SBIR-STTR Award

The kidney is a target of toxicity from drugs, and industrial and environmental chemicals because of its high blood flow, numerous transporters, and reliance on aerobic metabolism. Not surprisingly, mitochondria are a common intracellular target of chemicals in multiple organs, leading to decreased aerobic metabolism and ATP, and cell death. Current in vitro models of nephrotoxicity and mitochondrial damage are inadequate for many of the same reasons: cultured cells are very glycolytic with minimal aerobic metabolism, and there are no moderate or high-throughput real-time metabolomic assays. Consequently, new cellular models and metabolomic methodologies are needed to evaluate nephrotoxicity and mitochondrial damage. We have developed primary cultures of renal proximal tubular cells (RPTC) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions. In conjunction, we have a new technology (Seahorse Extracellular Flux Analyzer) to measure cell metabolism (oxygen consumption and acid extrusion) in real time in 24-well plates. The long-term goal of this proposal is to merge the RPTC model and the Seahorse technology to develop a quantitative high-throughput assay (qHTS) to measure the effects of toxicants on renal mitochondrial function. Phase I of the proposed research has two aims: (1) respiratory measurements for RPTC will be optimized for sensitivity and precision in a 96-well format;(2) the optimized metabolic assay integrated with automated imaging will be tested against a selection of clinically relevant nephrotoxicants and non-nephrotoxicants. Phase II of the research will use these results to develop a 96-well based qHTS format and validate it with 1400 TOXNET compound library. This assay system will identify nephrotoxicants with mechanism- based criteria for assessment of new drugs, consumer products, and environmental agents. , ,

Public Health Relevance:
The final results of the proposed research will be a quantitative high-throughput assay that can assess new drugs, consumer products, and environmental agents for their potential to cause kidney damage in humans.

Thesaurus Terms:
Acids;Address;Annexins;Apoptosis;Apoptosis Pathway;Assay;Binding;Binding (Molecular Function);Bioassay;Biologic Assays;Biological Assay;Blood Flow;Buffers;Calcimedins;Cell Death;Cell Death, Programmed;Cell Respiration;Cell Membrane;Cell Model;Cells;Cellular Respiration;Cellular Injury;Cellular Model;Chemicals;Concentration Measurement;Cultured Cells;Cytoplasmic Membrane;Dose;Drug Toxicity;Drugs;Electron Transport;Evaluation;Exhibits;Genomics;Genus Hippocampus;Goals;Health Sciences;High Throughput Assay;Human;Human, General;It Systems;Image;In Vitro;Information Systems;Information Technology Systems;Intermediary Metabolism;Kidney;Libraries;Lipocortins;Metbl;Man (Taxonomy);Man, Modern;Measurement;Measures;Medication;Metabolic;Metabolic Processes;Metabolism;Method Loinc Axis 6;Methodology;Methods;Mitochondria;Molecular Interaction;Nih;National Institutes Of Health;National Institutes Of Health (U.S.);Oligomycins;Organ;Oxygen Consumption;Pharmaceutic Preparations;Pharmaceutical Preparations;Phase;Plasma Membrane;Proxy;Reliance;Renal Cell;Research;Respiration;Screening Procedure;Seahorse;Staining Method;Stainings;Stains;Stress Tests;System;System, Loinc Axis 4;Systems, Data;Technology;Testing;Time;Toxic Effect;Toxicities;Toxicology Data Network;Tubular;Tubular Formation;United States National Institutes Of Health;Urinary System, Kidney;Validation;Aerobic Metabolism;Aerobic Respiration;Base;Cell Damage;Cell Imaging;Cell Injury;Cellular Imaging;Clinical Relevance;Clinically Relevant;Consumer Product;Drug/Agent;Electron Transfer;Environmental Agent;Environmental Chemical;Extracellular;Fluorophore;High Throughput Screening;Imaging;In Vitro Model;In Vivo;Instrument;Kidney Cell;Measurement Of Metabolism;Member;Metabolomics;Mitochondrial;Necrocytosis;Nephrotoxicity;New Technology;Novel;Oxidative Metabolism;Plasmalemma;Public Health Relevance;Renal;Respiratory;Respiratory Mechanism;Screening;Screenings;Toxicant

