Research Interests The folate deficiency of chronic alcoholism results from a combination of factors, including a direct effect of ethanol to deplete folate. Our studies have shown that acute or chronic treatment of rats with ethanol increases urinary folate excretion, in amounts that could explain the folate-depleting effects. In both the isolated perfused rat kidney and in cultured cells from the rat and human proximal tubule (PT), we have confirmed that ethanol blocks reabsorptive transport of 5-methyl-tetrahydrofolate (5MTHF). Currently, our studies in cultured cells are designed to determine the mechanism for the ethanol-induced increase in excretion. We have shown that two pathways are involved in reabsorptive transport of folate, endocytosis by the folate binding protein (folate receptor) or anion exchange by the reduced folate carrier. Our studies are investigating the regulation of these two proteins and how ethanol interferes with the activity of one of the pathways using pharmacological and kinetic methods. Ethylene glycol poisoning occurs infrequently, but produces severe morbidity and mortality. A typical feature of the poisoning syndrome is the development of renal failure, due to a toxic nephropathy (proximal tubular necrosis). Ethylene glycol is metabolized to two toxic metabolites, glycolate and oxalate. We have shown that the renal toxicity of ethylene glycol results from the accumulation of oxalate crystals in the proximal tubule. We are conducting studies to determine the mechanism by which oxalate affects the PT cell so as to produce cell death. Initial studies indicate that oxalate inhibits mitochondrial respiration, thereby inducing the mitochondrial permeability transition, and leading to loss of cellular energy production. Calcium oxalate also interacts with membrane phospholipids, which could contribute to cytotoxicity. Because of these membrane interactions, we are studying the potential therapeutic effects of aluminum citrate and related compounds, since these compounds have been shown to block oxalate crystal binding to cells as well as their cytotoxicity. A third project is examining the mechanism for the renal toxicity of diethylene glycol, a substance that has been involved in a number of mass poisonings world-wide. DEG is metabolized to several compounds and we have demonstrated that formation of these metabolites is necessary for DEG to produce toxicity. Current studies are examining the toxicity of the metabolites in cultured kindey cells as well as in animal models in order to determine which is the toxic metabolite. Subsequent studies will address the mechanisms by which the metabolite produces cell death.
Specialized Techniques/Procedures Epithelial cell culture Cytotoxicity assays HPLC Gas chromatography (GC) UV-visible spectrophotometry Differential scanning calorimetry Radiometabolic determinations Membrane transport kinetics Drug metabolism and kinetics Mitochondrial isolation and respiration experiments