC-5cell cultured in vitro as the cell model, to research whether high glucose and advanced glycation end products state can increase generation of micro vesicles from podocytes and its possible mechanism.Methods:We used MPC-5cell cultured in vitro as the cell model,30mM glucose as stimulate factor of high glucose group and200nM glycation end products as the stimulate factor of AGEs group(stimulate for20hours).We used antioxidants N-Acetyl-L-cysteine (NAC) and alpha lipoic acid(a-LA) as pretreatment factors(1hours in advance).1. ELISA was used to detect the MV content of MPC-5cells.2. Using CM-H2DCFDA as probes,flow cytometry instrument to detect the change of ROS levels after high glucose and glycation end products stimulating MPC-5cells for different time.3. Using MitoSOXT as probes,flow cytometry instrument to detect the change of ROS levels in mitochondria after high glucose and glycation end products stimulating MPC-5cells for different time.4. Western Blot was supplied to detect the impact of high glucose and glycation end products stimulating MPC-5cells on the protein expression of NOX4after different time points.5. Statistical methods:using SPSS17.0statistical software for data processing. Results with the mean+/-standard deviation (x±s) said. More than two groups compare using single factor analysis of variance (ANOVA). The inspection level is a=0.05. P<0.05for the difference was statistically significant.Results:1.The MV quantitative test results:compared with the control group, after30mM glucose and200nM glycation end products stimulation for20hours, the MV generated by MPC-5cells significantly increased (P<0.05).respectively4.6times.7.6times in the control group; After the antioxidant NAC pretreatment for1hour, the MV content in the high glucose+NAC group significantly was lower than the high glucose group (P＜0.05), decreased by52.9%.and AGE+NAC group significantly was also lower than the AGE group (P＜0.05). decreased by48.9%; After alpha lipoic acid pretreatment1hour, the MV content in the high glucose+α-LA group and the high glucose group had no significant change (P>0.05), the MV content in the glycation end products+α-LA group and the glycation end products group had no significant change (P>0.05) too.2. ROS of MPC-5cell test results:After30mM glucose stimulating MPC-5cells for48hour, the generation of ROS reached a peak of about4times compared with the control group(P＜0.05), and then demonstrated a downward trend; After glycation end products stimulating MPC-5cells for48hour, the generation of ROS reached a peak of about5times compared with the control group(P＜0.05), and then demonstrated a downward trend.3. ROS in mitochondria of MPC-5test results:After30mM glucose stimulating MPC-5cells for4hour, the generation of ROS reached a peak of about2times compared with the control group(P＜0.05), and then demonstrated a downward trend, returned to baseline levels at24hours; After glycation end products stimulating MPC-5cells for4hour, the generation of ROS reached a peak of about3times compared with the control group(P＜0.05), and then demonstrated a downward trend, returned to baseline levels at24hours.4. NOX4protein of podocyte test results:Compared with the control group, with high glucose stimulation time extended, the levels of NOX4protein were3.0-fold at4hours,1.9-fold at6hours,1.5-fold at6hours (P＜0.05). The NOX4protein was the strongest at4hours, and then demonstrated a downward trend, returned to baseline levels at24hours. Compared with the control group, with glycation end products stimulation time extended, the levels of NOX4protein were2.0-fold at12hours.3.0-fold at24hours,2.2-fold at48hours,2.1-fold at72hours (P＜0.05). The NOX4protein was the strongest at24hours, and then demonstrated a downward trend.Conclutions:1.High glucose and glycation end products can significantly increase the generation of MV from podocytes. 2.High glucose and glycation end products can get through the oxidative stress pathway to stimulate podocytes cells producing MV.3.High glucose and glycation end products promote the generation of MV from podocyte,which may mainly by increasing the level of ROS from NADPH oxidase(NOX4).