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.

中文翻译：

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发现新型药物诱发的肾损伤生物标志物的多平台方法

药物诱发的肾损伤（DIKI）是在药物开发过程中观察到的常见毒性。我们证明了无标记液相色谱-质谱（LC-MS）和多重液相色谱-单反应监测（LC-SRM）的使用，将其作为基于标准免疫测定的安全生物标记评估的实用扩展，可用于鉴定新的毒性标记候选物并用于增强了对机械的理解。由于其不同的肾毒性方式，选择了两种不同的抗癌药阿霉素（DOX）和顺铂（cis-diamminedichloridoplatinum，CDDP）作为有毒物质。比较了用有毒物质处理过的和未经处理的大鼠的尿液样品分析结果，以鉴定响应于有毒物质暴露而发生变化的生化分析物。发现（无标记LC-MS）和靶向蛋白质组学（多重LC-SRM）方法与成熟的免疫测定实验结合使用，用于鉴定一组与药物诱导的大鼠肾毒性相关的尿蛋白标记物。使用无标记的LC-MS发现筛选和基于ELISA的六种肾毒性生物标记蛋白分析，可以完成一组扩展标记的初始生成。统计上与常规肾毒性生物标志物相比，扩展了分析物组的诊断性能。虚假发现率（FDR）分析显示CDDP和DOX组分别有18和28种蛋白质，在媒介物和治疗组之间显示出显着差异。构建了多重SRM分析，以更精确地量化从发现筛选和免疫分析实验中选择的候选标记。为了评估每种候选生物标记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，然后对所选生物标记物进行受体操作特征（ROC）曲线分析。在考虑解剖定位和功能作用后，对性能最好的分析物的进一步检查显示了相关的生物学意义。总之，