Chronic alcoholism commonly results in deficiency of the vitamin folic acid, because of a folate-depleting effect of ethanol and the generally poor diet of the alcoholic. Studies in humans, monkeys and rats have shown that ethanol consumption produces an excess urinary loss of folate and that this is one of the major mechanisms for the folate depletion. The present application is designed to study the direct effects of ethanol on the kidney's handling of folate and to determine the mechanism of the effect on renal folate transport. Because rats show a marked increase in urinary folate excretion after ethanol treatment, the effect of ethanol on the excretion of 5-methyl-tetrahydrofolate (5-CH3-H2PteGlu), the physiologic, transportable form of folate, will be studied using the isolated perfused rat kidney (IPRK) as a model to eliminate any influences from non-renal factors. Initially, the reabsorption of various doses of 5-CH3-H4PteGlu and the metabolism of this reabsorbed folate by the control IPRK will be measured by high pressure liquid chromatographic (HPLC) analysis of folates in the urine and perfusate. The effects of ethanol on these parameters will be tested by direct administration in the perfusate as well as by acute treatment of the rats prior to preparation of the kidneys for perfusion. Changes in folate excretion will be correlated with changes in proximal tubule folate binding protein (FBP) activity. The mechanism of the increased urinary folate excretion will be studied in cell culture systems from the human and rat kidney proximal tubule (HPT and RPY). HPT and RPT cells will be grown to confluency on filter chambers that allow for separate manipulation of the transport incubation buffer on each side of the cell layer. Folate reabsorption will be modeled by measuring the transport of 5-CH3-H4PteGlu from the apical side through the cells to the basolateral side. Intracellular metabolism to other folates and to polyglutamates on passage through the cell will be measured by HPLC and enzymatic methods. The role of the apical membrane FBP will be studied by covalent or immunologic inactivation of the FBP prior to transport measurements. Studies with ethanol in the incubation buffers will be conducted to define the pathways by which ethanol interferes with ethanol interferes with folate transport pathways. The combination of IPRK and cell culture techniques will be extremely useful in delineating the mechanism by which ethanol increases urinary folate excretion. An understanding of the ethanol-induced increase in urinary folate excretion will help to combat one of the key factors by which folate deficiency is produced.
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