Effects of advanced glycation end products on renal fibrosis and oxidative stress in cultured NRK-49F cells

BACKGROUND: Advanced glycation end products (AGEs) play a critical role in the development of diabetic nephropathy. Reactive oxygen species (ROS) may play a critical role in AGEs induced growth factor expression. In this study, the effects of AGEs on transforming growth factor beta1 (TGF-beta1), connective tissue growth factor (CTGF) and fibronectin (Fn) mRNA expression and oxidative stress in cultured NRK-49F cells were examined. METHODS: NRK-49F cells were incubated with medium containing different doses of AGEs (50, 100 or 200 microg/ml) for 24 hours, or with AGEs 100 microg/ml for different times (0, 12, 24 or 48 hours). Cells in the serum-free medium or medium containing 25 mmol/L glucose were controls. Cells were treated with 25 mmol/L glucose and 100 microg/ml AGEs for 24 hours to determine the effects between AGEs and glucose. We clarified the role of antioxidant by pretreating cells with N-acetylcysteine (10 mmol/L), ginkgo biloba extract (50 or 100 mg/L) for 24 hours and with 100 microg/ml AGEs for further 24 hours. Alamarblue dye assay was used to analyze cell growth; intracellular ROS generation was measured by flow cytometry; intracellular glutathione by fluorescence spectrophotometry; expressions of TGF-beta1, CTGF and Fn mRNA by semiquantitative RT-PCR. RESULTS: AGEs significantly increased the expressions of TGF-beta1, CTGF, Fn mRNA and intracellular ROS generation, and decreased the glutathion level in NRK-49F cells in dose- and time-dependent manners. High glucose and AGEs together significantly increased the expression of TGF-beta1, CTGF and Fn mRNA, compared with AGEs and high glucose separately. Preincubation with N-acetylcysteine or ginkgo biloba extract increased GSH level, suppressed AGEs-induced oxidative stress and TGF-beta1, CTGF and Fn mRNA overexpression. CONCLUSIONS: AGEs can significantly increase expression of TGF-beta1, CTGF, Fn mRNA in NRK-49F cells through enhancement of oxidative stress. The accumulation of AGEs may play a pivotal role in the pathogenesis of tubulointerstitial fibrosis in diabetic nephropathy. Suppression of AGEs induced TGF-beta1, CTGF and Fn mRNA overexpression in renal fibroblasts through inhibition of oxidative stress may be a mechanism underlying effect of ginkgo biloba extract in diabetic nephropathy. In addition, antioxidant therapy may help prevent AGEs accumulation and its induced damage.