ABSTRACT
Diabetic nephropathy is characterized by an expansion of the glomerular mesangium, caused by mesangial cell proliferation and an excessive accumulation of extracellar matrix (ECM) proteins, which eventually leading to glomerulosclerosis. TGF-beta1 was found to play an important role in the accumulation of ECM in the kidney. In this study, TGF-beta1 RNA interference was used as an effective therapeutic strategy. The inhibitory effect of TGF-beta1 small interfering RNAs (siRNAs) on the high glucose-induced overexpression of TGF-beta1 in rat mesangial ceys (RMCs). A high levels of glucose induces TGF-beta1 mRNA and protein, and TGF-beta1 siRNAs reduce the ability of high glucose to stimulate their expression. We also examined the inhibitory effect of TGF-beta1 siRNAs on the expression of plasminogen activator inhibitor (PAI)-1 and Collagen Type I which are down-regulators of TGF-beta1. The expression of TGF-beta1, PAI-1 and Collagen Type I was increased in RMCs that were stimulated by 30 mM glucose. TGF-beta1 siRNAs reduces high glucose-induced TGF-beta1, PAI-1, and Collagen Type I mRNA and protein expression in a dose-dependent manner. In conclusion, the present study demonstrates that TGF-beta1 siRNAs effectively inhibits TGF-beta1 mRNA and protein expression in RMCs. These suggest that TGF-beta1 siRNAs through RNAi may be a useful tool for developing new therapeutic applications for the treatment of diabetic nephropathy.
Subject(s)
Rats , Male , Animals , Transforming Growth Factor beta1/metabolism , Rats, Sprague-Dawley , RNA, Small Interfering/metabolism , Microscopy, Fluorescence , Mesangial Cells/metabolism , Glucose/metabolism , Glomerular Mesangium/metabolism , Gene Expression Regulation , Diabetic Nephropathies/pathology , Collagen Type I/metabolism , Cells, Cultured , Cell ProliferationABSTRACT
Skin fibrotic disorders are understood to develop under the influence of various cytokines, such as transforming growth factor(TGF)-beta1, connective tissue growth factor(CTGF) and vascular endothelial growth factor (VEGF). To establish an appropriate animal model of skin fibrosis by exogenous application of growth factors, the author investigated the in vivo effects of growth factors by injecting recombinant TGF-beta1 protein and pCMV- Flag5-CTGF into the subcutaneous tissue of Sprague- Dawley rats. A single application of TGF-beta1 protein and CTGF DNA resulted in the formation of transient granulation tissue. Immunohistochemical finding showed increased expression of TGF-beta1 protein after injection of pCMV-Flag5-CTGF. In situ hybridization analysis revealed the expression of CTGF mRNA after injection of TGF-beta1 protein. VEGF expression was not affected by the TGF-beta1 and CTGF injection. These findings suggest TGF-beta1 and CTGF are deeply related with skin fibrosis and it appears that TGF-beta1 may cause the induction of CTGF expression. The animal model on skin fibrosis by exogenous application of TGF-beta1 protein and CTGF DNA developed in this study may be useful for future studies on fibrotic disorders.
