GLUTATHIONE AND MITOCHONDRIA IN TOXIC RENAL INJURY

Summary

Principal Investigator: Lawrence Lash
Abstract: DESCRIPTION: (Adapted from the investigator's abstract): The previous work focused on biochemical properties of glutathione (GSH) transport in isolated renal cells and subcellular organelles. The proposed research will extend those findings by investigating the molecular properties of GSH carrier proteins in kidney mitochondria. The investigators demonstrated previously that two of the known, organic anion carriers of the mitochondrial inner membrane, the dicarboxylate carrier (DCC) and the oxoglutarate carrier (OGC), account for most of the uptake of GSH from the cytoplasm into mitochondria. Specific Aim 1 will involve cloning, expression, purification, and functional characterization of the role of the DCC and OGC proteins in GSH transport. The DCC and OGC genes will be cloned from total rat kidney RNA by RT-PCR, will be expressed in bacteria, purified, and reconstituted into proteoliposomes. The kinetics and inhibitor and substrate specificity of the two carriers will be studies in detail. Specific Aim 2 will test the hypothesis that cellular and mitochondrial function differ in cells transfected with wild-type or mutant GSH carriers. DCC and OGC cDNA clones will be manipulated by site-directed mutagenesis using PCR. Wild-type and mutant genes for these carriers will be expressed in bacteria, purified, and reconstituted into proteoliposomes to assess their activity. Clones of wild-types and mutant carriers will be transfected into stable renal cell line, NRK-52E cells, and the effect of different activity levels of GSH transport on mitochondrial function will be assessed. Mitochondrial play a key role in cellular energetics and in the processes of cellular necrosis and apoptosis. Specific Aim 3 will test the hypothesis that cells transfected with wild-type or mutant mitochondrial GSH carriers have different susceptibilities to oxidant injury, apoptosis and necrosis. Oxidant injury in transfected NRK-52E cells will be induced by tert-butyl hydroperoxide. Cellular and mitochondrial function will be assessed by measurements of respiration, active transport, lipid peroxidation, and GSH status and subcellular distribution. Apoptosis will be quantitated by subdiploid DNA analysis with flow cytometry, measurement of cytochrome c release from mitochondria, activation of caspase-3, and the TUNEL assay for DNA fragmentation. Necrosis will be quantitated by measurements of lactate dehydrogenase release from cells. For each parameter, time and concentration dependent effects will be correlated with GSH transport activity to assess the role of the GSH carriers in the mitochondrial response to toxicants. Achievement of these aims will expand our knowledge of the function of these carriers. This information may have therapeutic applications for prevention of renal cellular injury or for understanding mitochondrial diseases or age-related decreases that occur in mitochondrial function.
Funding Period: 1988-04-01 - 2005-03-31
more information: NIH RePORT

Top Publications

  1. ncbi Renal membrane transport of glutathione in toxicology and disease
    L H Lash
    Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA
    Vet Pathol 48:408-19. 2011
  2. ncbi Role of glutathione transport processes in kidney function
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, Michigan 48201, USA
    Toxicol Appl Pharmacol 204:329-42. 2005
  3. pmc Role of organic anion and amino acid carriers in transport of inorganic mercury in rat renal basolateral membrane vesicles: influence of compensatory renal growth
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA
    Toxicol Sci 88:630-44. 2005
  4. ncbi Growth hormone administration to aged animals reduces disulfide glutathione levels in hippocampus
    Ashley N Donahue
    Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Medical Center Boulevard, Winston Salem, NC 27157 1083, USA
    Mech Ageing Dev 127:57-63. 2006
  5. pmc Modulation of expression of rat mitochondrial 2-oxoglutarate carrier in NRK-52E cells alters mitochondrial transport and accumulation of glutathione and susceptibility to chemically induced apoptosis
    Feng Xu
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Ave, Detroit, MI 48201, USA
    J Pharmacol Exp Ther 316:1175-86. 2006
  6. pmc Mitochondrial glutathione transport: physiological, pathological and toxicological implications
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA
    Chem Biol Interact 163:54-67. 2006
  7. ncbi Influence of compensatory renal growth on susceptibility of primary cultures of renal cells to chemically induced injury
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA
    Toxicol Sci 94:417-27. 2006
  8. pmc Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, United States
    Chem Biol Interact 170:124-34. 2007
  9. pmc Hepatic mitochondrial transport of glutathione: studies in isolated rat liver mitochondria and H4IIE rat hepatoma cells
    Qing Zhong
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201 1928, USA
    Arch Biochem Biophys 474:119-27. 2008

Scientific Experts

Detail Information

Publications9

  1. ncbi Renal membrane transport of glutathione in toxicology and disease
    L H Lash
    Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA
    Vet Pathol 48:408-19. 2011
    ....
  2. ncbi Role of glutathione transport processes in kidney function
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, Michigan 48201, USA
    Toxicol Appl Pharmacol 204:329-42. 2005
    ..Hence, membrane transport processes are critical to regulation of renal cellular and subcellular GSH pools and are determinants of susceptibility to cytotoxicity induced by oxidants and electrophiles...
  3. pmc Role of organic anion and amino acid carriers in transport of inorganic mercury in rat renal basolateral membrane vesicles: influence of compensatory renal growth
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA
    Toxicol Sci 88:630-44. 2005
    ..These results support the hypothesis that compensatory renal growth is associated with increased uptake of Hg(2+) in proximal tubular cells and we have identified specific transporters involved in the process...
  4. ncbi Growth hormone administration to aged animals reduces disulfide glutathione levels in hippocampus
    Ashley N Donahue
    Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Medical Center Boulevard, Winston Salem, NC 27157 1083, USA
    Mech Ageing Dev 127:57-63. 2006
    ..We conclude that the age-related decline in circulating growth hormone and IGF-1 contribute to increased oxidative stress in hippocampus with age...
  5. pmc Modulation of expression of rat mitochondrial 2-oxoglutarate carrier in NRK-52E cells alters mitochondrial transport and accumulation of glutathione and susceptibility to chemically induced apoptosis
    Feng Xu
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Ave, Detroit, MI 48201, USA
    J Pharmacol Exp Ther 316:1175-86. 2006
    ..These results provide further evidence that genetic manipulation of mitochondrial GSH transporter expression alters mitochondrial and cellular GSH status, resulting in markedly altered susceptibility to chemically induced apoptosis...
  6. pmc Mitochondrial glutathione transport: physiological, pathological and toxicological implications
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA
    Chem Biol Interact 163:54-67. 2006
    ..Several conditions, such as alcoholic liver disease, cirrhosis or other chronic biliary obstructive diseases, and diabetic nephropathy, are associated with depletion or oxidation of the mitochondrial GSH pool in liver or kidney...
  7. ncbi Influence of compensatory renal growth on susceptibility of primary cultures of renal cells to chemically induced injury
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA
    Toxicol Sci 94:417-27. 2006
    ..This is the first report validating the use of a mammalian renal cell culture model to study the toxicological effects of compensatory renal cellular hypertrophy...
  8. pmc Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione
    Lawrence H Lash
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, United States
    Chem Biol Interact 170:124-34. 2007
    ..We conclude that Oat3 can function in GSH uptake and that NRK-52E cells possess a low background rate of GSH uptake, making these cells a good model for overexpression of specific, putative GSH carriers...
  9. pmc Hepatic mitochondrial transport of glutathione: studies in isolated rat liver mitochondria and H4IIE rat hepatoma cells
    Qing Zhong
    Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201 1928, USA
    Arch Biochem Biophys 474:119-27. 2008
    ....