顺铂诱导的Beagle犬急性肾损伤模型中尿液生物标志物研究

Urinary biomarkers of cisplatin-induced acute kidney injury in Beagle dogs

目的:应用顺铂诱导Beagle犬急性肾损伤模型,评价新型尿液生物标志物的诊断效能。方法:通过对Beagle犬单次给予3 mg·kg^(-1)顺铂静脉注射(iv),建立了急性肾损伤模型,应用ELISA和Luminex液相芯片方法检测分析尿液中9种新型生物标志物浓度,并与传统血清指标尿素氮(BUN)和肌酐(Cr)进行比较,结合肾脏组织病理学分析结果,用受试者工作特征曲线(ROC)评价尿液生物标志物的诊断效能。结果:组织病理学分析结果显示Beagle犬单次给予3 mg·kg^(-1),iv,d 7肾脏发生轻度的肾小管变性、坏死或再生,至d 21肾脏仍存在一定程度的肾小管变性、再生。尿液生物标志物分析结果发现在给药d 2,尿液中丛生蛋白(clusterin)、单核细胞趋化蛋白-1(MCP-1)、视黄醇结合蛋白(RBP)和谷氨酰转肽酶(GGT)浓度就开始显著增加(P<0.05),上升幅度明显高于BUN和Cr,到了给药d 21,尿液中clusterin和MCP-1仍保持较高水平(P<0.05),且分析结果与其个体动物组织病理变化具有良好的相关性。ROC分析表明尿液clusterin和MCP-1的药时曲线下面积(AUC)值分别为0.931和0.909,高于传统生物标志物BUN和Cr,具有较好的诊断效能。选择代表性生物标志物基因分析结果也证实给药后clusterin基因mRNA表达显著提高,进一步支持其预测肾毒性的特异性。结论:研究结果表明尿液clusterin和MCP-1可作为一种药物肾毒性的候选生物标志物,在药物的Beagle犬模型临床前安全性研究中具有良好的应用前景。

Objective: To evaluate the diagnostic performance of new urinary biomarkers using the model of cisplatin-induced acute kidney injury in Beagle dogs. Methods: A single intravenous injection of 3 mg·kg;cisplatin was applied to establish an acute kidney injury model in Beagle dogs. Nine new urinary biomarkers were detected and analyzed by ELISA and Luminex methods, compared with traditional serum biomarkers, i.e., urea nitrogen(BUN) and creatinine(Cr). The diagnostic performance of urinary biomarkers was evaluated using receiver operating curve(ROC) analysis combined with renal histopathological findings. Results: Histopathological analysis showed that Beagle dogs had mild renal tubular degeneration, necrosis or regeneration on day 7 after a single intravenous injection of 3 mg·kg^(-1), and there was still a certain degree of renal tubular degeneration, necrosis or regeneration on day 21. The results further showed that urinary clusterin, monocyte chemoattractant protein-1(MCP-1), retinol binding protein(RBP) and GGT concentration increased significantly(P<0.05) on day 2 after administration, the tendency of which were more obvious than that of BUN and Cr. On day 21 of administration, clusterin and MCP-1 still remained high(P<0.05) with a good correlation with the pathological changes of individual animal kidneys. ROC analysis showed that the AUC values of urinary clusterin and MCP-1 were 0.931 and 0.909, respectively, which were higher than the traditional biomarkers BUN and Cr, suggesting better diagnostic efficiency. Gene analysis of the representative biomarkers also confirmed that mRNA expression of clusterin gene increased significantly after administration, further supporting its specificity in predicting nephrotoxicity. Conclusion: This study indicates that the urinary clusterin and MCP-1 can be used as a group of candidate biomarkers for nephrotoxicity caused by drug, which will have a good prospect of application in preclinical drug safety study in Beagle dog models.