A Role For AMP-activated Protein Kinase In High Glucose-induced Proliferation Of Glomerular Mesangial Cells

Objective:Changes in structure and function of glomerular mesangial cells contribut to the pathogenesis of diabetic nephrology.Therefore,to find some new drugs to prevent dysfunction of glomerular mesangial cells is very necessary in the treatment of diabetic nephrology.Recent studies indicated that activity of AMPK was significantly depressed in high glucose induced hyperplasia and hypertrophy of glomerular epithelial cells.However,whether this mechanism is involved in hyperplasia of mesangial cells in diabetes remains unclear.This study was designed to observe the expression and activity of AMPK in high glucose stimulated mesangial cells as well as the hyperplasia of mesangial cells.AMPK specific activator AICAR was used to further confirm the protect role of AMPK in high glucose environment.Methods:1.Establishment of high glucose environment:We applied the DMEM low and high carbohydrate cell-culture medium;glucose 5.6mmol/L is control group,glucose 22.0mmol/L is high glucose group.2.The groups:control group(glucose 5.6 mmol/L),high glucose group(glucose 22.0mmol/L), low AICAR group(22.0mmol/Lglucose+0.5mmol/LAICAR),high AICAR group (22.0mmol/Lglucose+1.0mmol/LAICAR).3.Detection of cell proliferation and cell cycle:The proliferation of GMCs was determined by MTT and the cell cycle of GMCs by flow cytometry.4.Detection of the expression and activity of AMPK:AMPKα1,α2 mRNA level was observed by RT-PCR and protein levels of AMPKα,P-AMPKαby Westernblot.Results:1.Effect of high glucose on proliferation and cell cycle of mesangial cells:MTT showed that,compared with control group,high glucose group cells displayed obvious proliferation tendency(P＜0.01);and flow cytometry showed that,compared with in control group,in high glucose G₁-stage cell percentage was lowered,S-stage cell percentage increased,G₂-stage cell percentage lowered,all comparisons of these three stages have significant differences(P＜0.01),which indicate that high glucose can promote cell DNA synthesis;2.Effect of high glucose on the expression and activity of AMPK:compared with control group,high glucose group has impaired AMPKα1,α2 mRNA level and impaired protein levels of AMPKα,P-AMPKα;which indicate that the expression and activity of AMPK are impressed in high glucose environment.3.Effect of AICAR on proliferation and cell cycle of mesangial cells in high glucose: MTT showed that AICAR can suppress high glucose-induced cell proliferation(P＜0.01),with concentration dependency;flow cytometry showed that, compared with high glucose group,AICAR group has lowerd S-stage cell percentage(P＜0.01) and elevated G₁-stage cell percentage(P＜0.01),this indicate that AICAR can suppress cell DNA synthesis;4.Effect of AICAR on the expression and activity of AMPK in high glucose: compared with high glucose group,AICAR group has enhanced AMPKα1,α2 mRNA level and impaired protein levels of AMPKα,P-AMPKα;which indicate that AICAR can enhance the expression and activity of AMPK in high glucose environment.Conclusions:1.High glucose can stimulate S-stage synthesis of rat glomerular mesangial cells and promote cell proliferation;the expression and activity of AMPKαare depressed by high glucose;2.AMPK specific activator AICAR can suppress high glucose-induced proliferation of mesangial cells,which may correlate with the enhanced expression and activity of AMPKαby AICAR; 3.High glucose-induced proliferation of mesangial cells is concerned with impaired expression and activity of AMPKαin this environment.