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Lawrence H. Lash, Ph.D.
Department of Pharmacology |
RESEARCH INTERESTS:
Overview. 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 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. We have also explored the impact of chronic diseases (e.g., diabetes) and pathological states (e.g., reduced nephron mass with compensatory renal hypertrophy) on susceptibility of the kidneys to chemically induced toxicity. 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 types of in vitro model systems have afforded us the opportunity to dissect biochemical and molecular mechanisms of action, to manipulate and specify incubation conditions and to validate them in terms of functional integrity and relevance to the normal, in vivo state.
Key research accomplishments.
- Discovery and characterization of a sodium-coupled GSH transporter on renal basolateral membrane that accounts for most of the clearance of GSH in the renal circulation.
Identification of mitochondria as a major subcellular site of action for nephrotoxic cysteine S-conjugates of halogenated solvents, such as trichloroethylene, in rat and human proximal tubular cells. - 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.
- Characterization of sex- and species-dependent differences in metabolism and sensitivity to trichloroethylene and perchloroethylene, providing data for human health risk assessment.
- Identification of the dicarboxylate carrier (DIC; Slc25a10) and the oxoglutarate carrier (OGC; Slc25a11) of the mitochondrial inner membrane as being responsible for transport of cytoplasmic GSH into renal and hepatic mitochondria.
- Characterized protein expression and transport function of a battery of organic anion, organic cation, and amino acid transporters and expression of Phase I and Phase II drug metabolism enzymes in primary cultures of human proximal tubular cells, validating these cells for use in drug development.
- Demonstrated adaptive changes in mitochondrial GSH transport and redox status in proximal tubular cells from kidney of diabetic or uninephrectomized rats.
Current Research Directions.
| “Mitochondrial Redox Status in Diabetic Nephropathy”: Our overall goals are to increase understanding of the underlying biochemistry of the diabetic renal proximal tubule and ultimately develop a novel therapeutic approach. To accomplish this, we focus primarily on what is considered the primary source of cellular dysfunction, the mitochondria, and on modulation of mitochondrial GSH status. Studies will use renal tissue and primary cultures of PT cells from streptozotocin (STZ)-induced diabetic and age-matched control Sprague-Dawley rats as a Type 1 diabetes model, and Zucker obese diabetic and Zucker lean control rats as a Type 2 diabetes model. We will test the hypothesis that overexpression of mitochondrial GSH transporters in proximal tubular cells of diabetic rats reverts the cells to a normal phenotype. |  |
We will test the hypothesis that overexpression of mitochondrial GSH transporters in proximal tubular cells of diabetic rats reverts the cells to a normal phenotype. We hypothesize that overexpression of the DIC and/or the OGC in primary cultures of renal PT cells from diabetic rats can produce a sustained improvement in mitochondrial function and effectively convert the cellular phenotype to that of the normal PT cell. We will then test the hypothesis that modification of specific proteins and signaling pathways are associated with the diabetic nephropathy phenotype and with reversion of the phenotype in cells overexpressing mitochondrial GSH transporters. We propose to identify specific proteins, largely mitochondrial in origin, that either undergo covalent or oxidative modification or are altered in abundance in renal PT cells as a consequence of diabetic nephropathy and that are responsive to overexpression of mitochondrial GSH transporters.
“Renal Mitochondria as Sentinels for Environmental Exposures”: Our overall hypothesis is that renal mitochondria can serve as sentinels for exposures to certain environmental toxicants and that both proteins and low-molecular-weight compounds are released from renal cells and can be used as sensitive biomarkers. We propose that changes in either proteins (abundance of or unique modifications in key mitochondrial proteins) or unique mitochondrial metabolites will occur in response to exposures to nephrotoxic chemicals that target renal mitochondria and that these will be released into the tubular lumen and ultimately secreted in urine. Two biological models will be used: 1) Primary cultures of proximal tubular cells from human and rat kidney (hPT and rPT) for discovery and mechanistic studies and 2) the male F344 rat for in vivo validation. Two classes of chemicals will be studied: 1) Cysteine conjugates that target renal mitochondria and are derived from nephrotoxic halogenated solvents, namely S-(1,2-dichlorovinyl)-L-cysteine (DCVC; penultimate nephrotoxic metabolite of trichloroethylene [TRI]) and 1,2,3,4,4-pentachloro-1,3-butadienyl-L-cysteine (PCBC; penultimate nephrotoxic metabolite of hexachloro-1,3-butadiene [HCBD]); and 2) two model toxicants, tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK), which cause renal mitochondrial toxicity and exemplify the two distinct mechanisms of oxidative stress induction by the cysteine conjugates, namely generation of reactive oxygen species (ROS) with lipid peroxidation (i.e., tBH) and thiol alkylation (i.e., MVK).
“Redox Therapy to Treat Environmentally-Induced Kidney Cancer”: Our overall hypothesis is that mitochondrial redox status is an appropriate therapeutic target for treatment of kidney cancer induced by exposure to the environmental contaminant trichloroethylene (TRI). Mitochondrial dysfunction is widely believed to be an underlying factor in numerous diseases and pathological states, including many cancers. Furthermore, oxidative stress is known to be an underlying mechanism that mediates this dysfunction. We propose that overexpression of mitochondrial GSH transporters in PT cells from human kidney will produce a sustained improvement in mitochondrial and cellular redox status, thereby correcting or inhibiting the deleterious effects of TRI.
“Polymyxin Antibiotic Nephrotoxicity”: Two research collaborations are underway to improve the therapeutic efficacy and safety of polymyxin antibiotics. One project involves collaboration with medicinal chemists to develop novel polymyxin derivatives in which efficacy is improved while adverse side effects (i.e., nephrotoxicity) are diminished. Primary cultures of hPT cells will be used to screen for nephrotoxicity. The other project involves modulation of renal transport to minimize renal accumulation of and toxicity induced by polymyxins, thereby enhancing their therapeutic efficacy.
Selected Recent References.
F. Xu, I. Papanayotou, D.A. Putt, J. Wang and L.H. Lash: Role of mitochondrial dysfunction in cellular responses to S-(1,2-dichlorovinyl)-L-cysteine in primary cultures of human proximal tubular cells. Biochem. Pharmacol. 76, 552-567 (2008). [PMID = 18602084]
L.H.Lash: Renal glutathione transport: Identification of carriers, physiological functions, and controversies. BioFactors 35, 500-508 (2009). [PMID: 19904718]
Q. Zhong, S.R. Terlecky, and L.H. Lash: Diabetes increases susceptibility of rat proximal tubular cells to chemical injury. Toxicol. Appl. Pharmacol. 241, 1-13 (2009). [PMID: 19682476]
B. Benipal, and L.H. Lash. Influence of renal compensatory hypertrophy on mitochondrial energetics and redox status. Biochem. Pharmacol. 81, 295-303 (2011). [PMID: 20959115]
D.A. Putt, Q. Zhong, and L.H.Lash. Adaptive changes in renal mitochondrial redox status in diabetic nephropathy. Toxicol. Appl. Pharmacol., doi: 10.1016/j.taap.2011.10.021 (2011). [PMID: 22085922]