The kidney is a target of toxicity from drugs, and industrial and environmental chemicals because of its high blood flow, numerous transporters, and reliance on aerobic metabolism. Not surprisingly, mitochondria are a common intracellular target of chemicals in multiple organs, leading to decreased aerobic metabolism and ATP, and cell death. Current in vitro models of nephrotoxicity and mitochondrial damage are inadequate for many of the same reasons: cultured cells are very glycolytic with minimal aerobic metabolism, and there are no moderate or high-throughput real-time metabolomic assays. Consequently, new cellular models and metabolomic methodologies are needed to evaluate nephrotoxicity and mitochondrial damage. We have developed primary cultures of renal proximal tubular cells (RPTC) that exhibit in vivo levels of aerobic metabolism, are not glycolytic and retain higher levels of differentiated functions. We previously developed primary cultures of renal proximal tubular cells (RPTC) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions. The goal of the Phase I proposal was to merge our novel and relevant RPTC model and the Seahorse technology to develop a high-throughput assay to accurately measure nephrotoxicity. In addition to completing the objectives described in the Phase I aims, we also developed a cheminformatic strategy in which chemical similarity is used to cluster molecules that are then modeled to define a potential ""toxicophore"" of similar physicochemical features in 3-dimesional space. These chemical entities/toxicophores that damage mitochondria are predicted to be nephrotoxicants. Our goals for Phase are to validate our integrated metabolic and imaging assay using the TOXNET &Toxcast land to use cheminformatic analyses to develop a toxicophore database. This RPTC/Seahorse platform will identify nephrotoxicants and mitochondrial toxicants and provide public companies and regulatory agencies with mechanism and chemical-based criteria for assessing and predicting nephrotoxicity and mitochondrial toxicity of new drugs, consumer products, and environmental agents, and shorten the overall time to identify potential problem chemicals. The commercialization plan is to offer these screening and cheminformatic services to pharmaceutical &federal agencies.

Public Health Relevance:
The final results of the proposed research will be a quantitative high-throughput assay that can assess new drugs, consumer products, and environmental agents for their potential to cause kidney damage in humans.

Thesaurus Terms:
Acids;Animal Testing;Assay;Benchmarking;Best Practice Analysis;Bioassay;Biochemical;Biologic Assays;Biologic Products;Biological;Biological Agent;Biological Assay;Biological Products;Blood Flow;Cell Death;Cell Respiration;Cell Survival;Cell Viability;Cell Model;Cells;Cellular Respiration;Cellular Injury;Cellular Model;Chemicals;Cultured Cells;Data;Data Banks;Data Bases;Databanks;Databases;Development;Domestic Rabbit;Drug Toxicity;Drugs;Electronic Databank;Electronic Database;Evaluation;Exhibits;Genus Hippocampus;Goals;High Throughput Assay;Human;Image;Informatics;Intermediary Metabolism;Kidney;Kidney Urinary System;Loinc Axis 2 Property;Libraries;Light;Man (Taxonomy);Measures;Medication;Metabolic;Metabolic Processes;Metabolism;Method Loinc Axis 6;Methodology;Mitochondria;Modeling;Modern Man;Organ;Oryctolagus Cuniculus;Oxygen Consumption;Pharmaceutic Preparations;Pharmaceutical Agent;Pharmaceutical Preparations;Pharmaceuticals;Pharmacologic Substance;Pharmacological Substance;Phase;Photoradiation;Property;Protocol;Protocols Documentation;Proxy;Quality Control;Rabbits;Rabbits Mammals;Reliance;Renal Tissue;Research;Risk;Screening Procedure;Seahorse;Services;Toxnet;Technology;Testing;Time;Toxic Effect;Toxicities;Toxicogenomics;Toxicology Data Network;Tubular;Tubular Formation;Validation;Variant;Variation;Aerobic Metabolism;Aerobic Respiration;Base;Biopharmaceutical;Biotherapeutic Agent;Cell Damage;Cell Injury;Chemical Informatics;Chemical Library;Cheminformatics;Clinical Data Repository;Commercialization;Comparative;Consumer Product;Data Repository;Developmental;Drug/Agent;Environmental Agent;Environmental Chemical;Exposed Human Population;Extracellular;High Throughput Screening;Human Exposure;Imaging;In Vitro Model;In Vivo;Metabolism Measurement;Metabolomics;Mitochondrial;Mitochondrial Dysfunction;Necrocytosis;Nephrotoxicity;New Technology;Novel;Novel Technologies;Oxidative Metabolism;Protein Expression;Renal;Respiratory;Screening;Screenings;Small Molecule Libraries;Tool;Toxicant