Yamamoto Soh, Wakui Hideki, Kubota Hiroshi, Komatsuda Atsushi, Itoh Hideaki, Yokota Shin-Ichi
Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan.
Chemotherapy. 2014;60(1):37-46. doi: 10.1159/000365880. Epub 2014 Oct 29.
The mechanism of aminoglycoside (AG)-induced nephrotoxicity has not been fully elucidated. We previously reported that gentamicin suppresses the functions of HSC70, a heat shock cognate protein, which is a cytosolic molecular chaperone in mammalian cells. However, the relationship between HSC70 dysfunction and nephrotoxicity has not been clarified yet.
The effects of 11 AGs, including gentamicin, on the function, protein refolding and substrate binding of HSC70 were examined in vitro. The protein refolding activity of HSC70 was examined by enzymatic activity restoration of the denatured firefly luciferase. Substrate binding activity of HSC70 was examined by the binding of fluorescent-dye-labeled reduced carboxymethlylated α-lactalbumin determined by HSC70/substrate complex formation by native polyacrylamide gel electrophoresis.
Strong inhibitory activity of AGs on the protein refolding activity of HSC70 was observed for arbekacin, gentamicin, neomycin and sisomicin, but not spectinomycin, streptomycin, ribostamycin and paromomycin, and inhibition was weak for tobramycin, amikacin and kanamycin. Neomycin strongly suppressed the formation of the HSC70/substrate complex and had the lowest concentration that resulted in 50% inhibition (IC50; in the order of 10(-4) M). Arbekacin, gentamicin, sisomicin and tobramycin had IC50 values in the order of 10(-3) M. Amikacin, ribostamycin and paromomycin had no effective potencies.
The inhibition efficacies of AGs for protein refolding of HSC70 were as follows: neomycin >> gentamicin, arbekacin, and sisomicin > tobramycin, amikacin and kanamycin > spectinomycin, streptomycin, ribostamycin and paromomycin. Amino groups and/or hydroxyl groups located at 2', 3', 4', and 6' of amino sugar residue II, as well as the number of amino sugar rings were closely associated with the inhibition efficacies of AGs. Inhibition efficacies of AGs towards the function of HSC70 correlated well with the nephrotoxicity of AGs deduced from the in vivo studies previously reported. Amino groups/hydroxyl groups and the number of amino sugar rings of AGs were shown to be important for inhibition efficacies. The results strongly suggest that the AG-induced dysfunction of HSC70 is involved in the nephrotoxicity.
氨基糖苷类（AG）诱导肾毒性的机制尚未完全阐明。我们之前报道过庆大霉素会抑制热休克同源蛋白HSC70的功能，HSC70是哺乳动物细胞中的一种胞质分子伴侣。然而，HSC70功能障碍与肾毒性之间的关系尚未明确。
在体外检测了包括庆大霉素在内的11种AG对HSC70的功能、蛋白质重折叠及底物结合的影响。通过变性的萤火虫荧光素酶的酶活性恢复来检测HSC70的蛋白质重折叠活性。通过天然聚丙烯酰胺凝胶电泳检测HSC70/底物复合物形成来确定荧光染料标记的还原羧甲基化α-乳白蛋白的结合，以此检测HSC70的底物结合活性。
阿贝卡星、庆大霉素、新霉素和西索米星对HSC70的蛋白质重折叠活性有较强抑制活性，而大观霉素、链霉素、核糖霉素和巴龙霉素则没有，妥布霉素、阿米卡星和卡那霉素的抑制作用较弱。新霉素强烈抑制HSC70/底物复合物的形成，且导致50%抑制的浓度最低（IC50；约为10⁻⁴M）。阿贝卡星、庆大霉素、西索米星和妥布霉素的IC50值约为10⁻³M。阿米卡星、核糖霉素和巴龙霉素没有有效效力。
AG对HSC70蛋白质重折叠的抑制效力如下：新霉素>>庆大霉素、阿贝卡星和西索米星>妥布霉素、阿米卡星和卡那霉素>大观霉素、链霉素、核糖霉素和巴龙霉素。氨基糖残基II的2'、3'、4'和6'位的氨基和/或羟基，以及氨基糖环的数量与AG的抑制效力密切相关。AG对HSC70功能的抑制效力与先前体内研究推断的AG肾毒性密切相关。AG的氨基/羟基和氨基糖环数量对抑制效力很重要。结果强烈表明AG诱导的HSC70功能障碍与肾毒性有关。
