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