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Lawrence H. Lash, Ph.D. |
RESEARCH INTERESTS:
Dr. Lash’s research program over the past nearly three decades has focused on various aspects of determining how chemicals produce injury to the kidneys and how we can design approaches to preventing or correcting such injury. The kidneys are critical for the maintenance of electrolyte and acid-base balance in the body and for reabsorption of nutrients and excretion of waste products. Because of the manner by which these functions are accomplished, the kidneys are very susceptible to injury from many types of chemicals. Consequently, a good understanding of how the kidneys handle and respond to drugs and other chemicals (in physiological, biochemical, and molecular terms) is necessary. Kidney injury can take the form of acute toxicity with failure of organ or cellular function, or chronic toxicity, which may be characterized by decreased organ or cellular function or transformation of kidney cells into tumor cells. Acute toxicity typically occurs with exposures to relatively high doses of chemicals over short periods of time whereas chronic toxicity typically involves exposures to relatively lower doses of chemicals over longer periods of time. The first situation is analogous to overdose exposures to either a drug or environmental chemical or to an accidental exposure to a high amount of an environmental and/or industrial chemical in the workplace. In contrast, the second situation is analogous to a continual or long-term exposure to a relatively low dose of an environmental or industrial chemical.
The chemicals that we have used to produce kidney toxicity fall into one of three categories: 1) Model chemicals that are used to study specific mechanisms of action; 2) pharmacologic agents, such as analgesics or antibiotics; and 3) environmental chemicals, such are trichloroethylene and perchloroethylene. Although some of our studies have been conducted in intact, experimental animals, most of our studies have involved a variety of in vitro systems, including freshly isolated and primary cultures of kidney cells from rats and humans, subcellular fractions, or purified proteins. These type of in vitro model systems have afforded us the opportunity to dissect biochemical and molecular mechanisms of action and to manipulate and specify incubation conditions. Moreover, it is important to note that we have validated these in vitro model systems in terms of their functional integrity and relevance to the normal, in vivo state.
Major Research Accomplishments (1981-2008)
Scientific findings from Dr. Lash’s research, first as a Graduate student with Dr. Dean P. Jones at Emory University School of Medicine in Atlanta, GA (1981 to 1984), then as a postdoctoral fellow with Dr. M.W. Anders at the University of Rochester School of Medicine and Dentistry in Rochester, NY (1985 to 1988), and then as an independent investigator and faculty member at Wayne State University School of Medicine (1988 to present), has been reported in 106 peer-reviewed, primary publications and 51 book chapters and reviews. Some major findings are listed below:
Thiol Transport and Oxidation in Rat Kidney and Small Intestine:
- Discovery and characterization of a sodium-coupled glutathione transporter on renal basolateral membrane that accounts for most of the clearance of glutathione in the renal circulation.
- Demonstrated that this sodium-glutathione cotransport process is a general attribute of epithelial cells, not just the renal proximal tubule, and can function to provide these cells with extracellular glutathione for protection against injury due to oxidants and electrophiles.
- Identification, subcellular localization, purification, and biochemical characterization of a thiol oxidase protein that likely plays a key role in the maintenance of disulfide bonds in extracellular proteins and peptides.
Biochemical and Cellular Mechanisms of Cysteine Conjugate Nephrotoxicity:
- Identification of mitochondria as a major subcellular site of action for nephrotoxic cysteine S-conjugates of halogenated solvents, such as trichloroethylene.
- Purification of the cysteine conjugate b-lyase activity from human kidney cytoplasm and determination that its kinetic efficiency (i.e., Vmax/Km) is approximately 10-fold lower than that of the enzyme from rat kidney cytoplasm. This suggests that human kidney would generate significantly less reactive metabolite than rat kidney from this pathway. This has important implications for human health risk assessment for chemicals such as trichloroethylene, because much of the available data are from studies in rats. Extrapolation of nephrotoxicity data from rats to humans may thus overestimate the risk for toxicity in humans.
Development of In Vitro Model Systems to Study Renal Cell Type Specificity of Toxicity:
- Development of in vitro procedures to isolate cell suspensions from proximal and distal tubular regions of the rat nephron, to enable investigation of factors that determine cell type specificity of susceptibility to pathological and chemically induced injury.
- Demonstration that renal proximal tubular cells are markedly less susceptible than renal distal tubular cells to injury from oxidants, thiol alkylating agents, or agents or conditions that inhibit mitochondrial function and deplete cellular ATP.
- Extension of in vitro cell models to primary culture for rat and human proximal and distal tubules. Development of primary culture procedures whereby the cells retain their differentiated function allows investigation of processes and responses that occur over a longer time period than is possible with the freshly isolated cell models. Detailed quantitation of expression of protein, mRNA, and activity of several key drug metabolism enzymes. This is particularly important if these models are to be used to study chemically induced injury and as predictors of risk for injury, particularly in human kidney.
