Advanced glycation end products (AGEs)-induced expression of TGF-beta 1 is suppressed by a protease in the tubule cell line LLC-PK1.
BACKGROUND
Advanced glycation end products (AGEs) are assumed to play a key role in diabetic nephropathy (DN). Since little is known about their action in tubule cells, we investigated in LLC-PK1 cells: (i) whether AGE-bovine serum albumin (AGE-BSA) affects cell proliferation and expression of transforming growth factor-beta (TGF-beta 1); and (ii) whether the AGE-induced effects can be modulated by trypsin due to interference with its binding proteins at the cell surface.


METHODS
Arrested cells were exposed to vehicle (control), AGE-BSA (19--76 microM) and BSA (38 microM) in the presence or absence of trypsin (0.625--5.0 microg/ml) (2.5 microg/ml) for 24 h. We evaluated cell proliferation by cell count and by [(3)H]thymidine incorporation, TGF-beta 1 expression by reverse transcription-polymerase chain reaction (RT-PCR), and TGF-beta 1 protein by ELISA. In addition, cell accumulation of AGEs was studied by immunohistochemical staining of the AGE imidazolone.


RESULTS
AGE-BSA inhibited [(3)H]thymidine incorporation, lowered cell number and increased cell protein content as well as TGF-beta 1 mRNA and protein as compared with control and BSA. Immunohistochemical staining revealed a marked intracellular accumulation of the AGE imidazolone. Co-incubation of AGE-BSA with trypsin ameliorated the impaired thymidine incorporation, the decreased cell count and the enhanced cell protein content. TGF-beta 1 overexpression was normalized, while TGF-beta 1 protein declined insignificantly. Intracellular imidazolone accumulation was strikingly suppressed.


CONCLUSIONS
In the tubule cell line LLC-PK1, AGE-BSA exerts an antiproliferative effect, most probably due to TGF-beta 1 overproduction. The co-administration of trypsin abrogated this alteration, very likely as a result of an interaction with AGE-binding protein(s), which is supported by the decreased intracellular AGE accumulation. These findings may be the starting point for the development of specific proteolytic enzymes to interfere with the interaction between AGEs and their receptors/binding proteins.
Xiang
G
G
Department of Internal Medicine and Institute of Physiological Chemistry, University of Würzburg, Germany.
Schinzel
R
R
Simm
A
A
Münch
G
G
Sebekova
K
K
Kasper
M
M
Niwa
T
T
Schmitz
C
C
Heidland
A
A
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D010450:Endopeptidases / Q000502:physiology*; D017127:Glycation End Products, Advanced / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D007150:Immunohistochemistry / Q000379:methods; D007425:Intracellular Membranes / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018374:LLC-PK1 Cells; D009500:Neutralization Tests; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013194:Staining and Labeling; D013552:Swine; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D053773:Transforming Growth Factor beta1; D014357:Trypsin / Q000494:pharmacology
D000906:Antibodies; D004338:Drug Combinations; D017127:Glycation End Products, Advanced; D011506:Proteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C081628:advanced glycation end products-bovine serum albumin; D012710:Serum Albumin, Bovine; D010450:Endopeptidases; D014357:Trypsin