Chen Liuxi, Smith James, Mikl Jaromir, Fryer Ryan, Pack Frank, Williams Brad J, Phillips Jonathan A, Papov Vladimir V
Boehringer Ingelheim Pharmaceuticals , Ridgefield, Connecticut 06877, United States.
Waters Corporation, Milford, Massachusetts 01757, United States.
Chem Res Toxicol. 2017 Oct 16;30(10):1823-1834. doi: 10.1021/acs.chemrestox.7b00159. Epub 2017 Sep 27.
Drug-induced kidney injury (DIKI) is a common toxicity observed in pharmaceutical development. We demonstrated the use of label-free liquid chromatography-mass spectrometry (LC-MS) and multiplex liquid chromatography-single reaction monitoring (LC-SRM) as practical extensions of standard immunoassay based safety biomarker assessments for identification of new toxicity marker candidates and for improved mechanistic understanding. Two different anticancer drugs, doxorubicin (DOX) and cisplatin (cis-diamminedichloridoplatinum, CDDP), were chosen as the toxicants due to their different modes of nephrotoxicity. Analyses of urine samples from toxicant treated and untreated rats were compared to identify biochemical analytes that changed in response to toxicant exposure. A discovery (label-free LC-MS) and targeted proteomics (multiplex LC-SRM) approach was used in combination with well established immunoassay experiments for the identification of a panel of urinary protein markers related to drug induced nephrotoxicity in rats. The initial generation of an expanded set of markers was accomplished using the label-free LC-MS discovery screen and ELISA based analysis of six nephrotoxicity biomarker proteins. Diagnostic performance of the expanded analyte set was statistically compared to conventional nephrotoxicity biomarkers. False discovery rate (FDR) analysis revealed 18 and 28 proteins from the CDDP and DOX groups, respectively, exhibiting significant differences between the vehicle and treated groups. Multiplex SRM assays were constructed to more precisely quantify candidate markers selected from the discovery screen and immunoassay experiments. To evaluate the sensitivity and specificity for each of the candidate biomarkers, histopathology severity scores were used as a benchmark for renal injury followed by receiver-operating characteristic (ROC) curve analysis on selected biomarkers. Further examination of the best performing analytes revealed relevant biological significance after consideration of anatomical localization and functional roles. In summary, the inclusion of mass spectrometry together with conventional ELISA based assays resulted in the identification of an expanded set of biomarkers with a realistic potential for providing additional beneficial information in mechanistic investigations of drug induced kidney injury and with similar responsiveness to conventionally applied indicators of renal injury.
药物性肾损伤（DIKI）是药物研发中常见的一种毒性反应。我们展示了使用无标记液相色谱 - 质谱联用（LC-MS）和多重液相色谱 - 单反应监测（LC-SRM）作为基于标准免疫分析的安全性生物标志物评估的实际扩展方法，用于鉴定新的毒性标志物候选物以及增进对作用机制的理解。由于两种不同的抗癌药物阿霉素（DOX）和顺铂（顺二氨二氯铂，CDDP）具有不同的肾毒性模式，因此选择它们作为毒物。比较了毒物处理组和未处理组大鼠尿液样本的分析结果，以鉴定因毒物暴露而发生变化的生化分析物。采用了一种发现性（无标记LC-MS）和靶向蛋白质组学（多重LC-SRM）方法，并结合成熟的免疫分析实验，来鉴定一组与大鼠药物性肾毒性相关的尿蛋白标志物。使用无标记LC-MS发现性筛选和基于酶联免疫吸附测定（ELISA）对六种肾毒性生物标志物蛋白的分析，初步生成了一组扩展的标志物。将扩展分析物集的诊断性能与传统肾毒性生物标志物进行了统计学比较。错误发现率（FDR）分析显示，CDDP组和DOX组分别有18种和28种蛋白质在溶剂对照组和处理组之间表现出显著差异。构建了多重SRM分析方法，以更精确地定量从发现性筛选和免疫分析实验中选择的候选标志物。为了评估每种候选生物标志物的敏感性和特异性，将组织病理学严重程度评分用作肾损伤的基准，随后对选定的生物标志物进行受试者操作特征（ROC）曲线分析。对表现最佳的分析物进行进一步检查，在考虑了解剖定位和功能作用后揭示了相关的生物学意义。总之，将质谱分析与传统的基于ELISA的检测方法相结合，导致鉴定出一组扩展的生物标志物，这些生物标志物具有在药物性肾损伤的机制研究中提供额外有益信息的现实潜力，并且对传统应用的肾损伤指标具有相似的反应性。
