Advanced glycation end products impair protein turnover in LLC-PK1: amelioration by trypsin.
BACKGROUND
Advanced glycation end products (AGEs) are assumed to play a key role in the pathogenesis of diabetic nephropathy (DN) and other diabetic complications. While AGEs have been shown to exert marked effects on mesangial and endothelial cells as well as on monocytes/macrophages, little is known about their effects on tubule cells. Therefore, we addressed the questions of (1) whether AGE-bovine serum albumin (AGE-BSA) impairs the protein metabolism in the tubule cells, and if so, (2) whether the AGE-induced effects are mediated via a protease sensitive mechanism.


METHODS
Arrested LLC-PK1 cells were exposed to a medium containing the vehicle (control, serum free), AGE-BSA (38 micromol/L), or BSA (38 micromol/L) in the presence or absence of trypsin (2.5 microg/mL) for 24 hours. We evaluated cell number, cell size, and cell protein content, as well as protein synthesis 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). [3H]-thymidine incorporation declined to 66% of control (P < 0.05), while BSA was without any effect. The same AGE-BSA dose reduced protein degradation (P < 0.05) and stimulated total protein synthesis slightly, as determined by L-[14C]Phe incorporation into acidic-insoluble proteins. These effects resulted in a rise in cell protein content (AGE-BSA vs. control, 21.9 +/- 6.7%; AGE-BSA vs. BSA, 11.1 +/- 6.0%, P < 0.05) and cell volume (AGE-BSA vs. control 9.4 +/- 3.2%, AGE-BSA vs. BSA 18.4 +/- 3.7%, P < 0.05). Coincubation with AGE-BSA and trypsin was associated with an amelioration of all investigated parameters concerning cell number, cell proliferation, raised cell protein content, decreased protein degradation, and enhanced protein synthesis.


CONCLUSIONS
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 interference of this serine protease with the AGE-binding proteins on cell surfaces is assumed.
Xiang
G
G
Department of Internal Medicine, Institute of Physiological Chemistry, University of Würzburg, Würzburg, Germany.
Schinzel
R
R
Simm
A
A
Sebekova
K
K
Heidland
A
A
D000818:Animals; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D003928:Diabetic Nephropathies / Q000209:etiology / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000037:antagonists & inhibitors / Q000633:toxicity*; D006801:Humans; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018374:LLC-PK1 Cells; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000037:antagonists & inhibitors / Q000633:toxicity*; D013552:Swine; D014357:Trypsin / Q000494:pharmacology*
D017127:Glycation End Products, Advanced; D011506:Proteins; C081628:advanced glycation end products-bovine serum albumin; D012710:Serum Albumin, Bovine; D014357:Trypsin