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

摘要： 暴露在广泛的环境污染物中，包括卤化溶剂、多环芳烃 碳氢化合物和重金属会导致肾脏损伤。急性肾损伤(AKI)也是剂量限制的一个方面 几类治疗药物的效果，包括多种抗生素、抗病毒药物、抗癌药物 化疗药物和非甾体抗炎药。尽管一些高度敏感的蛋白质生物标志物已经被 近年来得到证实的是，它们仍然与某种程度的肾脏损害有关。标记可以 表明在任何轻微伤害之前或之后仍可检测到暴露。另外， 识别与作用机制更密切相关的新标记将加强理解 机制和改进治疗的方法。我们识别这种机械标记的方法是 主要集中在线粒体作为近端肾小管细胞常见的、早期的和敏感的靶点 一系列环境污染物和治疗药物。我们将使用近端肾小管的原代培养 取人肾细胞(HPT细胞)作为实验模型系统。我们的总体假设是 HPT细胞暴露于环境或治疗相关浓度的毒物或治疗性药物 药物将分别修饰线粒体和其他细胞成分，并导致选定的 蛋白质和类脂以及代谢物模式的改变。尽管线粒体功能障碍与 各种形式的肾脏损伤和疾病都是有目共睹的，我们的应用这一中心，潜在的 识别机械生物标记物的概念是新的。HPT细胞将用两种环境处理 污染物(三氯乙烯代谢物S-(1，2-二氯乙烯基)-L-半胱氨酸和氯化汞)或三 治疗药物(富马酸替诺福韦、顺铂和多粘菌素B)均以肾脏线粒体为靶点。 此外，抗霉素A将作为阳性对照。初步研究帮助完善了这一假设 并鉴定了三种特定的蛋白质，一种是线粒体(亚硫酸盐氧化酶)，一种是细胞骨架(角蛋白)，还有一种 细胞质(HSP90)，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 采取有针对性的方法来确定从HPT细胞释放的蛋白质作为生物标记物的效用。我们将测试 线粒体亚硫酸盐氧化酶、细胞骨架角蛋白和胞浆热休克蛋白90的释放是否反映了 肾毒物的暴露和近端肾小管毒性。蛋白质的释放将与 肾细胞功能和存活率的参数以及其他公认的生物标志物。《特定目标2》将聚焦于 HPT细胞中修饰(加成或氧化)蛋白作为生物标志物的研究。具体目标3将重点放在发布 心磷脂、其他脂质和中间代谢物作为暴露于和近端的敏感指标 肾毒物引起的肾小管毒性。最后，具体目标4将侧重于潜在的蛋白质鉴定。 在曝光体中，包括在AIMS 1和2中确定的那些，作为另一个潜在的敏感标记的来源 暴露和受伤。

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.