Many drugs, chemical carcinogens and other toxic substances are eliminated from the body primarily by transporters in the kidney. Renal transporters also influence the pharmacokinetics of drugs and other xenobiotics by selective reabsorption. The overall goal of the studies in this continuing renewal application is to understand the mechanisms by which drugs are transported in the kidney. Studies will focus primarily on organic cations. In addition, we will pursue our recent exciting findings of the mechanism of transport of nucleosides and nucleoside analogs in the kidney.
The specific aims of the studies of organic cation transport are (a) to determine the molecular structure of OCPA1, the organic cation/proton antiporter responsible for the transport of many basic drugs across the renal brush border membrane; (b) to determine the tissue distribution of the mRNA transcript encoding OCPA1; (c) to determine the levels of the specific OCPA1 mRNA transcript in renal cells in which OCPA1 has been """"""""induced"""""""" by selected organic cations; (d) to determine whether OCPA2, a second organic cation proton antiporter, is present in the human renal brush border membrane and to elucidate its characteristics and (e) to determine the mechanisms by which two model organic cations, tetraethylammonium (TEA) and N-1- methylnicotinamide (NMN), are transported across the human renal basolateral membrane.
The specific aims of the studies of nucleoside transport are to determine the molecular structure and tissue distribution of N4, the novel Na+-nucleoside cotransporter that we recently identified in the human renal brush border membrane. The experimental approach that will be used to elucidate the molecular structure of N4 and OCPA1 is (i) to clone the transporters by expression cloning in Xenopus laevis oocytes; and (ii) to sequence the cDNA's and deduce the amino acid sequences. Exciting preliminary data in our laboratory suggest that both transporters can be functionally expressed in Xenopus laevis oocytes. Northern blotting will be used to determine the tissue distribution of the specific mRNA transcripts. The molecular mechanisms involved in the induction of OCPA1 will be studies in cultured renal cells. Isotopic uptake studies in isolated brush border and basolateral membrane vesicles will be used to determine the cellular mechanisms of organic cation transport in the human kidney. Transporters in the kidney are critical in drug disposition and ultimately control the exposure of renal cells to potentially nephrotoxic drugs. The proposed studies are significant to our understanding of the disposition and nephrotoxicities of many clinically important basic drugs (e.g., cimetidine, pindolol, and amiloride) and nucleosides and nucleoside analogs used to treat viral infections, neoplasms and cardiac arrhythmias. These studies have important implications for drug targeting and delivery. The studies will pave the way for future studies of genetic polymorphisms in renal transporters; the molecular events involved in the transepithelial flux of drugs; and the mechanisms and signals involved in the regulation of drug transporters in the kidney.
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