AGEs activate mesangial TGF-beta-Smad signaling via an angiotensin II type I receptor interaction.
BACKGROUND
The renin-angiotensin system (RAS) and the accumulation of advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic nephropathy. Whether there is a functional interaction between the RAS and AGEs in diabetic nephropathy is not known. In this study, we investigated whether AGEs could activate autocrine angiotensin II (Ang II) signaling and subsequently induce transforming growth factor-beta (TGF-beta)-Smad signaling in cultured rat mesangial cells.


METHODS
The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H2DCFDA. Ang II was measured by radioimmunoassay. TGF-beta released into media was quantitatively analyzed in an enzyme-linked immunosorbent assay (ELISA). Smad2, p27(Kip1) (p27), fibronectin, and receptor for AGEs (RAGE) protein expression were determined by Western blot analysis. TGF-beta-inducible promoter activity was analyzed by a luciferase assay. DNA synthesis was evaluated by 5-bomo-2'-deoxyuridine (BrdU) incorporation and de novo protein synthesis was determined by [3H]leucine incorporation.


RESULTS
AGEs increased intracellular ROS generation in mesangial cells, and this effect was significantly inhibited by an antiserum against RAGE. AGEs also were found to stimulate Ang II production in a time- and dose-dependent manner, which was completely prevented by an antioxidant, N-acetylcysteine (NAC). AGE-induced TGF-beta overproduction was completely blocked by candesartan, an Ang II type 1 receptor (AT1R) antagonist. Both candesartan and neutralizing antibody against TGF-beta completely prevented AGEs-induced Smad2 phosphorylation and TGF-beta-inducible promoter activity. Furthermore, AGEs were found to inhibit DNA synthesis and to stimulate de novo protein synthesis and fibronectin production in association with up-regulation of p27. All of these phenomena were completely prevented by candesartan or a polyclonal antibody against TGF-beta.


CONCLUSIONS
The present study suggests that AGE-RAGE-mediated ROS generation activates TGF-beta-Smad signaling and subsequently induces mesangial cell hypertrophy and fibronectin synthesis by autocrine production of Ang II. This pathway may provide an important link between metabolic and haemodynamic factors in promoting the development and progression of diabetic nephropathy.
Fukami
Kei
K
Department of Nephrology, Internal Medicine III, the Cardiovascular Research Institute, Department of Pathology, Kurume University School of Medicine, Kurume, Japan.
Ueda
Seiji
S
Yamagishi
Sho-ichi
S
Kato
Seiya
S
Inagaki
Yosuke
Y
Takeuchi
Masayoshi
M
Motomiya
Yoshihiro
Y
Bucala
Richard
R
Iida
Shuji
S
Tamaki
Kiyoshi
K
Imaizumi
Tsutomu
T
Cooper
Mark E
ME
Okuda
Seiya
S
D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D006801:Humans; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051785:Smad Proteins; D015534:Trans-Activators / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005353:Fibronectins; D017127:Glycation End Products, Advanced; D017382:Reactive Oxygen Species; D000067759:Receptor for Advanced Glycation End Products; D044140:Receptor, Angiotensin, Type 1; D011971:Receptors, Immunologic; D051785:Smad Proteins; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D000804:Angiotensin II; D004247:DNA