MOLECULAR MECHANISM OF PROXIMAL TUBULE ION TRANSPORT

其中一个最一致的异常离子转运发现， 原发性高血压患者及其一级亲属 红细胞Na+-Li+反向转运蛋白的过度活性。鉴于 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 一直不为人知该项目的主要目标是确定 介导Na+-Li+逆向转运的蛋白质和 编码它。然后就有可能描述动力学 这种转运蛋白的性质，以确定在什么其他组织，它可能 表达，从而获得新的洞察其可能的 高血压的病理生理作用。此外，一旦基因编码 一旦确定了Na+-Li+反向转运蛋白， 是否直接导致转运活性增加的突变 与人类高血压有关。我们的实验方法将是 根据Na+-Li+反向转运蛋白是同种型的假设， 一价阳离子交换剂的NHE（Na+-H+交换剂）家族。在 除了表征Na+-Li+反向转运蛋白及其在 高血压，我们将测试是否在NHE亚型介导的Na+突变 肾脏中的重吸收（例如，NHE 3）与人类 高血压 我们将进行以下一系列研究。首先，我们将生成 合适的抗体，以确定是否NHE 4，唯一一个已知的 数据未显示在介导红细胞凋亡中起作用的NHE亚型 Na+-Li+反向转运不太可能，在人红细胞中表达， 兔，后者是具有高反转运活性的物种。如果 NHE 4在人和兔红细胞中表达，我们将进行 功能表达研究，以测试这种同种型是否能够 介导阿米洛利不敏感的Na+-Li+逆向转运，并评估 运输更多的生理相关性，如能力， 介导Na+-H+交换。在这些研究的同时， 人骨髓文库以克隆编码新NHE同种型的cDNA。 对于鉴定的任何新的NHE同种型，我们将产生同种型- 特异性抗血清，以确认其在红细胞中的表达，并确定 其在其他器官中表达的细胞和亚细胞位点， 组织中此外，还将进行功能表达研究， 测试新亚型介导Na+-Li+的能力 反向转运，并表征其生理相关性 输运性质对于发现介导红细胞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：NHE 3），我们将测试与高血压的联系，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。我们将从而 能够直接检验NHE突变导致 增加的转运活性与高血压有关。

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.