庆大霉素致大鼠肾毒性模型中早期生物标志物的表达

摘要

通过研究肾毒性早期生物标志物肾损伤分子-1（kidney inj ury molecule-1，KIM-1）和中性粒细胞明胶酶相关脂质运载蛋白（neutrophil gelatinase-associated lipocalin，NGAL）在庆大霉素诱导的大鼠肾毒性模型中的表达，探讨其在氨基糖苷类药物安全性评价中的应用价值.将45只SPF级雄性Sprague-Dawley（SD）大鼠随机分为对照组、庆大霉素低剂量组（50 mg/kg）和庆大霉素高剂量组（100 mg/kg），每组15只，连续肌内注射7 d，对照组给予等体积0.9%氯化钠注射液.给药第1、3和7天分别处死各组5只大鼠，腹主动脉采血检测血清肌酐（serum creatinine， SCr）和血尿素氮（blood urea nitrogen，BUN）水平，肾组织 HE染色观察病理改变，实时荧光定量 PCR法和免疫组织化学检测KIM-1和 NGAL的表达.结果表明，血清肌酐和尿素氮水平仅在给药第7天明显升高；病理组织学观察发现，肾损伤严重程度随时间及剂量依赖性增加，高剂量组给药7d后近端小管刷状缘消失，上皮细胞脱落坏死，出现蛋白管型及间质炎性细胞浸润.KIM-1和NGAL 的 mRNA 及蛋白表达水平均在给药第1天就显著上调（P<0.05），之后呈时间剂量依赖性升高，与病理组织学改变过程相一致，且优先于临床生化检测指标.支持这2个生物标志物作为庆大霉素引起的急性肾损伤的敏感指标.%Summary Gentamicin is a member of aminoglycosides which has represented highly effective antimicrobial agents especially in gram-negative infections despite their toxic effects in a kidney.Rapid diagnosis is vital to preserve renal function and to slow down renal inj ury.Owing to the poor sensitivity and specificity of serum creatinine (SCr) and blood urea nitrogen(BUN),there is a strong need for the identification and validation of more sensitive and reliable biomarkers.The aim of the study is to prove whether kidney inj ury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin(NGAL) could be useful to predict or detect acute kidney injury (AKI) caused by gentamycin. In this study,45 male Sprague-Dawley(SD)rats were injected with a range of doses of gentamicin which was administered at 0,50 or 100 mg/(kg.d)(n=5 rats/dose group/time point) and the animals were necropsied on days 1,3 or 7 for toxicity evaluation.Heparinized blood was analyzed for SCr and BUN using a standard clinical chemistry analyzer.Kidney tissue samples were embedded in paraffin sections processing for hematoxylin-eosin staining and immunohistochemistry.Real-time fluorescent quantitative PCR was used for relative quantitation of KIM-1/NGAL mRNA levels. In the gentamicin dose and time-response study,traditional indicators for nephrotoxicity,SCr and BUN levels in rats were significantly above control values after treatment for 7 days.Tubular cell degenerations,necrosis, tubular dilatation,hyaline cast tubules and inflammation were observed in the proximal tubules in the rats at the highest dose for 7 days.Repeated administration of gentamicin to animals resulted in a dose-and time-dependent increase in the expression of KIM-1 and NGAL which were evident as early as 1 day in both low-dose and high-dose animals(P<0.05).The degrees of mRNA increase were correlated well with the extent of tissue damage in the kidney.Consistent with gene expression analyses,KIM-1 and NGAL were undetectable in proximal tubules of control kidneys,but they were observed in epithelial cells that were affected by gentamicin toxicity as seen by cell degeneration and regeneration. In sum,the changes in gene and protein expressions of KIM-1 and NGAL were found to be correlated with the progressive histopathological alterations and preceded effects on traditional clinical parameters indicative of impaired kidney function.Time and dose-related effects on these two biomarkers expressions were observed at the target site of gentamicin toxicity,supporting the use of these two biomarkers as sensitive indicators of acute kidney inj ury caused by gentamicin.