Methylglyoxal-induced fibronectin gene expression through Ras-mediated NADPH oxidase activation in renal mesangial cells.
BACKGROUND
The formation of methylglyoxal (MGO), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. Although recent studies have suggested that apoptotic cell death is involved in diabetic nephropathy, the precise mechanism of MGO-induced renal fibrosis remains to be elucidated.


METHODS
Rat kidney mesangial cells with or without pretreatment with inhibitors, including superoxide dismutase, catalase, L-NAME, diphenylene iodonium, rotenone, allopurinol, PD98059, SB203580 and SP600125 were cultured in medium containing 100 microM MGO. In the MGO-treated cell culture system, fibrosis-related signalling pathway was assessed by enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction and western blotting.


RESULTS
Expression of fibronectin induced by MGO was highest after 48 h treatment. Superoxide production rapidly increased after 2 h and remained at a high level for 24 h. Scavenging O(2) (-) reversed transforming growth factor beta 1 (TGF-beta1) and fibronectin mRNA level. Pretreatment with diphenylene iodonium significantly suppressed MGO-induced superoxide, TGF-beta1 expression and fibronectin gene expression, indicating that NADPH oxidase is responsible for inducing superoxide formation and subsequently induced renal fibrosis. High MGO rapidly enhanced Ras activation in 1 h and progressively increased cytosolic p38 activation. Additionally, SB203580 pretreatment reduced MGO promotion of fibronectin gene activation suggesting that cytosolic p38 activation might affect MGO-induced renal mesangial fibrosis. Inhibiting Ras activity with manumycin A significantly reduced the promoting effect of MGO on superoxide synthesis, and fibronectin expression.


CONCLUSIONS
Induction of superxoide by Ras via p38 pathway activates fibrotic gene transcription of mesangial cells. Reduction of oxidative stress by scavenging superoxide may offer an alternative strategy for controlling MGO-induced renal fibrosis.
Ho
Cheng
C
Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, Chiayi, Taiwan.
Lee
Pei-Hsien
PH
Huang
Wei-Jan
WJ
Hsu
Yen-Chen
YC
Lin
Chun-Liang
CL
Wang
Jeng-Yi
JY
D000818:Animals; D002478:Cells, Cultured; D005353:Fibronectins / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects*; D011905:Genes, ras / Q000235:genetics*; D005920:Glomerular Mesangium / Q000166:cytology* / Q000187:drug effects*; D019255:NADPH Oxidases / Q000502:physiology*; D011765:Pyruvaldehyde / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D005353:Fibronectins; D011765:Pyruvaldehyde; D019255:NADPH Oxidases