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.
Yan
Hai-dong
HD
Department of Nephrology, Eastern Hospital, Tongji University, Shanghai 200120, China. yhdcmu@sina.com
Li
Xue-zhu
XZ
Xie
Jun-mei
JM
Li
Man
M
D000975:Antioxidants / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D003928:Diabetic Nephropathies / Q000209:etiology*; D005353:Fibronectins / Q000235:genetics; D005355:Fibrosis; D005978:Glutathione / Q000032:analysis; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D006801:Humans; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects*; D053773:Transforming Growth Factor beta1 / Q000235:genetics
D000975:Antioxidants; C526317:CCN2 protein, human; D005353:Fibronectins; D017127:Glycation End Products, Advanced; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D005978:Glutathione