Advanced glycation end products impair protein turnover in LLC-PK1: Amelioration by trypsin

Background. Advanced glycation end products (AGEs) are assumed to play a ke y role in the pathogenesis of diabetic nephropathy (DN) and other diabetic complications. While AGEs have been shown to exert marked effects on mesang ial and endothelial cells as well as on monocytes/macrophages, little is kn own about their effects on tubule cells. Therefore, we addressed the questi ons of (1) whether AGE-bovine serum albumin (AGE-BSA) impairs the protein m etabolism in the tubule cells, and if so, (2) whether the AGE-induced effec ts are mediated via a protease sensitive mechanism. Methods. Arrested LLC-PK1 cells were exposed to a medium containing the veh icle (control, serum free), AGE-BSA (38 mu mol/L), or BSA (38 mu mol/L) in the presence or absence of trypsin (2.5 mug/mL) for 24 hours. We evaluated cell number, cell size, and cell protein content, as well as protein synthe sis and protein degradation. Results. After an incubation period of 24 hours, AGE-BSA decreased the cell number to 84.5 +/- 5.5% of control and 82.5 +/- 5.6% of BSA-treated cells (P < 0.05). [H-3]-thymidine incorporation declined to 66% of control (P < 0 .05), while BSA was without any effect. The same RGE-BSA dose reduced prote in degradation (P < 0.05) and stimulated total protein synthesis slightly, as determined by L-[C-14]Phe incorporation into acidic-insoluble proteins. These effects resulted in a rise in cell protein content (AGE-BSA vs. contr ol, 21.9 +/- 6.7%; AGE-BSA vs. BSA, 11.1 +/- 6.0%, P < 0.05) and cell volum e (AGE-BSA vs. control 9.4 +/- 3.2%, AGE-BSA vs. BSA 18.4 +/- 3.7%, P < 0.0 5). Coincubation with AGE-BSA and trypsin was associated with an ameliorati on of all investigated parameters concerning cell number, cell proliferatio n, raised cell protein content, decreased protein degradation, and enhanced protein synthesis. Conclusion. These data indicate that AGE-BSA impairs cell proliferation and protein turnover in LLC-PK1 cells with a consequent rise in cell protein. Since these alterations were abrogated by coincubation with trypsin, an int erference of this serine protease with the AGE-binding proteins on cell sur faces is assumed.