Antisense Smad2 inhibits high glucose-stimulated production of extra cellular matrix in cultured rat glomerular mesangial cells

摘要

Objective: Hyperglycemia stimulates secretion of transforming growth factor-β1 (TGF-β1) in cultured glomerular mesangial cells, thereby increases production of extracellular matrix (ECM). We examined the effect of antisense mRNA for Smad2 on high glucose-induced ECM production in rat mesangial cells. Methods: A mammalian expression vector, pES2a, which expresses antisense Smad2 mRNA and green fluorescent protein (EGFP) , was transfected into mesangial cells. Following incubation in high glucose medium, EGFP expression and Smad2 mRNA level were determined by fluorescence microscopy and PCR, respectively. Secreted fibronectin and type Ⅳ collagen were assessed by enzyme-linked immunosor-bent assay. Results: Within 48 h of incubation in high glucose medium, Smad2 mRNA level significantly increased by 1. 6 fold in association with increases in production of both fibronectin (from [45.86 ± 2.73] to [84.19 ± 6.81] ng/ml) and type Ⅳ collagen (from [16. 28 ± 0. 90] to [55. 27 ± 4. 75] ng/ml) in nontransfected cells (P < 0. 05). In pES2a-transfected cells, the high glucose-induced increase in Smad2 mRNA was abrogated completely, in parallel with significant suppression of the high glucose-induced increase in fibronectin production ([54. 44 ± 4. 99] ng/ml) and type Ⅳ collagen ([20. 96 ± 2. 47] ng/ml). An empty vector was without effects. Conclusion: These findings demonstrate that Smad2 plays a critical role in mediating high glucose-stimulated ECM production in mesangial cells, indicating that inhibition of Smad2 activity by antisense Smad2 mRNA may be an effective means to attenuate glomerular matrix accumulation in diabetic nephropathy.