目的 在顺铂诱导的大鼠急性肾损伤模型中,系统评价短时间采尿下系列尿液肾毒性生物标志的诊断性能。方法 Wistar大鼠单次ip 6 mg·kg^-1顺铂构建急性肾损伤模型,在检疫期、给药后第3和6天分别收集给药后5 h对照组和顺铂组动物尿液和血样,使用日立7180全自动生化仪测定血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、总胆红素(TBIL)、尿素氮(BUN)、肌酐(CRE)以及尿液样本中尿葡萄糖苷酶(NAG)、尿总蛋白(uTP)、尿肌酐(uCr)水平;使用Luminex仪器测定尿液中丛生蛋白(CLU)、谷胱甘肽S转移酶-α(GST-α)、干扰素诱导蛋白-10(IP-10)、肾损伤因子-1(KIM-1)、骨桥蛋白(OPN)、组织金属蛋白酶抑制剂-1(TIMP-1)、血管内皮生长因子-A(VEGF-A)、酸性糖蛋白(AGP)、白蛋白(Alb)、β₂微球蛋白(β₂M)、半胱氨酸蛋白酶抑制剂C(CysC)、表皮生长因子(EGF)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)水平。结合动物肾脏组织病理学检查,制作受试者操作特性曲线(ROC)进行肾毒性标志物的灵敏度和特异性分析。结果 顺铂组动物AST、ALT、TBIL值与各自对照组比较无统计学差异,且解剖大体检查并未发现除肾脏以外其他组织器官病变,排除其他组织器官病变对肾生物标志物变化的影响。组织病理学结果证实顺铂诱导动物模型均出现典型急性肾损伤:外髓质外带肾小管上皮细胞变性/坏死和髓质蛋白管型等。与对照组比较,传统标志物血清BUN和CRE在顺铂给药第6天、动物严重肾损伤时才出现显著升高(P<0.05);而IP-10、KIM-1、Alb、β2M和CLU生物标志物则在给药第3天已出现显著增加(P<0.05),且持续升高,增幅明显高于传统指标。ROC分析结果表明,IP-10、CysC、KIM-1和Alb的曲线下面积(AUC)明显优于BUN和CRE,具有更高的灵敏度和特异性。结论 尿液IP-10、CysC、KIM-1和Alb的肾毒性诊断性能优于传统生物标志物BUN和CRE,建议可作为药物致急性肾毒性早期诊断的生物标志物。

Objective To systematically evaluate the diagnostic performance of a series of newly reported urine nephrotoxicity biomarkers in a rat model of acute kidney injury induced by cisplatin. Methods A single ip injection with 6 mg·kg^-1 cisplatin in Wistar rats was used to construct an acute kidney injury (AKI) model. During the quarantine period and on the 3rd and 6th day following the administration, animal urine and blood samples in control and cisplatin group were collected five hours after administration. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), urea nitrogen (BUN), creatinine (CRE) in serum and urinary glucoside enzyme (NAG), total urinary protein (uTP), urinary creatinine (uCr) in urine samples were determined by Hitachi 7180 automatic biochemical analyzer. Clumping protein (CLU), glutathione S-transferase -α (GST- α), interferon inducible protein-10 (IP-10), kidney injury factor-1 (KIM-1), osteopontin (OPN), tissue metalloproteinase-1 (TIMP-1), vascular endothelial growth factor-A (VEGF-A), acid glycoprotein (AGP), albumin (Alb), β₂ microglobulin (β₂M), cysteine protease inhibitor C (CysC), epidermal growth factor (EGF), and neutrophil gelatinase-associated lipid carrier protein (NGAL) were determined by Luminex instrument. Combined with animal kidney histopathological examination, a receiver operating characteristic curve (ROC) were establised for sensitivity and specificity analysis of nephrotoxicity markers. Results Histopathological results confirmed that all animal models induced by cisplatin had typical acute kidney injury: Degeneration/ necrosis of renal tubule epithelial cells and myeloid protein tubule type. Compared with control group, the traditional markersserum BUN and CRE did not increase significantly until the 6th day following administration with cisplatin when the animals suffered severe kidney injury (P<0.05), while IP-10, KIM-1, Alb, β2M and CLU biomarkers were found to be significant increased on the third day of administration (P<0.05), and continues to increase. The degree of increase was significantly higher than the traditional biomarkes. ROC results showed that the area under the curve (AUC) of IP-10, CysC, KIM-1 and Alb were significantly better than BUN and CRE, suggesting these biomarkers had more high sensitivity and specificity. Conclusion Diagnostic performance of urine IP-10, CysC, KIM-1 and Alb were better than the traditional biomarkers BUN and CRE. It is suggested that they can be used as candidate biomarkers for the early diagnosis of acute nephrotoxicity caused by drugs

R965

国家自然科学基金项目(81603210);国家“重大新药创制”科技重大专项(2018ZX09201017-001)