Expression of MMP-2, TIMP-2, CTGF and MT1-MMP in rat glomerular mesangial cells stimulated by high-glucose and its significance

Objective　To observe the dynamic changes of matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2), membrane-type 1 matrix metalloproteinase (MT1-MMP) and connective tissue growth factor (CTGF) expression in high glucose-stimulated glomerular mesangial cells (GMCs) in rats, and investigate the mechanism of the pathogenesis of diabetic nephropathy. Methods　Rat HBZY-1 GMCs were cultured and divided into 3 groups: low concentration (5.5mmol/L) D-glucose (LG) group, high concentration (30mmol/L) D-glucose (HG) group and 24.5mmol/L mannitol plus 5.5mmol/L D-glucose (LG+M) group (served as osmotic pressure control). The mRNA and protein expressions of MMP-2, TIMP-2, MT1-MMP and CTGF were detected with semi-quantitative RT-PCR and Western blotting, and the secreted collagen Ⅳ in supernatants of the GMCs was detected by ELISA after cultured for 24, 48, 72 and 96h. Results　Compared with LG group, after exposure to high glucose for 24h, MMP-2 expression was slightly increased (increased by 10%±4%) in GMCs in HG group (P<0.05). But when the exposure time last from 48h to 96h, the expression of MMP-2 was decreased by 42%±2% to 78%±2% (P<0.01). Compared with LG group, high-glucose incubation resulted in down-regulation of MT1-MMP (decreased by 29%±3% at 24h to 78%±9% at 96h, P<0.01) whereas up-regulation of TIMP-2 (increased by 55%±3% at 24h, P<0.01) and CTGF (increased by 201%±24% at 24h to 484%±51% at 96h, P<0.01). RT-PCR revealed consistent dynamic changes of MMP-2, TIMP-2, MT1-MMP and CTGF with their protein changes listed above. Compared with LG group, the secreted collagen Ⅳ in supernatants of HG group was increased by 201%±24% at 24h to 1232%±198% at 96h (P<0.01). There was no significant difference of above indexes between LG group and LG+M group. Conclusions　High glucose may induce and activate MMP-2 transiently, but can inhibit the expression of MMP-2 and MT1-MMP for a long term whereas induce the expressions of TIMP-2 and CTGF in GMCs. MMP-2, TIMP-2, MT1-MMP and CTGF may contribute to the metabolism imbalance of extracellular matrix then induce the accumulation of extracellular matrix in diabetic nephropathy. DOI: 10.11855/j.issn.0577-7402.2013.10.006