Superoxide Mediates Through ERK Signaling TGF-β1 Expression in Rat Kidney Proximal Tubule Epithelial Cells Treated with Advanced Glycation End Products

Background: The degree of renal tubulointerstitial fibrosis correlates best with renal function deterioration. Diabetic nephropathy is attributed to extracellular matrix accumulation and renal hypertrophy; previous studies have shown that advanced glycation end products (AGEs), oxidative stress and fibrosis stimulatory pathways are central to diabetic renal injury. Methods: Rat kidney proximal tubule epithelial cells pretreated with or without inhibitors-500 U/ml superoxide dismutase (SOD), 500 U/ml catalase, 100 μM N-nitro-L-arginine methyl ester (L-NAME,), 20 μM diphenylene iodonium (DPI), 30 μM rotenone, 30 μM allopurinol, 10 μM PD98059, 10 μM SB203580, or 10 μM SP600125-were cultured in medium containing 10 μg/ml glycated bovine serum albumin (AGE-BSA). Following incubation for 48 hrs, enzyme-linked immunosorbent assays (ELISAs) were applied to determine transforming growth factor-beta 1 (TGF-β1) protein levels; TGF-β1 gene expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Results: The AGEs produced the strongest promotion effect on TGF-β1 expression after 48 hrs of treatment. Superoxide production rapidly increased at 1 hr and elevated O2(superscript -) formation persisted for 6 hrs. Scavenging O2(superscript -) and not nitrogen oxide (NO) or hydrogen peroxide (H2O2) reversed TGF-β1 mRNA levels. Pretreatment with DPI significantly limited TGF-β1 expression, suggesting that NADPH oxidase is responsible for inducing superoxide formation. Moreover, extracellular response kinases (ERK) activation likely plays a role in AGEs induced over-expression of TGF-β1 on NRK-52E cells. Conclusion: In summary, renal proximal tubule epithelial cells responded to AGEs treatment by increasing NADPH oxidase-dependent superoxide production, subsequently causing cytosolic ERK activation that resulted in fibrotic pathogenesis. Regulation of redox reactions or modulation of ERK signaling cascades may be an alternative strategy for preventing diabetes-induced renal injury in the future.