Effect Of P38MAPK On Expression Of MMP-2/TIMP-2 And Collagen â…£ Induced By Glucose In Mesangial Cells

Objective Diabetic nephropathy (DN) is an important microvascular complication in diabetes, about 20%-30% of diabetic patients complicate with kidney disease. Diabetic nephropathy has become a major cause of death in diabetic patients. The pathological features of DN include glomerular mesangial broadening and diffuse thickening of glomerular basement membrane. The histological basis for this change is extracellular matrix (ECM) accumulation. Under normal circumstances, ECM production and degradation in body is under precise control. Matrix metalloproteinases (MMPs) and tissue inhibitor metalloproteinases (TIMPs) is the main regulator of ECM degradation. How high glucose regulating the expression of MMPs and TIMPs is not clear. The relationship between the expression of MMPs/ TIMPs and the p38MAPK should be explored. How p38MAPK signal pathway regulating the expression of collagen IV in diabetic nephropathy is inconclusive. In this study, we investigated the effect of high glucose and SB203580, inhibitor of p38MAPK, on the expression of p38MAPK and MMP-2/TIMP-2 in cultured SD rat glomerular mesangial cell and discussed the mechanism of p38MAPK signal transduction pathways in DN as well as the relation between p38MAPK and MMP-2/TIMP-2.Methods The cultured SD rat mesangial cells were divided into six groups, normal glucose group(5.6mmol/L), high glucose group(including different concentration gluclose:10 mmol/L,20mmol/L,30 mmol/L), SB203580 group(30 mmol/L glucose plus 10μmol/L SB203580) and mannitol group(5.6mmol/L glucose+24.4mmol/L mannitol). After the cells were incubated in different conditions for 48 hours, total RNA was isolated with Trizol reagent, the expression of p38MAPK, MMP-2 and TIMP-2 mRNA were examined by Real time Quantitative PCR. Total protein was isolated with RIPA, the expression of MMP-2/TIMP-2 and collagenⅣprotein were examined by western blot.Results (1) p38MAPK, MMP-2 and TIMP-2 mRNA express in normal glucose group; (2) The expression of p38MAPK mRNA is increased in high glucose group and mannitol group than that in normal glucose group, but the expression of p38MAPK mRNA in high glucose groups is much higher than mannitol group, which is in a concentration-dependent manner; (3) Compared with normal glucose group, the expression of TIMP-2 mRNA and protein is increased, but MMP-2 mRNA and protein and MMP-2/TIMP-2 mRNA and protein ratio in high glucose groups and in mannitol group are decreased; these changes in high glucose groups are more significantly than that in mannitol group, which are dependent of glucose concentration; (4) Compared with normal glucose group, the expression of collagen IVmRNA and protein is increased, these changes in high glucose groups are more significantly than that in mannitol group, the expression of collagenⅣmRNA are dependent of glucose concentration, but the expression of collagenⅣprotein are independent of glucose concentration,(5) The mRNA and protein expression of MMP-2 and MMP-2/TIMP-2 mRNA and protein ratio are higher in SB203580 group, and TIMP-2 mRNA and protein is lower than that in high glucose group (30 mmol/L), whereas there is no significant difference between high glucose group and SB203580 group in the expression of p38MAPK mRNAConclusion (1) High glucose can increase significantly the mRNA and protein expression of p38MAPK and TIMP-2, and decrease the mRNA and protein expression of MMP-2, and also decrease MMP-2/TIMP-2 mRNA and protein ratio in cultured mesangial cells, and these changes are dependent of glucose concentration; (2) The expression of collagen IV mRNA are dependent of glucose concentration, but the expression of collagen IV protein are independent of glucose concentration; (3) Mannitol increase the expression of p38MAPK mRNA in mesangial cells, high osmitic pressure at least partly participates in increasing of p38MAPK mRNA induced by high glucose; (4) The inhibitor of p38MAPK can reverse the changes of MMP-2, TIMP-2 mRNA,protein and MMP-2/TIMP-2 mRNA and protein ratio induced by high glucose, but can not inhibit the increased expression of p38MAPK mRNA induced by high glucose; (5) P38MAPK is related to the mRNA and protein expression of MMP-2,TIMP-2 and collagen IV in diabetic nephropathy.