Megalin Traffic in Dent Disease

项目摘要/摘要 组成近端小管(PT)的极化上皮细胞有一个特化的高容量的顶端。 内吞途径是恢复必要的营养物质和保持无蛋白尿液所必需的途径。蛋白质 在超滤液中与PT细胞顶端表面的多配体受体megalin和cubilin结合，并 通过受体介导的内吞作用内化。尽管这一途径在维持PT中起着关键作用 功能，参与PT细胞分类和回收的隔室的分子特性，从而 监管它们的机制在很大程度上是未知的。 PT内吞作用受损导致尿中滤过蛋白排出[称为低分子量(LMW)] 蛋白尿]。齿状神经病是一种X连锁疾病，由编码ClC-5的CLCN5基因突变引起， 一种电生2Cl-/H+交换剂。Dent病患者表现为低分子量蛋白尿，典型的 进展为肾功能衰竭。CLC-5的缺失已被证明降低了细胞内对过滤的配体的摄取 齿状神经病的小鼠模型。来自CLCN5基因敲除小鼠的PTS显示巨蛋白蛋白显著减少 表达(没有改变的RNA水平)，这可能是这些细胞内吞能力降低的原因。它 已经提出，ClC-5的缺失会导致巨蛋白的降解增强，但这并不是直接的 经过测试，这种情况可能发生的机制尚不清楚。 理解受体介导的过滤蛋白顶端内吞作用调节的一个重要障碍 一直缺乏高度差异化的细胞培养模式，以保持组织和高能力 PT顶端内吞途径。Weisz实验室优化了分化的PT细胞的细胞培养模型， 形成与体内PT相似的形态特化、新陈代谢和内吞能力。使用这个 系统后，我们现在可以确定PT顶端内吞运输的路线、动力学和调节。为此，我 我将利用从以下方面获得的数据开发一个模型来描述PT细胞中巨蛋白流量的路径和动力学 成像和生化方法，以及巨蛋白运输的路线将在小鼠近端得到验证 小管。这个模型将被用来对巨蛋白交易的变化如何导致 Dent病患者的低分子蛋白尿。我将在Dent病细胞培养模型中测试这些预测，生成 使用CRISPR/CAS9技术。我假设失去CLC-5改变了巨蛋白交易，通过损害 循环并将受体转移到降解途径，导致巨蛋白表达减少，并 低分子量蛋白尿。完成拟议的研究将加强我们对疾病如何导致 LMW蛋白尿，并提供了对操纵PT内吞能力以保存 蛋白尿症患者的肾功能。

Project Summary/ Abstract The polarized epithelial cells that comprise the proximal tubule (PT) have a specialized and high capacity apical endocytic pathway that is necessary to recover essential nutrients and to maintain a protein-free urine. Proteins in the ultrafiltrate bind to the multiligand receptors megalin and cubilin on the apical surface of PT cells and are internalized via receptor-mediated endocytosis. Despite the critical role of this pathway in maintaining PT function, the molecular identities of the compartments involved in sorting and recycling in PT cells, and thus the mechanisms by which they are regulated, are largely unknown. Impaired PT endocytosis results in urinary excretion of filtered proteins [termed low molecular weight (LMW) proteinuria]. Dent disease is an X-linked disorder caused by mutations in the CLCN5 gene that encodes CLC-5, an electrogenic 2Cl-/H+ exchanger. Patients with Dent disease present with LMW proteinuria and typically progress to renal failure. Loss of CLC-5 has been shown to decrease the endocytic uptake of filtered ligands in mouse models of Dent disease. PTs from CLCN5 knockout mice exhibit a marked reduction in megalin protein expression (without altered RNA levels) that likely accounts for the reduced endocytic capacity of these cells. It has been suggested that loss of CLC-5 causes enhanced degradation of megalin, but this has not been directly tested and the mechanism by which this might occur is unclear. A significant barrier to understanding the regulation of receptor-mediated apical endocytosis of filtered proteins has been the lack of a highly differentiated cell culture model that retains the organization and high capacity of the PT apical endocytic pathway. The Weisz lab has optimized a cell culture model of differentiated PT cells that develops morphological specializations, metabolism, and endocytic capacity similar to the PT in vivo. Using this system, we can now determine the itinerary, kinetics, and regulation of PT apical endocytic traffic. To this end, I will develop a model to describe the route and kinetics of megalin traffic in PT cells using data acquired from imaging and biochemical approaches, and the itinerary of megalin trafficking will be verified in mouse proximal tubules. This model will be used to make testable predictions for how changes in megalin trafficking result in LMW proteinuria in Dent disease. I will test these predictions in a Dent disease cell culture model, generated using CRISPR/Cas9 technology. I hypothesize that loss of CLC-5 alters megalin trafficking, by impairing recycling and shifting the receptors toward degradative pathways, leading to reduced megalin expression and to LMW proteinuria. Completing the proposed studies will enhance our understanding of how disease can lead to LMW proteinuria and provide insight into new approaches to manipulate PT endocytic capacity to preserve kidney function in proteinuric diseases.