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

描述（由申请方提供）：功能性肾脏滤过器可能由毛细血管内皮、肾小球基底膜和“狭缝隔膜”或位于内脏上皮细胞（足细胞）交错足突之间的专门细胞间连接形成。包括糖尿病肾病在内的肾小球疾病表现为尿中的蛋白渗漏（蛋白尿）。导致蛋白尿性肾病的几种足细胞特异性基因突变，包括NPHS 1和NPHS 2，强调足细胞是滤过屏障的主要组成部分，对正常肾小球功能很重要。最广泛接受的模型是肾小球滤过屏障功能为具有增加的尺寸选择性的一系列筛。然而，目前尚不清楚为什么滤器不会被进入肾小球基底膜的大蛋白质常规堵塞。最近的研究表明，足细胞在内吞过程中非常活跃，该过程常规地去除积累在过滤屏障处的蛋白质如免疫球蛋白。足细胞表达FcRn受体，其被报道为白蛋白和免疫球蛋白转运蛋白。缺乏FcRn受体的小鼠随着年龄的增长而积累IgG，并在示踪剂研究中显示IgG的清除延迟。在最近的一项研究中，在嘌呤霉素氨基胍侧损伤模型中观察到足细胞摄取伊文思蓝标记的白蛋白。在肾病综合征患者肾脏活检的TEM研究中经常观察到足细胞囊泡。这些研究支持足细胞在从GBM中清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伤（被蛋白质泄漏量压倒）可能是促进肾小球疾病的常见机制。为了更好地理解内吞作用在维持过滤屏障中的作用，我们产生了一种小鼠模型来破坏足细胞中的内吞活性。Vps 34（PI 3 K III类）是磷脂酰肌醇3-激酶（PI 3 K）家族的成员，并且在调节内体/溶酶体系统中的囊泡运输中是重要的。正如预期的，以足细胞特异性方式缺失vps 34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，我们想进一步研究内吞作用在足细胞稳态中的作用。在前期工作的基础上，我们将重点研究这一机制的几个方面.具体而言，我们将：（1）分析足细胞特异性缺失vsp 34的体内效应。我们将分析足细胞中观察到的空泡的特征。我们还将在诱导型模型中分析vps 34缺失的影响。(2)使用这个模型系统，我们将研究足细胞的作用，积累在狭缝隔膜的蛋白质的内吞。我们预期，通过破坏细胞内吞/囊泡运输过程，将使我们能够可视化正在进行的内吞的基础水平，使足细胞保持清洁的过滤器。 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，占美国开始血液透析患者的55%以上。我们对肾小球疾病患者的治疗以减缓或预防疾病进展并不足够有效，并且没有精确地针对肾小球疾病的原因。足细胞是肾小球的主要组成部分，对于肾脏过滤器的正常功能至关重要。足细胞损伤导致蛋白质渗漏，这是肾小球疾病存在的主要临床体征，并根据其严重程度预测结果。该提案旨在了解足细胞健康受蛋白尿影响的机制。最近的研究表明，足细胞吸收了泄漏的蛋白质，这一过程可能对其整体健康有害。该提案与 NIDDK的目标是促进基础肾脏生物学的研究，包括肾脏细胞和分子生物学（蛋白质运输和细胞极性）以及肾脏疾病病理学（肾小球细胞的正常生物学和蛋白尿）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).