Role of Endocytosis and Vesicular Trafficking in Podocyte Health and Filter Integ

描述(申请人提供)：功能性肾滤器可能是由毛细血管内皮细胞、肾小球基底膜和位于内脏上皮细胞(足细胞)交错的足突之间的“裂隙隔膜”或特殊的细胞间连接形成的。包括糖尿病肾病在内的肾小球疾病表现为尿中蛋白泄漏(蛋白尿)。一些足细胞特有的基因突变，包括NPHS1和NPHS2，导致蛋白尿性肾脏疾病，强调足细胞是滤过屏障的主要成分，对正常的肾小球功能至关重要。最被广泛接受的模型是，肾小球滤过屏障作为一系列筛子发挥作用，增加大小的选择性。然而，目前尚不清楚为什么过滤器不会常规地堵塞进入肾小球基底膜的大蛋白。最近的研究表明，足细胞在内吞过程中非常活跃，内吞过程通常会移除聚集在过滤屏障上的免疫球蛋白等蛋白质。足细胞表达FcRN受体，据报道FcRN是一种白蛋白和免疫球蛋白转运体。缺乏FcRN受体的小鼠随着年龄的增长积累了免疫球蛋白，在示踪剂研究中显示出免疫球蛋白的延迟清除。在最近的一项研究中，在嘌呤霉素氨基核苷损伤模型中观察到足细胞摄取伊文思蓝标记的白蛋白。在对肾病综合征患者的肾活检进行的透射电子显微镜研究中，经常可以观察到足细胞囊泡。这些研究支持足细胞在从基底膜中去除蛋白质方面发挥积极作用的观点，并表明清除机制的遗传或获得性损害(被蛋白质泄漏的量所淹没)可能是促进肾小球疾病的常见机制。为了更好地了解内吞作用在维持滤过屏障中的作用，我们建立了一个破坏足细胞内吞活动的小鼠模型。Vps34(PI3K III类)是磷脂酰肌醇3-激酶(PI3K)家族中的一员，在调节内体/溶酶体系统中的囊泡运输中起重要作用。正如预期的那样，以足细胞特有的方式缺失Vps34的小鼠出现早期蛋白尿，足细胞中可见大空泡。利用这个模型，我们希望进一步研究内吞作用在足细胞动态平衡中的作用。在本项目前期工作的基础上，我们将重点研究这一机制的几个方面。具体地说，我们将：(1)分析足细胞特异性缺失vsp34在体内的影响。我们将分析足细胞中观察到的空泡的特征。我们还将在可诱导模型中分析Vps34缺失的影响。(2)利用这个模型系统，我们将研究足细胞在蛋白质内吞中的作用，这些蛋白质聚集在狭缝隔膜上。我们预计，通过扰乱细胞内吞作用/囊泡运输过程，我们将能够可视化正在进行的内吞作用的基础水平，从而使足细胞保持干净的Fitler。 公共卫生相关性：肾小球疾病是发病率、死亡率和公共卫生支出的主要原因，在美国开始血液透析的患者中，肾小球疾病占55%以上。我们对肾小球疾病患者进行的延缓或防止疾病进展的治疗效果不够有效，也没有准确地针对肾小球疾病的病因。足细胞是肾小球的主要组成部分，对肾脏过滤器的正常功能是必不可少的。足细胞损伤导致蛋白泄漏，这是肾小球疾病存在的主要临床标志，并根据其严重程度预测预后。这项建议试图了解蛋白尿影响足细胞健康的机制。最近的研究表明，足细胞吸收了泄漏的蛋白质，这一过程可能对它们的整体健康有害。该提案与以下内容相关 NIDDK的目标是促进基础肾脏生物学的研究，包括在肾脏细胞和分子生物学(蛋白质运输和细胞极性)和肾脏疾病病理生物学(肾小球细胞的正常生物学和蛋白尿)中的应用。

DESCRIPTION (provided by applicant): The functional kidney filter is likely created by the capillary endothelium, the glomerular basement membrane, and the "slit diaphragm" or specialized intercellular junction that lies between the interdigitating foot processes of the visceral epithelial cells (podocyte). Glomerular diseases including diabetic nephropathy present with protein leak (proteinuria) in the urine. Several podocyte specific gene mutations including NPHS1 and NPHS2 that result in proteinuric renal diseases have placed the emphasis on podocyte as primary component of the filtration barrier and important for normal glomerular function. The most widely accepted model is that the glomerular filtration barrier functions as a series of sieves with increasing size selectivity. It is not clear, however, why the filter does no routinely clog with large proteins that enter the glomerular basement membrane. Recent investigations have suggested that the podocytes are very active in the process of endocytosis that routinely remove proteins like immunoglobulin that accumulate at the filtrations barrier. Podocytes express FcRn receptor which is reported as an albumin and immunoglobulin transporter. Mice lacking the FcRn receptor accumulated IgG as they aged and showed delayed clearance of IgG in tracer studies. In a more recent study, podocyte uptake of Evans Blue labeled albumin was observed in puromycin aminonucleoside injury model. Podocyte vesicles have often been observed in TEM studies on kidney biopsies from patients with nephrotic syndrome. These studies support the idea that podocytes play an active role in removing proteins from the GBM and suggest that genetic or acquired impairment (overwhelmed by the amount of protein leak) of the clearance machinery is likely to be a common mechanism promoting glomerular diseases. To better understand the role of endocytosis in maintaining the filtration barrier we generated a mouse model to disrupt the endocytic activity in the podocytes. Vps34 (PI3K class III) is member of the phosphoinositides 3-kinsae (PI3K) family of lipid kinases and is important in regulation of vesicular trafficking in the endosomal/lysosomal system. As anticipated, mice deleted of vps34 in a podocyte specific manner develop early proteinuria and large vacuoles are visible in the podocytes. Using this model we would like to further investigate the role of endocytosis in podocyte homeostasis. Based on the preliminary work this project we will focus on several aspects of this mechanism. Specifically we will: (1) Analyze the effect of podocyte specific deletion of vsp34 in vivo. We will analyze the characteristics of the vacuoles observed in the podocytes. We will also analyze the effect of vps34 deletion in an inducible model. (2) Using this model system we will investigate the role of podocyte in endocytosis of proteins that accumulate at the slit diaphragm. We anticipate by disrupting the cellular endocytosis/vesicular trafficking processes will enable us to visualize basal levels of ongoing endocytosis that enable the podocytes to maintain a clean fitler. PUBLIC HEALTH RELEVANCE: Glomerular diseases are a major cause of morbidity, mortality, and public health expenditure, accounting for over 55% of patients starting hemodialysis in the United States. Our treatments for patients with glomerular disease to slow or prevent progression of disease are not sufficiently effective, and do not precisely target the causes of glomerular disease. A major component of the glomeruli, the podocyte are essential for the proper function of the kidney filter. Injury to the podocyte result in protein leak which i a major clinical sign of the presence of glomerular disease and predicts outcome based on its severity. This proposal seeks to understand the mechanisms by which podocyte health is affected by proteinuria. Recent investigations have suggested that podocytes take up the leaked proteins, a process that might be detrimental to their overall health. This proposal is relevant to the goals of the NIDDK to promote research on basic kidney biology, including applications within Cell and Molecular Biology of the Kidney (protein trafficking and cell polarity) and within the Pathobiology of Kidney Disease (normal biology of glomerular cells, and proteinuria).