MOLECULAR MECHANISM OF PROXIMAL TUBULE ION TRANSPORT

在离子传输中发现的最一致的异常之一 高血压病患者及其一级亲属 红细胞Na+-Li+逆向转运蛋白过度活跃。鉴于这样一个事实 这种转运蛋白通常介导红细胞中的Na+-Na+交换 在生理条件下，其在高血压中的病理生理作用 仍然默默无闻。该项目的主要目标是确定 介导Na+-Li+反向转运的蛋白质和参与Na+-Li+反向转运的基因 对其进行编码。然后，就有可能表征这个动力学 这种转运蛋白的特性，以确定它可能在哪些其他组织中 被表达出来，从而获得对其可能性的新见解 高血压的病理生理学作用。此外，一旦编码基因 确定了Na+-Li+反向转运体，我们将能够测试 直接导致运输活动增加的突变是否 与人类高血压有关。我们的实验方法将是 以Na+-Li+反向转运蛋白为异构体的假说为指导 属于单价阳离子交换剂NHE(Na+-H+交换器)家族。在……里面 Na~+-Li~+逆向转运蛋白的特性及其在体内的作用 高血压，我们将测试NHE亚型突变是否介导Na+ 肾脏的重吸收(例如。NHE3)与人类 高血压。 我们将进行以下一系列的研究。首先，我们将生成 合适的抗体来确定NHE4是否是已知的唯一一种 数据没有在调节红细胞中起作用的异构体 Na+-Li+反向转运不太可能，在人和人的红细胞中表达 兔，后者是一种具有高反向转运蛋白活性的物种。如果 NHE4在人和兔的红细胞中表达，我们将进行 功能表达研究，以测试该异构体是否能够 介导阿米洛利不敏感的Na~+-Li~+反向转运及评价 具有更多生理相关性的运输特性，例如 介导Na+-H+交换。在这些研究的同时，我们将筛选 克隆编码新的NHE亚型的人骨髓文库(S)。 对于已鉴定的任何新的NHE亚型，我们将生成亚型- 以确定其在红细胞中的表达，并确定其在红细胞中的表达 其在其他器官和组织中表达的细胞和亚细胞位置 纸巾。此外，还将进行功能表达研究，以 检测新型异构体对Na~+-Li~+的调节能力 反运输，并表征其生理相关性 传输属性。对于发现的介导红细胞Na+-Li+的NHE亚型 反向转运或介导顶端Na+进入肾小管细胞 (例如，NHE3)，我们将测试与高血压的关联，并将筛选 突变。将进行功能表达研究，以比较 野生型和突变型NHE蛋白的动力学性质。因此，我们将 能够直接测试NHE突变引起的假设 运输活动增加与高血压有关。

One of the most consistent abnormalities in ion transport found in patients with essential hypertension and their first degree relatives is overactivity of the red cell Na+-Li+ countertransporter. Given the fact that this transporter normally mediates Na+-Na+ exchange in red cells under physiologic conditions, its pathophysiologic role in hypertension has remained obscure. The principal goal of this project is to identify both the protein mediating Na+-Li+ countertransport and the gene that encodes it. It will then be possible to characterize the kinetic properties of this transporter, to determine in what other tissues it may be expressed, and thereby to gain new insight into its possible pathophysiologic role in hypertension. Moreover, once the gene encoding the Na+-Li+ countertransporter is identified, we will be able to test directly whether mutations leading to increased transport activity are associated with human hypertension. Our experimental approach will be guided by the hypothesis that the Na+-Li+ countertransporter is an isoform of the NHE (Na+-H+ exchanger) family of monovalent cation exchangers. In addition to characterizing the Na+-Li+ countertransporter and its role in hypertension, we will test whether mutations in NHE isoforms mediating Na+ reabsorption in the kidney (eg. NHE3) are associated with human hypertension. We will pursue the following sequence of studies. First, we will generate suitable antibodies to determine whether NHE4, the only one of the known NHE isoforms for which data have not rendered a role in mediating red cell Na+-Li+ countertransport unlikely, is expressed in red cells of human and rabbit, the latter a species with high countertransporter activity. If NHE4 is expressed in human and rabbit red cells, we will perform functional expression studies to test whether this isoform is capable of mediating amiloride-insensitive Na+-Li+ countertransport, and to evaluate transport properties of more physiologic relevance, such as the ability to mediate Na+-H+ exchange. In parallel with these studies, we will screen human bone marrow libraries to clone cDNAs encoding novel NHE isoform(s). For any novel NHE isoform that is identified, we will generate isoform- specific antisera to confirm its expression in red cells, and to determine the cellular and subcellular sites of its expression in other organs and tissues. In addition, functional expression studies will be performed to test the novel isoform for its ability to mediate Na+-Li+ countertransport, and to characterize its physiologically relevant transport properties. For NHE isoforms found to mediate red cell Na+-Li+ countertransport or to mediate apical Na+ entry into renal tubular cells (eg. NHE3), we will test for linkage to hypertension and will screen for mutations. Functional expression studies will be performed to compare the kinetic properties of wild-type and mutant NHE proteins. We will thereby be able to directly test the hypothesis that NHE mutations causing increased transport activity are associated with hypertension.