Renal Physiology of Pendrin

哺乳动物的肾脏有很大的排泄碱性负荷的能力。例如，在饮用水中给予等渗NaHCO3的啮齿动物只会发生轻微的代谢性碱中毒，尽管有如此巨大的碱性负荷。肾脏排出大量碱性负荷的能力部分是通过B嵌入细胞顶端阴离子交换介导的皮质集合管(CCD)中的HCO3-分泌来实现的。虽然顶端阴离子交换在功能上已被描述，但介导这一运输过程的基因产物仍然是一个有争议的领域。然而，我们的实验室和我们的合作者的工作已经证明，在大鼠、人和小鼠的非A插入细胞的顶膜上有一种阴离子交换器--垂垂蛋白。此外，我们的实验室已经证明，吊环蛋白介导了小鼠CCD中HCO3-的分泌。这个转运蛋白是否代表推测的顶端阴离子交换器，还有待充分的测试。基因敲除小鼠的建立极大地促进了垂垂蛋白在体内肾脏生理的研究。由于目前尚无有效的Pendrin或心尖阴离子交换的特异性抑制剂，基因敲除小鼠可作为生物抑制剂，用于检测Pendrin在体外肾脏中的转运特性。此外，使用这些动物进行的平衡研究将使我们能够确定这些垂垂蛋白缺乏的小鼠是否在碱性负荷的排泄方面存在缺陷。因此，本提案将测试Pendrin是否代表推测的电荷耦合器件的阴离子交换器。此外，这些研究将有助于我们了解这种转运蛋白的生理作用。为了回答这些问题，我们的实验室和我们的合作者将使用平衡研究、体外灌流小管、实时定量RT-PCR、免疫金细胞化学和免疫组织化学研究野生型和侧膜蛋白缺陷小鼠。本项目的具体目标是：1.确定垂垂蛋白在天然肾脏组织中的转运特性；2.确定代谢性碱中毒时垂垂蛋白是否在肾脏中表达上调；3.确定垂垂蛋白的肾脏表型；4.确定垂垂蛋白在肾脏中的定位。这些研究将有助于我们了解哺乳动物肾脏是如何排泄碱性负荷的。

The mammalian kidney has a substantial capacity to excrete an alkaline load. For example, rodents given isotonic NaHCO3 in their drinking water develop only a mild metabolic alkalosis despite this huge base load. The kidney's ability to excrete large alkaline loads occurs in part through active HCO3- secretion in the cortical collecting duct (CCD) mediated by apical anion exchange in the B intercalated cell. While apical anion exchange has been characterized functionally in the CCD, the gene product, which mediates this transport process has remained an area of controversy. However the work of our laboratory and that of our collaborators has demonstrated that pendrin, an anion exchanger, localizes to the apical membrane of non-A intercalated cells in rat, human and mouse. Moreover, our laboratory has demonstrated that pendrin mediates secretion of HCO3- in the mouse CCD. Whether this transporter represents the putative apical anion exchanger remains to be tested fully. Development of knockout mice has greatly facilitated the study of the renal physiology of pendrin in vivo. Because there are no specific inhibitors of either pendrin or apical anion exchange available, knockout mice can be exploited as a biological inhibitor and used to test transport characteristics of pendrin in kidney in vitro. Moreover, balance studies using these animals will allow us to determine if these pendrin-deficient mice have a defect in excretion of an alkaline load. Thus, the present proposal will test if pendrin represents the putative anion exchanger of the CCD. Moreover, these studies will contribute to our understanding of the physiological role of this transporter. To answer these questions our laboratory and that of our collaborators will employ balance studies, tubules perfused in vitro, real-time quantitative RT-PCR, immunogold cytochemistry and immunohistochemistry studies in wild type and pendrin-deficient mice. The Specific Aims of the project are: 1. To determine the transport characteristics of pendrin in native renal tissue, 2. To determine if pendrin is upregulated in kidney during metabolic alkalosis, 3. To determine the renal phenotype of pendrin and 4. To determine the localization of pendrin in kidney. These studies will contribute to our understanding of how the mammalian kidney excretes an alkaline load.