The long term goal of the proposed program of research is to understand the cellular mechanisms associated with the entrance and exit from renal cells of organic conjugates and chelates of heavy metals. Because these complexes carry a net negative charge, it has been assumed that the classical 'renal organic anion secretory pathway' plays a central role in the renal elimination of these compounds. While this may be true, there is no direct evidence that uptake or elimination of metal-containing complexes involves this process. Indeed, the cellular basis for clearance of anionic metal chelates from intoxicated cells is unknown. The mechanism by which the kidney secretes organic anions (OAs) has, in fact, received considerable attention, and a cellular model of OA secretion, for which p-aminohippurate (PAH) is considered the prototypic substrate, has been widely accepted. New evidence, however, suggests that renal OA secretion involves several distinct transporters with overlapping selectivity. Consequently, the overarching goal of the this study, i.e., establishing the mechanism of renal transport of anionic metal complexes, must be placed into the following context: the several, distinct mechanisms involved in renal transport of organic anions (OAs) have only recently begun to be identified, and the relative role played by each in renal secretion is largely unknown. We outline here, in two Specific Aims, experiments that examine characteristics of individual OA transporters, and their integrated behavior when working in concert with multiple processes in native renal tubules. We focus on several transporters: OAT1 (renal organic anion transporter); OAT-K2 (a renal homologue of the organic anion transporting polypeptide); and Mrp2 (the apical membrane homologue of the family of multidrug resistance-associated transport proteins). These transporters were selected because current evidence on their substrate specificity and their distribution within renal proximal tubules suggests that they can interact with anionic chelators and/or their heavy metal chelates.
In Aim 1, we will determine, using heterologous expression systems, the extent to which these OA transporters interact with a common set of test substrates and inhibitors (including the anionic chelator 2,3-dimercapto-l-propanesulfonate [DMSP] and its mercury and arsenic-containing chelates). The information obtained in these experiments will be applied in Aim 2, which will examine the integrated behavior of these processes in (i) a model cell culture system containing selected combinations of these processes; and (ii) intact proximal tubules in which OA secretion is the product of a suite of transporters working in the physiological contex of the intact cell. The studies with intact tubules will include direct tests of the hypothesis that DMPS-metal chelates are exported from renal cells through interaction with Mrp2 and OAT-K2. The proposed studies will result in a new, general model of the cellular strategy for secretion of a diverse array of xenobiotic OAs, and a more specific understanding of the action of heavy metal chelators on renal cells.
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