利用离体大鼠肾脏灌流模型鉴定肾脏损伤相关的代谢生物标志物

The low abundant biomarkers that originate from kidney tend to be more associated with the renal pathophysiology. In previous metabolomics studies based on profiling differential mass spectrometric techniques, the high abundant plasma-derived metabolic biomarkers were repeatedly identified, however the low abundant kidney origin biomarkers were difficult to be identified due to the presence of high abundance suppression. Multiple reaction monitoring (MRM)-based targeted metabolomics technology has higher sensitivity, but it need to know in advance the candidate biomarkers. Therefore, an effective strategy is as follows: the low abundant candidate biomarkers associated with renal damage are firstly identified by excluding of the interference from plasma metabolite based on animal models, then are screened in the human using the MRM-based target metabolic techniques. This study used isolated rat kidney perfusion model to exclude the interference of plasma-derived metabolite, the perfusion-driven urines under varying degrees of renal impairment were acquired by perfused the kidney with and without oxygen supplement in succession. Then the mass spectrometry technology was used to study the changes of metabolites in the perfusion-driven urines and to identify the metabolic biomarkers associated with hypoxic-ischemic renal injury. Finally the MRM based quantitative analysis methods for these metabolic biomarkers were established, and would be used for the targeted screening and validation of these biomarkers in the large-scale clinical patients’ samples in the future, and finally would to lay the foundation for the diagnosis and prognosis of patients with renal injury.

肾脏来源的低丰度生物标志物往往与肾脏病理生理变化更相关。既往研究采用全谱分析的差异代谢组学技术鉴定尿液中肾脏疾病的代谢生物标志物，由于高丰度抑制现象，血浆来源的高丰度代谢生物标志物被重复鉴定，而肾脏来源的低丰度生物标志物很难被鉴定到。以多反应监测（MRM）为基础的靶向代谢组学技术有更高的灵敏度，但是需要预先知道候选生物标志物。因此有效的策略是通过动物模型排除尿液中来源于血浆的代谢物干扰，鉴定到肾损伤相关的候选生物标志物后再采用MRM技术在人类疾病样本中进行靶向筛选。本课题采用离体大鼠肾脏灌流模型排除掉血浆干扰，先后进行有氧和无氧灌流获得不同肾损伤程度下肾脏产生的尿液，运用质谱技术研究尿液中代谢物的变化，鉴定缺血缺氧肾损伤相关的代谢生物标志物。最后建立这些代谢生物标志物的MRM定量分析方法，用于以后大规模临床病人样本中代谢生物标志物的靶向筛选和验证，为临床病人肾损伤的诊断和预后评估打下基础。

本项目旨在鉴定尿液中肾脏来源的低丰度代谢物。肾脏来源的低丰度生物标志物往往与肾脏病理生理变化更相关。由于高丰度抑制现象，既往的差异代谢组学研究中，血浆来源的高丰度代谢生物标志物被重复鉴定，而肾脏来源的低丰度生物标志物很难被鉴定到。本课题采用离体大鼠肾脏灌流模型排除掉血浆干扰，运用质谱技术研究灌流产生尿液中代谢物的变化，鉴定缺血缺氧肾损伤相关的肾脏来源尿液代谢生物标志物。首先优化了离体肾脏灌流模型的手术方法，提高了手术的成功率。其次通过定量分析离体灌流过程中6个时间点灌流产生尿液代谢物，鉴定随时间延长具有升高趋势的代谢物。由于排除了血浆的干扰，在灌流过程中表达升高的代谢物是肾脏来源的尿液代谢物。本研究进一步对这些代谢物进行二级质谱鉴定，所获得的MS/MS质谱图为建立MRM方法提供离子对信息。另外，本研究在原项目目标之外，同时对正常大鼠尿液蛋白、离体肾脏灌流产生尿液蛋白进行定量分析，鉴定了肾脏来源的尿液蛋白，并对肾脏来源的尿液代谢物和尿液蛋白进行多组学生物信息学分析，为肾脏缺血缺氧损伤的机制研究提供了线索，同时为肾脏疾病生物标志物研究筛选肾脏来源的尿液生物标志物提供了参照数据库。