Effects Of High Glucose On The Expressions Of MMPs And TIMPs In Rat Glomerular Mesangial

Objective: To observe the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metaloproteinases-1 (TIMP-1) in rat glomerular mesangial cells exposed to high glucose and to explore further the mechanism of glomerular extracellular matrix accumulation caused by high glucose in diabetic nephropathy.Methods: The culture system of rat glomerular mesangial cells in vitro was established. The rat glomerular mesangial cells were separated into two groups and cultured separately in high glucose medium and normal glucose medium for 12,24,48,72hours. The sheep polyclonal antibodies for MMP-9 and TIMP-1 against rat serum and the rabbit polyclonal antibody for TGF-J31 against rat serum were used to detect the expressions of MMP-9, TIMP-1, and TGF-J3, in the mesangial cells by immunocytochemical staining and western blotting analysis. The mouse monoclonal antibody for IV collagen against rat serum was used tomeasure quantity of IV collagen secreted by the mesangial cells using a solid-phase enzyme -linked immunosorbent assay (ELISA).Results: Immunocytochemically, the positive reactions for MMP-9, TIMP-1 and TGF-(31 were observed in cytoplasm of the mesangial cells cultured in the medium with normal level and high-level glucose. Positive signals were not observed in the nucleus of the cells. Positive reactions were observed in every period of culture. Compared with those in the mesangial cells cultured in the medium with normal level glucose, the stainings were strengthened for TIMP-1 and TGF-J31, weakened for MMP-9 in the mesangial cells exposed to high glucose. Western blotting analysis showed that above effects of high glucose were enhanced along with the culture times whereas the expressions of MMP-9, TIMP-1 and TGF-J31 in mesangial cells cultured continuously in normal glucose medium were maintained on steady levels. The increase of TGF-31 in the mesangial cells exposed to high glucose was observed from 12 to 72 hours, while the alteration of MMP-9 and TIMP-1 were detected from 24 to 72 hours. ELISA could not detect significant difference of IV collagen secreted by the mesangial cells between normal glucose group and high glucose group.Conclusions: The increasing synthesis, of manymatrix components was not the only reason for the accumulation of glomerular extracellar matrix (ECM) in diabetic nephropathy. The decreasing degradation of matrix also plays a very important role on the accumulation of ECM. The decrease of expression for MMP-9 and the increase of expression for TIMP-1 in the mesangial cells exposed to high glucose observed in this study suggest that high glucose may alter the expression of MMPs/TIMPs in mesangial cell, thus influence the degradation of ECM in diabetic nephropathy. TGF-3 can inhibit the expression of MMPs and induce the expression of TIMPs. The expression of TGF-3 was increased in mesangial cells cultured in high glucose medium and the alteration of TGF-p occurred earlier than that of MMP-9/TIMP-1. These facts suggest that the effects of high glucose on MMP-9/TIMP-1 may be mediated by increasing level of TGF-P .