Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE)
Matthew D. Oldfield, Leon A. Bach, Josephine M. Forbes, David Nikolic-Paterson, Anne McRobert, Vicki Thallas, Robert C. Atkins, Tanya Osicka, George Jerums, Mark E. Cooper
Matthew D. Oldfield, Leon A. Bach, Josephine M. Forbes, David Nikolic-Paterson, Anne McRobert, Vicki Thallas, Robert C. Atkins, Tanya Osicka, George Jerums, Mark E. Cooper
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Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE)

Abstract

Tubulointerstitial disease, a prominent phenomenon in diabetic nephropathy, correlates with decline in renal function. The underlying pathogenic link between chronic hyperglycemia and the development of tubulointerstitial injury has not been fully elucidated, but myofibroblast formation represents a key step in the development of tubulointerstitial fibrosis. RAGE, the receptor for advanced glycation end products (AGEs), induces the expression of TGF-β and other cytokines that are proposed to mediate the transdifferentiation of epithelial cells to form myofibroblasts. Here we report specific binding of 125I-AGE-BSA to cell membranes prepared from a rat proximal tubule cell line and show that the binding site was RAGE. AGE exposure induced dose-dependent epithelial-myofibroblast transdifferentiation determined by morphological changes, de novo alpha smooth-muscle actin expression, and loss of epithelial E-cadherin staining. These effects could be blocked with neutralizing Ab’s to RAGE or to TGF-β. Transdifferentiation was also apparent in the proximal tubules of diabetic rats and in a renal biopsy from a patient with type 1 diabetes. The AGE cross-link breaker, phenyl-4,5-dimethylthiazolium bromide (ALT 711) reduced transdifferentiation in diabetic rats in association with reduced tubular AGE and TGF-β expression. This study provides a novel mechanism to explain the development of tubulointerstitial disease in diabetic nephropathy and provides a new treatment target.

Authors

Matthew D. Oldfield, Leon A. Bach, Josephine M. Forbes, David Nikolic-Paterson, Anne McRobert, Vicki Thallas, Robert C. Atkins, Tanya Osicka, George Jerums, Mark E. Cooper

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Time- and dose-dependent α-SMA expression by NRK 52E cells. (a) Cells cu...
Time- and dose-dependent α-SMA expression by NRK 52E cells. (a) Cells cultured in the presence of increasing concentrations of AGE-BSA or unglycated BSA, in the presence or absence of Ab’s to RAGE, TGF-β, or control Ab’s, were counted in three or more separate experiments. Represented are α-SMA +ve cells counted in ten or more high-power fields (×200) expressed as a percentage of total cell number (mean ± SEM). **P < 0.01, ***P < 0.001 compared with untreated cells. The observed increase in α-SMA staining with AGE-BSA (40 μM) was prevented by Ab’s to either RAGE (R) or to TGF-β (T), but not by control Ab’s (C). ##P < 0.01, ###P < 0.001 compared with AGE-BSA (40 μM). (b) Effects of exposure of NRK cells to AGE-BSA (40 μM) over time. Cells were examined at 2, 4, and 6 days in three or more separate experiments; **P < 0.01, ***P < 0.001 compared with 0 days. (c) Effects of exposure of NRK cells to CML-modified BSA. Variously, CML-modified BSA was prepared by incubating BSA with sodium cyanoborohydride, in the presence or absence of glyoxylic acid. Incubation of NRK cells in the presence of CML-BSA (200 μM) caused a modification-dependent increase in α-SMA staining. Shown are results of three or more independent experiments; degree of lysine modification versus SMA positive cells as percentage of total cell number (mean ± SEM). *P < 0.05, **P < 0.01.