INCREASED EXPRESSION OF HEAT SHOCK PROTEIN (HSP)72 IN A HUMAN PROXIMAL TUBULAR CELL LINE (HK-2) WITH GENTAMICIN-INDUCED INJURY

Abstract

Gentamicin (GM) has been widely used as an antibiotic and its nephrotoxicity has been recognized. However, the alternation of heat shock protein (HSP) 72 as an inductive protein in proximal tubular cells treated with GM is still unclear. In this study, GM cytotoxicity and its effect on the expression of HSP72 in human kidney proximal tubular (HK-2) cells were measured. HK-2 cells were incubated for 24 hr, 48 hr, 72 hr, and 96 hr with GM only and GM plus MnCl₂, respectively. Cytotoxicity was determined by the release of lactate dehydrogenase (LDH). Activity of N-acetyl-β-D-glucosaminidase (NAG) and effects of GM on oxidation in HK-2 cells were investigated by measurements of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, and the ability of viable cells to reduce a tetrazolium-based compound (MTT). The expression of HSP72 was measured by immunocytochemistry, Western blotting and RT-PCR. Cells were exposed to GM at a concentration of 100 μg/ml. After 24 hr MTT uptake decreased significantly and then gradually until 96 hr. LDH release increased time-dependently from 24 hr to 72 hr, but decreased at 96 hr compared with the data at 72 hr when cells were treated with GM only. Both results of NAG and SOD activities and results of MDA content were similar to that of the LDH release. The amount of HSP72 positive cells increased at 24 hr after exposure to GM up to 72 hr. HSP72 expression increased significantly from 24 hr, and reached its peak at 72 hr when cells were treated with GM only. Furthermore, the change of the HSP72 gene transcription was similar to the expression of HSP72.
These results demonstrated that GM treatment could induce damage to HK-2 cells and that the expression of HSP72 increased when cells were injured by GM.