Study Of The Mechanism Of High Glucose Provokes The Generation Of Microvesicles From Podocytes

Objective:To investigate the influence of high glucose provokes the generation of microvesicle from cultured mouse glomerular podocytes and preliminary discussion on its mechanism.Methods:Mouse podocyte clone5(MPC-5) were cultured in vitro.The Zymuphen MP-Activity ELISA kit was used to quantify total PS-positive MVs generated from podocytes. The rate of podocyte apoptosis was evaluated by flow cytometry. The intracellular ROS of podocytes were measured by fluorescence microscopy.While the expression of p-ERK1/2/ERKl/2, p-p38/p38and p-JNK/JNK in podocyte were studied by Western blotting method.All the data were analyzed by SPSS16.0. Comparisons amongst three or more groups were performed using one-way analysis of variance (ANOVA),followed by the Tukey’s Honestly Significant Difference(Tukey)test, with p<0.05considered significant. Image-Pro Plus6.0was used to analysis fluorescence intensity. The western blotting images were analyzed by Image J.Result:1. The effects of high glucose on the production of MVs from podocytes:with extended incubation time of high glucose, also increased the MVs, which in a time-dependent manner. The numbers of MVs released from podocytes were4-fold at24h,12-fold at48h compared with that for11.1mM D-glucose, respectively (p<0.01).2. The effects of high glucose on apoptosis of podocytes:Treatment of differentiated mouse podocytes with high glucose increased the rates of apoptosis significantly (almost increased by1-fold) compared with the control level of D-glucose. Unlike D-glucose, mannitol did not increase the rates of apoptosis of podocytes.3. The effects of high glucose on production of ROS in podocytes:treatment of differentiated mouse podocytes with30mM high D-glucose increased DCF-sensitive intracellular ROS significantly compared with the control level of D-glucose. D-glucose induced intracellular ROS in a time-dependent manner. Semi-quantitative analysis revealed that ROS generation by high glucose was1.4-fold at12h(p<0.01),1.7-fold at24h (p<0.01),2.3-fold at48h (p<0.01)and1.7-fold at72h (p<0.01) compared with that for11.1mM D-glucose as measured by fluorescence microscopy.4. The effects of glucose on the protein expression of p-ERKl/2/ERK1/2, p-p38/p38and p-JNK/JNK in podocytes:(1) High glucose stimulated a strong increase in relative expression of p-ERKl/2in podocytes in a time-dependent manner. Semi-quantitative analysis revealed that the relative expression of p-ERK1/2was2-fold at0.5h,2-fold at2h,2.3-fold at4h,2.7-fold at6h, and2.2-fold at12h, respectively, compared with that for11.1mM D-glucose.(2)the relative expression of p-p38was7.6-fold at0.5h,8.8-fold at2h,7.1-fold at4h,6.3-fold at6h, respectively,compared with that for the control level of D-glucose (p<0.01),and (3) High D-glucose did not significantly increased the relative level of p-JNK compared with that for the control level of D-glucose of podocytes (p>0.05)5. The effects of ERK inhibitor on the production of MVs from podocytes:ERK inhibitor did not significantly decreased total MVs generation from podocytes (p=0.232).Conclusion:1. High glucose stimulated total MVs generation and production of ROS from podocytes, which is in a time-dependent way, accompanied by apoptosis of podocytes.2. The ERK and p38MAPK pathways were activatied in high glucose concentration of podocytes.3. ERK inhibitor U0126failed to lower the total MVs generation from podocytes, the ERKl/2pathway may not participate in the MVs generation from podocytes stimulated by high glucose. As to whether the p38MAPK pathway involved, further experimental researches are required.