RENAL TRANSPORT OF ORGANIC CHELATORS OF HEAVY METALS

描述：(改编自申请者摘要)： 提出的研究计划是了解细胞机制 与有机结合物进入和离开肾细胞有关 以及重金属的络合物。因为这些复合体带有净负值 电荷，人们一直假设经典的‘肾脏有机阴离子分泌’ 途径在这些化合物的肾脏消除过程中起着核心作用。 虽然这可能是真的，但没有直接证据表明摄取或消除 含金属络合物的研究涉及到这一过程。事实上，细胞基础 对于阴离子金属络合物从中毒细胞中的清除尚不清楚。这个 事实上，肾脏分泌有机阴离子(OAS)的机制， 得到了相当大的关注，并建立了OA分泌的细胞模型， 哪种对氨基马尿酸(PAH)被认为是原型底物， 被广泛接受。然而，新的证据表明，肾脏的OA分泌 涉及几个不同的转运蛋白，具有重叠的选择性。 因此，这项研究的总体目标，即建立 阴离子金属络合物的肾转运机制，必须放入 以下背景：肾脏涉及的几种不同的机制 有机阴离子(OA)的传输直到最近才开始被识别， 而且，它们在肾脏分泌中扮演的相对角色在很大程度上尚不清楚。我们 在这里，在两个具体的目标中，勾勒出检验 个体OA转运体及其在工作时的整合行为 与天然肾小管中的多个突起一致。我们的重点是几个 转运体：OAT1(肾脏有机阴离子转运体)；OAT-K2(肾脏同系物 有机阴离子转运多肽)；以及mrp2(顶膜 多药耐药相关运输蛋白家族的同源物)。 之所以选择这些转运蛋白，是因为它们底物上的现有证据 特异性及其在肾近端小管中的分布表明 它们可以与阴离子螯合剂和/或它们的重金属螯合物相互作用。在……里面 目标1，我们将使用异源表达系统来确定 这些OA转运蛋白与一组共同的测试底物相互作用 缓蚀剂(包括阴离子螯合剂2，3-二巯基-L-丙磺酸盐[DMSP] 及其含汞和砷的螯合物)。在这些文件中获得的信息 实验将在目标2中应用，它将检查这些 (I)模型细胞培养系统中的过程，该模型细胞培养系统包含这些的选定组合 突起；和(Ii)完整的近端小管，其中OA的分泌是一套产品 在完整细胞的生理环境中工作的转运蛋白。这项研究与 完整的小管将包括对DMPS-金属络合物是 通过与MRP2和OAT-K2相互作用从肾细胞输出。建议数 研究将产生一种新的、通用的细胞分泌a策略的模型。 不同种类的异生组织，并对其作用有更具体的了解 肾细胞上的重金属螯合剂。

DESCRIPTION: (Adapted from the Applicant's Abstract): 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.