目的 在順鉑誘導(dǎo)的大鼠急性腎損傷模型中,系統(tǒng)評(píng)價(jià)短時(shí)間采尿下系列尿液腎毒性生物標(biāo)志的診斷性能。方法 Wistar大鼠單次ip 6 mg·kg^-1順鉑構(gòu)建急性腎損傷模型,在檢疫期、給藥后第3和6天分別收集給藥后5 h對(duì)照組和順鉑組動(dòng)物尿液和血樣,使用日立7180全自動(dòng)生化儀測(cè)定血清中丙氨酸氨基轉(zhuǎn)移酶(ALT)、天冬氨酸氨基轉(zhuǎn)移酶(AST)、總膽紅素(TBIL)、尿素氮(BUN)、肌酐(CRE)以及尿液樣本中尿葡萄糖苷酶(NAG)、尿總蛋白(uTP)、尿肌酐(uCr)水平;使用Luminex儀器測(cè)定尿液中叢生蛋白(CLU)、谷胱甘肽S轉(zhuǎn)移酶-α(GST-α)、干擾素誘導(dǎo)蛋白-10(IP-10)、腎損傷因子-1(KIM-1)、骨橋蛋白(OPN)、組織金屬蛋白酶抑制劑-1(TIMP-1)、血管內(nèi)皮生長因子-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劑C(CysC)、表皮生長因子(EGF)、中性粒細(xì)胞明膠酶相關(guān)脂質(zhì)運(yùn)載蛋白(NGAL)水平。結(jié)合動(dòng)物腎臟組織病理學(xué)檢查,制作受試者操作特性曲線(ROC)進(jìn)行腎毒性標(biāo)志物的靈敏度和特異性分析。結(jié)果 順鉑組動(dòng)物AST、ALT、TBIL值與各自對(duì)照組比較無統(tǒng)計(jì)學(xué)差異,且解剖大體檢查并未發(fā)現(xiàn)除腎臟以外其他組織器官病變,排除其他組織器官病變對(duì)腎生物標(biāo)志物變化的影響。組織病理學(xué)結(jié)果證實(shí)順鉑誘導(dǎo)動(dòng)物模型均出現(xiàn)典型急性腎損傷:外髓質(zhì)外帶腎小管上皮細(xì)胞變性/壞死和髓質(zhì)蛋白管型等。與對(duì)照組比較,傳統(tǒng)標(biāo)志物血清BUN和CRE在順鉑給藥第6天、動(dòng)物嚴(yán)重腎損傷時(shí)才出現(xiàn)顯著升高(P<0.05);而IP-10、KIM-1、Alb、β2M和CLU生物標(biāo)志物則在給藥第3天已出現(xiàn)顯著增加(P<0.05),且持續(xù)升高,增幅明顯高于傳統(tǒng)指標(biāo)。ROC分析結(jié)果表明,IP-10、CysC、KIM-1和Alb的曲線下面積(AUC)明顯優(yōu)于BUN和CRE,具有更高的靈敏度和特異性。結(jié)論 尿液IP-10、CysC、KIM-1和Alb的腎毒性診斷性能優(yōu)于傳統(tǒng)生物標(biāo)志物BUN和CRE,建議可作為藥物致急性腎毒性早期診斷的生物標(biāo)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

國家自然科學(xué)基金項(xiàng)目(81603210);國家“重大新藥創(chuàng)制”科技重大專項(xiàng)(2018ZX09201017-001)