尿microRNA作为药物肾毒性评价生物标志物的筛选及其分子机制研究

Currently, drug nephrotoxicity has become the main cause of the drug research suspension and clinical adverse reactions from drug. Due to traditional preclinical renal function markers with low sensitivity, it is urgent necessary for discovering more sensitive and reliable biomarkers for detection of kidney injuries induced by drug. In this study, new urine microRNA biomarkers will be investigated in gentamicin-induced acute kidney injuries (AKI) in rats using high-throughput microarray analysis. The sensitivity and specificity of microRNA biomarkers for detection of renal injuries are compared with that of conventional and novel protein biomarker. Furthermore, the performances of these microRNA biomarkers are also evaluated in rat with AKI induced by distinct nephrotoxicants, and nonrodent species (dog and monkey) with gentamicin-induced AKI. On this basis, two representative candidate microRNAs are selected, and the related target genes and the signaling pathways involved are analyzed using a variety of biological technologies and methods. The molecular mechanisms of microRNAs involved in drug-induced AKI are investigated. The data will provide theoretical basis for urinary microRNA as biomarkers of nephrotoxicity, and facilitate new biomarkers application in preclinical drug toxicity study.

药物肾毒性已成为当前药物中止研发和药品不良反应的主要原因。由于传统的临床前肾毒性评价指标缺乏敏感性，迫切需要寻找更加灵敏、可靠的生物标志物来预测和诊断药物引起的肾损伤。本研究将综合运用高通量芯片分析技术，在庆大霉素诱导的大鼠急性肾损伤动物模型中筛选新的尿microRNA生物标志物，与传统肾功能评价指标和各种新的蛋白类生物标记物进行灵敏度和特异性比较；并在不同作用机制肾毒性药物大鼠模型和不同动物模型（犬和食蟹猴）中比较分析候选microRNA表达水平，深入探讨尿microRNA生物标志物的实用性；以此为基础，选择2种代表性候选microRNA，应用多种生物技术和方法分析其相关靶基因及参与调控的信号通路，试图揭示microRNA参与调控药物诱导肾毒性的内在分子机制，从而为尿microRNA作为药物肾毒性评价的生物标志物提供理论依据，推进新的生物标志物在临床前药物毒性研究中的应用。

药物肾毒性已成为药物中止研发和药品临床不良反应的主要原因。由于传统的临床前肾毒性评价指标缺乏敏感性，迫切需要寻找更加灵敏、可靠的生物标志物来预测和诊断药物引起的肾损伤。本研究将综合运用高通量芯片分析方法和RNA测序技术，系统分析庆大霉素诱导的大鼠、Beagle犬和食蟹猴急性肾损伤动物模型中尿microRNA表达特征，获得庆大霉素诱导肾损伤最常用三种动物模型中尿液miRNA表达谱；并与传统BUN、sCr、肾损伤蛋白新生物标志物以及组织病理学分析结果比较，系统评价miRNA在药物肾损伤模型中诊断性能；证实了尿液miRNA表达在不同种属动物中存在差异性；以此为基础，选择2种代表性候选miRNA(miR-15b和miR-16)，应用采用细胞内双荧光素酶活性检测方法进行其特定靶基因验证，并经Western Blotting分析，发现这两种候选miRNA可通过调节SMAD2和SMAD5的表达影响TGF-β信号通路的活性参与药物性肾损伤过程，这些研究结果为后续尿miRNA作为药物肾毒性评价的生物标志物提供理论依据，从而进一步推进新的生物标志物在临床前药物毒性研究中的应用。