Role Of AMPK In Over-expression Of Collagen Ⅳ Induced By High Glucose In Rat Glomerular Mesangial Cells

Objective:Hyperplasia of glomerular mesangial matrix is one of pathological changes of diabetic nephropathy (DN). One of important methods to prevent and cure DN is to block and reverse this pathological change. Recent studies indicated that activity of AMPK was significantly depressed in glomerular epithelial cells in high glucose. However, whether this mechanism is involved in hyperplasia of glomerular mesangial matrix in diabetes remains unclear. This study was designed to observe the expression and activity of AMPK in high glucose stimulated mesangial cells, and to explore the relationship between this change and hyperplasia of glomerular mesangial matrix. AMPK-activator, AICAR, was used to further explore the protective role of AMPK to glomerular mesangial cells in high glucose environment.Methods:1. Establishment of high glucose environment:The DMEM cell-culture mediums with low and high glucose were applied; glucose 5.6mmol/L was used for control group, glucose 22.0mmol/L for high glucose group.2. The groups:Control group(glucose 5.6 mmol/L), high glucose group (glucose 22.0 mmol/L), low AICAR group (22.0mmol/L glucose+0.5 mmol/L AICAR), high AICAR group (22.0mmol/L glucose+1.0 mmol/L AICAR). GMCs were incubated in these four groups for 24 hours, and then correlative changes were detected. 3. Detection of the expression and activity of AMPK:AMPKα1 mRNA level was observed by RT-PCR and protein levels of AMPKa, p-AMPKa by Western blot.4. Detection of the expression of COL4a5:The protein level of COL4a5 was observed by Western blot.Results:1. Effect of high glucose on the expression and activity of AMPK in mesangial cells:RT-PCR showed that, compared with control group, high glucose increased AMPKal mRNA synthesis of GMCs distinctly (P＜0.01); and western blot showed that, compared with control group, high glucose increased total AMPKa and p-AMPKa protein synthesis of GMCs obviously (P＜0.01).2. Effect of high glucose on the expression of COL4a5:Compared with control group, high glucose enhanced COL4a5 protein level distinctly (P＜0.01).3. Effect of AICAR on the expression and activity of AMPK in high glucose:Compared with high glucose group, AICAR group enhanced AMPKal mRNA level (P＜0.01) and protein levels of AMPKa (P<0.01), p-AMPKa (P<0.01).4. Effect of AICAR on the expression of COL4a5 in high glucose: Compared with high glucose group, AICAR enhanced protein level of COL4a5 distinctly (P＜0.01).Conclusions:1. High glucose can suppress the expression and activity of AMPKa.2. High glucose can enhance the expression of COL4a5.3. AMPK activator, AICAR, can suppress high glucose induced over-expression of COL4a5, which may correlate with the enhanced expression and activity of AMPKa by AICAR. 4. High glucose induced over-expression of COL4a5 may be concerned with the impaired expression and activity of AMPKa in the environment.