Sex-, Species-, and Tissue-Dependent Differences in Metabolism and Acute Toxicity of Trichloroethylene and Perchloroethylene:
- Quantitation of metabolism by both glutathione conjugation and cytochrome P450 pathways for trichloroethylene and perchloroethylene in liver and kidney from rats, mice, and humans. This provides information that can be used to improve pharmacokinetic models that are part of human health risk assessment efforts.
- Demonstration of sex- and species-dependent differences in sensitivity to trichloroethylene and perchloroethylene metabolites in model systems for study of acute toxicity.
- Identification of S-(1,2-dichlorovinyl)glutathione (DCVG), the glutathione conjugate of trichloroethylene, in the blood of human volunteers exposed by inhalation to subtoxic levels of trichloroethylene. This finding indicates that the glutathione conjugation pathway for metabolism of trichloroethylene, which is believed to be associated with renal toxicity and renal cancer, occurs in humans. Males exhibited two- to threefold higher levels of DCVG in blood than did females, suggesting that males will have a higher risk for renal toxicity or cancer from exposure to trichloroethylene.
- Characterization in primary cultures of human proximal tubular cells of time and dose dependence of two forms of cell death (apoptosis and necrosis) induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC) (the cysteine conjugate of trichloroethylene that is the penultimate nephrotoxic metabolite).
- Demonstration that S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and DCVC sulfoxide, two penultimate nephrotoxic metabolites of trichloroethylene, cause cytotoxicity by both necrosis and apoptosis in human proximal tubular cells and that sublethal injury of these cells can be followed by repair and renal cellular regeneration.
Compensatory Renal Cellular Hypertrophy and Mercury-Induced Nephrotoxicity:
- Development and validation of several in vitro models from the rat kidney (i.e., freshly isolated and primary cultures of proximal tubular cells, isolated mitochondria, isolated plasma membrane vesicles) to study the influence of compensatory renal cellular hypertrophy, which occurs after a reduction of functional renal mass (e.g., uninephrectomy), on glutathione status and the disposition and toxicity of inorganic mercury. This is a highly significant project with clinical relevance because reduced, functional renal mass is common with aging and various diseases or conditions that damage kidney function.
- Concentrations of glutathione in renal cortical cells are significantly increased, primarily in proximal straight tubule, as a consequence of renal cellular hypertrophy or exposure to subtoxic concentrations of inorganic mercury. This increase is due to a concomitant increase in activity and mRNA expression of the rate-limiting enzyme for synthesis of glutathione.
- Although one might surmise that increased cellular concentrations of glutathione would be protective against nephrotoxicity and cytotoxicity induced by inorganic mercury, compensatory hypertrophy is actually associated with increased susceptibility. The mechanism of this somewhat surprising effect relates to the transport of inorganic mercury into renal proximal tubular cells as a mercury-thiol conjugate.
- Studies conducted both in vivo and in vitro in isolated renal cells and plasma membrane vesicles showed that inorganic mercury is transported predominantly as the cysteine conjugate.
Biochemical and Molecular Mechanisms of Renal Mitochondrial Transport of Glutathione:
- Identification of the dicarboxylate carrier (DIC) and the oxoglutarate carrier (OGC) of the mitochondrial inner membrane as being responsible for transport of cytoplasmic glutathione into renal mitochondria. This is an important finding because it explains how mitochondrial glutathione levels are determined and can allow us to determine its regulation. The significance of this is that glutathione likely plays a central role in maintenance of redox status in mitochondria and prevention of oxidative injury.
- PCR amplification, bacterial expression, purification, and functional reconstitution of the DIC protein: This allows us to characterize the biochemical function of the transporter in great detail and to manipulate the genetics of the protein so that its function can be explored in depth.
- Successfully overexpressed the DIC and OGC in an immortalized renal cell line derived from normal rat kidney proximal tubules, NRK-52E cells.
- Demonstration that expression of either the DIC or the OGC in NRK-52E cells protects those cells from oxidative injury or mitochondrial toxicants.
- Mutant DIC and OGC proteins, generated by site-directed mutagenesis and overexpressed in NRK-52E cells, exhibited markedly reduced transport activities and diminished ability to protect cells from toxicants.
Human Kidney Cells as a Model for Study of Drug Metabolism and Transport:
- Characterized protein expression and transport function of a battery of organic anion, organic cation, and amino acid transporters in primary cultures of human proximal tubular (hPT) cells.
