Advanced glycation end products inhibit de novo protein synthesis and induce TGF-beta overexpression in proximal tubular cells.
BACKGROUND
We have shown previously that OPB-9195, a novel inhibitor of advanced glycation end products (AGE), significantly prevented renal tubular injury and tubulointerstitial fibrosis in spontaneous diabetic rats. However, the molecular mechanisms underlying this have not been fully elucidated.


METHODS
Three immunochemically distinct AGE were prepared by incubating bovine serum albumin (BSA) with glucose, glyceraldehyde, or methylglyoxal. Then, the effects of AGE on human proximal tubular epithelial cells were examined. The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H2DCFDA. DNA synthesis was evaluated by thymidine uptake, and de novo protein synthesis was determined by [3H]leucine incorporation. Prostaglandin E2 (PGE2) and transforming growth factor-beta (TGF-beta) released into media were quantitatively analyzed in an enzyme-linked immunosorbent assay. TGF-beta gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR).


RESULTS
When these AGE-BSA were administered to tubular cells, each of them increased generation of intracellular ROS. All of the AGE-BSA, but not non-glycated BSA, were found to induce statistically significant decreases in de novo protein synthesis and PGE2 secretion by tubular cells. Furthermore, AGE-BSA up-regulated the levels of mRNAs for TGF-beta in tubular cells. The structural epitope designated glucose-derived AGE was found to have the greatest cytopathic effects on tubular cells. These AGE-induced inhibition of protein synthesis and PGE2 secretion as well as the up-regulation of TGF-beta mRNA were found to be completely prevented by N-acetylcysteine. Furthermore, H2O2 was shown to inhibit protein synthesis and PGE2 secretion by proximal tubular cells in a dose-dependent manner.


CONCLUSIONS
The results suggest that AGE inhibits de novo protein synthesis and stimulates TGF-beta mRNA expression in proximal tubular epithelial cells through overgeneration of intracellular ROS. Thus, AGE are involved in the pathogenesis of tubular injury in diabetic nephropathy.
Yamagishi
Sho-Ichi
S
Division of Endocrinology and Metabolism, Department of Medicine, Kurume University School of Medicine, Kurume, Japan. shoichi@med.kurume-u.ac.jp
Inagaki
Yosuke
Y
Okamoto
Tamami
T
Amano
Shinjiro
S
Koga
Kohachiro
K
Takeuchi
Masayoshi
M
D002478:Cells, Cultured; D003920:Diabetes Mellitus / Q000097:blood; D015232:Dinoprostone / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D017127:Glycation End Products, Advanced; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016212:Transforming Growth Factor beta; C081628:advanced glycation end products-bovine serum albumin; D012710:Serum Albumin, Bovine; D006861:Hydrogen Peroxide; D007930:Leucine; D015232:Dinoprostone; D013936:Thymidine