Advanced glycation end products (AGEs)-induced expression of TGF-beta 1 issuppressed by a protease in the tubule cell line LLC-PK1

Background. Advanced glycation end products (AGEs) are assumed to play a ke y role in diabetic nephropathy (DN). Since little is known about their acti on in tubule cells, we investigated in LLC-PK1 cells: (i) whether AGE-bovin e serum albumin (AGE-BSA) affects cell proliferation and expression of tran sforming growth factor-beta (TGF-beta1); and (ii) whether the AGE-induced e ffects can be modulated by trypsin due to interference with its binding pro teins at the cell surface. Methods. Arrested cells were exposed to vehicle (control), AGE-BSA (19-76 m uM) and BSA (38 muM) in the presence or absence of trypsin (0.625-5.0 mug/m l) (2.5 mug/ml) for 24 h. We evaluated cell proliferation by cell count and by [H-3]thymidine incorporation, TGF-beta1 expression by reverse transcrip tion-polymerase chain reaction (RT-PCR), and TGF-beta1 protein by ELISA. In addition, cell accumulation of AGEs was studied by immunohistochemical sta ining of the AGE imidazolone. Results. AGE-BSA inhibited [H-3]thymidine incorporation, lowered cell numbe r and increased cell protein content as well as TGF-beta1 mRNA and protein as compared with control and BSA. Immunohistochemical staining revealed a m arked intracellular accumulation of the AGE imidazolone. Co-incubation of A GE-BSA with trypsin ameliorated the impaired thymidine incorporation, the d ecreased cell count and the enhanced cell protein content. TGF-beta1 overex pression was normalized, while TGF-beta1 protein declined insignificantly. Intracellular imidazolone accumulation was strikingly suppressed. Conclusions. In the tubule cell line LLC-PK1, AGE-BSA exerts an antiprolife rative effect, most probably due to TGF-beta1 overproduction. The co-admini stration of trypsin abrogated this alteration, very likely as a result of a n interaction with AGE-binding protein(s), which is supported by the decrea sed intracellular AGE accumulation. These findings may be the starting poin t for the development of specific proteolytic enzymes to interfere with the interaction between AGEs and their receptors binding proteins.