Effect of heparin on high glucose induced proliferation and expression of matrix metalloproteinases in normal human mesangial cells

Abstract

Background The pathogenesis of diabetic nephropathy (DN) is a complex pathophysiological process. Its precise mechanism is not fully known. In recent years it has been recognized that synthesis of various extracelluar matrix (ECM) components may increase, and that degradation of ECM may decrease in DN. It was reported heparin could inhibit mesangial cells proliferation in vitro. The main aim of this study is to explore whether heparin inhibits proliferation of mesangial cells grown in high glucose concentration and to measure the effect of heparin on matrix metalloproteinases (MMPs) expression in mesangial cells. Methods The medium contained either low glucose (5 mmol/L) or high glucose (25 mmol/L). The concentrations of heparin in the culture medium were 0, 25, 50, 100, 200 or 400 µg/mL. A metabolic (WST-1) assay was used to measure mesangial cell proliferation and Western blot analysis was used to measure MMPs expression of mesangial cells. Results Normal human mesangial cell (NHMC) proliferation was higher in high glucose (HG) medium than in low glucose (LG) medium. They showed a 1.93 fold expansion after 72 h in high glucose in contrast to a 1.63 fold expansion in low glucose. In the presence of heparin, mesangial cells proliferation was inhibited, which was more obvious at high glucose concentrations than at low glucose concentrations. In high glucose, with heparin concentration of 50, 100, 200 and 400 µg/mL, the mesangial cells showed a 0.61 fold, 0.52 fold, 0.52 fold and 0.41 fold reductions in cell number compared to cells grown without heparin. In low glucose, only concentrations of 200 µg/mL and 400 µg/mL showed reduction in cell number, namely 0.54 fold and 0.45 fold, when compared to cells grown without heparin. In Western blot analysis, MMP₁, MMP₂, MMP₃ and MMP₉ was expressed by mesangial cells expressed in both high and low glucose concentrations, which was more prominent in high glucose medium. Incubation of heparin further increased expression of MMP₁, MMP₂, MMP₃ and MMP₉. Conclusions This study suggests that glucose can accelerate mesangial cell proliferation while heparin can reduce proliferation, being more obvious at high glucose concentrations. Higher glucose concentrations led to increased MMP expression, which may take part in the regulation of mesangial matrix synthesis and degradation. Addition of heparin resulted in a corresponding increase in MMP expression, most notably at high glucose concentrations, indicating a potentially renoprotective role in DN.

Keywords

matrix metalloproteinases / normal human mesangial cell / glucose / heparin

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