Zhipeng Wang, Li Liu, Qibing Mei, Linna Liu, Yuhua Ran, Rong Zhang
Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi' an, Shaanxi, 710032, China.
J Toxicol Sci. 2006 Feb;31(1):61-70. doi: 10.2131/jts.31.61.
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-beta-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 microg/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.
庆大霉素（GM）作为一种抗生素已被广泛使用，其肾毒性也已得到公认。然而，热休克蛋白（HSP）72作为GM处理的近端肾小管细胞中的一种诱导蛋白，其变化情况仍不清楚。在本研究中，测定了GM的细胞毒性及其对人肾近端小管（HK - 2）细胞中HSP72表达的影响。HK - 2细胞分别仅用GM以及GM加MnCl₂孵育24小时、48小时、72小时和96小时。通过乳酸脱氢酶（LDH）的释放来测定细胞毒性。通过测量丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性，以及活细胞还原基于四氮唑的化合物（MTT）的能力，研究了N - 乙酰 - β - D - 氨基葡萄糖苷酶（NAG）的活性以及GM对HK - 2细胞氧化的影响。通过免疫细胞化学、蛋白质印迹法和逆转录 - 聚合酶链反应（RT - PCR）测定HSP72的表达。细胞暴露于浓度为100μg/ml的GM中。24小时后MTT摄取显著下降，然后逐渐下降直至96小时。仅用GM处理细胞时，LDH释放从24小时到72小时呈时间依赖性增加，但在96小时时与72小时的数据相比有所下降。NAG和SOD活性结果以及MDA含量结果与LDH释放结果相似。暴露于GM后24小时至72小时，HSP72阳性细胞数量增加。仅用GM处理细胞时，HSP72表达从24小时开始显著增加，并在72小时达到峰值。此外，HSP72基因转录的变化与HSP72的表达相似。这些结果表明，GM处理可诱导HK - 2细胞损伤，并且当细胞受到GM损伤时HSP72的表达增加。