- Demonstrated protein expression that was generally maintained in culture for OAT1-4, OCT2, OCTN2, MRP2, MRP5, P-gp (MDR1), PepT2, and amino acid System L.
- Showed OAT3 was the primary OAT in hPT cells, which contrasts with rat PT cells, where Oat1 is the primary OAT.
- Demonstrated protein expression and activity of a large battery of Phase I and Phase II drug metabolism enzymes in hPT cells, including several cytochrome P450s (CYP), flavin-containing monooxygenases (FMO), GSH S-transferases (GST), UDP-glucuronosyltransferases (UGT), and sulfotransferases (SULT).
Current Research
Current research in Dr. Lash’s laboratory focuses on three projects, one funded by a grant from the Department of Defense Congressionally Directed Medical Research Program and one by a grant from the National Institutes of Health. The first project, “Diabetic Nephropathy and Mitochondrial Function,” is funded by the DOD from 2007 to 2011, and currently focuses on characterization of renal cellular energetics and redox status in diabetic rats, and tests two key hypotheses:
1) Primary cultures of proximal tubular (PT) cells from diabetic rats exhibit enhanced susceptibility to chemically induced oxidative stress and that this is associated with alterations in mitochondrial GSH status, and
2) enhancement of mitochondrial GSH transport function in primary cultures of PT cells from diabetic rats improves cellular function and produces reversion of the cellular phenotype from a diabetic to a non-diabetic state.
These studies grew out of our previous work characterizing the mechanisms of GSH transport in renal mitochondria. In those studies, we demonstrated that two carrier proteins on the mitochondrial inner membrane, the dicarboxylate carrier (DIC) and 2-oxoglutarate carrier (OGC), were responsible for transporting GSH from cytoplasm into mitochondrial matrix and that overexpression of these proteins in a rat renal PT cell line, NRK-52E cells, protected from chemically induced toxicity. The current work applies these findings to development of novel therapeutic approaches to various renal diseases, such as diabetic nephropathy, in which mitochondrial dysfunction and oxidative stress are central components of the disease etiology.
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The second project, “Molecular Toxicology in Human Kidney Cells,” has been funded by the National Institute of Environmental Health Sciences since 1999, and focuses on studying mechanisms of DCVC-induced cell death and proliferation in primary cultures of human proximal tubular (hPT) cells. Trichloroethylene is a significant environmental contaminant and is an established animal carcinogen. Human health risk assessment for trichloroethylene is difficult because of marked sex- and species-dependent differences in metabolism, toxicity, and target organ specificity. Toxic and carcinogenic effects of trichloroethylene in the kidneys are due to its metabolism by glutathione conjugation, subsequent metabolism to the cysteine conjugate DCVC, and metabolism of DCVC by the cysteine conjugate b-lyase or possibly other enzymes to reactive compounds. Rats are the most susceptible species to trichloroethylene-induced kidney toxicity, but there is much disagreement about the kidney as a target organ in humans. We have tested the hypothesis that hPT cells are less susceptible than rat proximal tubular cells to trichloroethylene-induced kidney toxicity because of lower rates of metabolism and/or transport and different toxic responses. In terms of toxic responses, we defined conditions under which DCVC produces hPT cell death by necrosis and apoptosis and assessed the relationship between the well-characterized mitochondrial toxicity of DCVC and DCVC-induced apoptosis. Studies underway are examining signaling pathways, in particular the MAP kinase and protein kinase C pathways, that may mediate renal cellular repair and proliferation that occurs after sublethal injury by DCVC exposure. These studies will enhance our understanding of how DCVC produces renal cell injury in the human kidney and should serve as a model for analysis of species differences in responses to other nephrotoxic chemicals and should enhance our ability to evaluate human susceptibility to chemically induced renal injury.
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Selected Recent References.
L.H. Lash, D.A. Putt, S.E. Hueni and B.P. Horwitz: Molecular markers of trichloroethylene-induced toxicity in human kidney cells. Toxicol. Appl. Pharmacol. 206, 157-168 (2005). PubMed
L.H. Lash: Mitochondrial glutathione transport: Physiological, pathological and toxicological implications. Chem.-Biol. Interact. 163, 54-67 (2006). PubMed
L.H. Lash, D.A. Putt and H. Cai: Membrane transport function in primary cultures of human proximal tubular cells. Toxicology 228, 200-218 (2006). PubMed
Q. Zhong and L.H. Lash: Mitochondrial glutathione transport in diabetic nephropathy. Nephroprevention 2, http://www.nephroprevention.org/ (2007).
L.H. Lash, D.A. Putt and H. Cai: Drug metabolism enzyme expression and activity in primary cultures of human proximal tubular cells. Toxicology 244, 56-65 (2008). PubMed
