Mitochondrial and Cellular Biomarkers of Renal Injury from Environmental and Therapeutic Agents

摘要： 暴露于各种环境污染物，包括卤化溶剂、多环芳烃 碳氢化合物和重金属会导致肾损伤。急性肾损伤（阿基）也是一个剂量限制性方面 几类治疗药物的作用，包括许多抗生素，抗病毒剂，抗癌药， 化疗剂和NSAID。尽管一些高度敏感的蛋白质生物标志物已经被 尽管近年来得到证实，但它们仍与一定程度的肾损伤有关。标记可以 优选在任何损伤之前或仅在最小损伤之后检测到指示暴露。此外，本发明还 确定与作用机制更密切相关的新标志物将增进了解， 机制和改善治疗方法。我们鉴定这种机械标记物的方法 主要集中在线粒体作为常见的，早期的，和敏感的目标，在近端肾小管细胞， 一系列环境污染物和治疗药物。我们将使用原代培养的近端肾小管 来自人肾的细胞（hPT细胞）作为实验模型系统。我们的总体假设是 将hPT细胞暴露于环境或治疗相关浓度的毒物或治疗剂 试剂将分别修饰线粒体和其他细胞组分，并导致释放选定的 蛋白质和脂质以及改变的代谢物模式。虽然线粒体功能障碍与 各种形式的肾损伤和疾病是公认的，我们的应用这一中心，基础 识别基于机械的生物标志物的概念是新颖的。hPT细胞将用两种环境 污染物（三氯乙烯代谢产物S-（1，2-二氯乙烯基）-L-半胱氨酸[DCVC]和HgCl 2）或三种 治疗剂（富马酸替诺福韦酯、顺铂和多粘菌素B）均靶向肾线粒体。 此外，抗霉素A将用作阳性对照。初步研究有助于完善这一假设 并确定了三种特异性蛋白质，一种是线粒体蛋白（亚硫酸氧化酶），一种是细胞骨架蛋白（角蛋白）， 细胞质（HSP 90），作为潜在的生物标志物，并采取有针对性的方法进行验证。具体目标1将 采取靶向方法来确定从hPT细胞释放的蛋白质作为生物标志物的效用。我们将测试 线粒体亚硫酸盐氧化酶、细胞骨架角蛋白和细胞质HSP 90的释放是否反映了 暴露于肾毒物和近端肾小管毒性。蛋白质的释放将与 肾细胞功能和存活力的参数以及其他公认的生物标志物。第2章重点 作为生物标志物的hPT细胞中修饰的（加合或氧化的）蛋白质。具体目标3将集中在释放 心磷脂、其他脂质和中间代谢物作为暴露和近端的敏感指标 肾毒物的肾小管毒性。最后，具体目标4将侧重于蛋白质的潜在鉴定 在染色体中，包括在目的1和2中鉴定的那些，作为敏感的标记物的另一个潜在来源， 暴露和伤害。

Abstract: Exposure to a broad range of environmental contaminants, including halogenated solvents, polycyclic aromatic hydrocarbons, and heavy metals, can cause kidney injury. Acute kidney injury (AKI) is also a dose-limiting side effect of several classes of therapeutic drugs, including many antibiotics, antiviral agents, anticancer chemotherapeutic agents, and NSAIDs. Although some highly sensitive protein biomarkers have been validated in recent years, they are still associated with some degree of renal damage. Markers that can indicate exposure yet be detected prior to any or after only minimal injury are preferable. Additionally, identification of new markers that are more closely linked to mechanism of action will enhance understanding of mechanism and improve therapeutics. Our approach to identification of such mechanistic markers has focused primarily on the mitochondria as common, early, and sensitive targets in proximal tubular cells for an array of environmental contaminants and therapeutic drugs. We will use primary cultures of proximal tubular cells from human kidneys (hPT cells) as the experimental model system. Our overall hypothesis is that exposure of hPT cells to environmentally or therapeutically relevant concentrations of toxicants or therapeutic agents, respectively, will modify mitochondria and other cellular components and result in release of selected proteins and lipids and altered patterns of metabolites. Although association of mitochondrial dysfunction with various forms of kidney injury and disease is well-established, our application of this central, underlying concept to identify mechanistically-based biomarkers is novel. hPT cells will be treated with two environmental contaminants (the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine [DCVC] and HgCl2) or three therapeutic agents (tenofovir disoproxil fumarate, cisplatin, and polymyxin B) that all target renal mitochondria. Additionally, antimycin A will be used as a positive control. Preliminary studies helped refine the hypothesis and identified three specific proteins, one mitochondrial (sulfite oxidase), one cytoskeletal (keratins), and one cytoplasmic (HSP90), as potential biomarkers and take a targeted approach for validation. Specific Aim 1 will take a targeted approach to determine the utility of released proteins from hPT cells as biomarkers. We will test whether release of mitochondrial sulfite oxidase, cytoskeletal keratins, and cytoplasmic HSP90 reflect exposure to and proximal tubular toxicity from nephrotoxicants. Release of proteins will be correlated with parameters of renal cell function and viability and other well-established biomarkers. Specific Aim 2 will focus on modified (adducted or oxidized) proteins in hPT cells as biomarkers. Specific Aim 3 will focus on release of cardiolipins, other lipids, and intermediary metabolites as sensitive indicators of exposure to and proximal tubular toxicity from nephrotoxicants. Finally, Specific Aim 4 will focus on the potential identification of proteins in exposomes, including those identified in Aims 1 and 2, as another potential source of sensitive markers of exposure and injury.