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

内吞作用和囊泡运输在足细胞健康和过滤器完整性中的作用

• 批准号: 8355212
• 负责人: PUNEET GARG
• 金额: $ 7.73万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-15 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8355212
• 关键词: Accounting; Affect; Albumins; Autophagocytosis; Biochemistry; Biological Models; Biology; Biopsy; Biopsy Specimen; Blood capillaries; Cell Polarity; Cell surface; Cells; Cellular biology; Characteristics; Clinical; Diabetic Nephropathy; Disease Progression; Electron Microscopy; Endocytosis; Endothelium; Evans blue stain; Family; Filtration; Foot Process; Gene Mutation; Generations; Genetic; Goals; Health; Health Expenditures; Hemodialysis; Homeostasis; Immunoglobulin G; Immunoglobulins; Impairment; Injury; Intercellular Junctions; Invertebrates; Investigation; Kidney; Kidney Diseases; LDL-Receptor Related Protein 2; Label; Lipids; Mammals; Modeling; Molecular Biology; Morbidity - disease rate; Mus; NPHS2 protein; National Institute of Diabetes and Digestive and Kidney Diseases; Nephrotic Syndrome; Outcome; Patients; Perfusion; Phenotype; Phosphatidylinositols; Phosphotransferases; Play; Process; Proteins; Proteinuria; Public Health; Puromycin Aminonucleoside; Regulation; Renal function; Renal glomerular disease; Reporting; Research; Role; Series; Serum; Severities; Specific qualifier value; Staining method; Stains; System; Testing; Tracer; United States; Urine; Vacuole; Vesicle; Work; aged; base; capillary; glomerular basement membrane; glomerular filtration; glomerular function; in vivo; member; mortality; mouse model; neonatal Fc receptor; nephrin; podocyte; prevent; protein transport; receptor; slit diaphragm; tool; trafficking; uptake

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).

描述（由申请人提供）：功能性肾脏滤波器可能是由毛细管内皮，肾小球基底膜和“缝隙隔膜”或专门的细胞间交界处创建的，该连接位于等体上皮细胞的相互构造足过程（Podocycyte）。肾小球疾病，包括尿液中蛋白渗漏（蛋白尿）的糖尿病性肾病。导致蛋白尿性肾脏疾病的几种足细胞特异性基因突变（包括NPHS1和NPHS2）将强调足细胞作为过滤屏障的主要成分，对正常肾小球功能很重要。最广泛接受的模型是，肾小球滤过屏障作为一系列筛子的作用，其大小选择性增加。但是，尚不清楚为什么过滤器不常规地堵塞大蛋白，这些蛋白质进入肾小球基底膜。最近的研究表明，足细胞在内吞作用的过程中非常活跃，该过程常规去除在过滤屏障上积累的免疫球蛋白等蛋白质。足细胞表达FCRN受体，据报道是白蛋白和免疫球蛋白转运蛋白。在示踪剂研究中，缺乏FCRN受体累积的IgG的小鼠会累积IgG，并显示出延迟的IgG清除率。在最近的一项研究中，观察到紫摩霉素氨基核苷损伤模型的埃文斯蓝色标记白蛋白的足细胞吸收。在来自肾病综合征患者的肾脏活检的TEM研究中，经常观察到足细胞囊泡。这些研究支持这样一种观念，即足细胞在去除GBM中的蛋白质中起积极作用，并表明清除机械的遗传或获得性损伤（因蛋白质泄漏的量淹没）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过滤屏障中的作用，我们生成了小鼠模型，以破坏足细胞中的内吞作用。 VPS34（PI3K III类）是脂质激酶的磷酸肌醇3-KINSAE（PI3K）家族的成员，对于调节内体/溶酶体系统中水泡贩运很重要。如预期的那样，以足细胞为特定方式删除VPS34的小鼠早期蛋白尿，并且在足细胞中可见大液泡。使用该模型，我们想进一步研究内吞作用在足细胞稳态中的作用。基于初步工作，我们将重点关注该机制的几个方面。具体而言，我们将：（1）分析体内VSP34的Podocyte特异性缺失的影响。我们将分析在足细胞中观察到的液泡的特征。我们还将分析VPS34缺失在诱导模型中的效果。 （2）使用此模型系统，我们将研究足细胞在裂缝隔膜处积聚的蛋白质内吞作用。我们可以通过破坏细胞内吞作用/囊泡运输过程来预料，这将使我们能够可视化基础持续的内吞作用水平，从而使足细胞能够维持干净的脂肪。 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的患者的55％以上。我们对肾小球疾病患者的治疗缓慢或预防疾病的进展不足以有效，并且不能精确地针对肾小球疾病的原因。肾小球的主要成分是足细胞对于肾脏过滤器的正确功能至关重要的。足细胞的损伤导致蛋白质泄漏，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结果。该提案旨在了解蛋白尿影响足细胞健康的机制。最近的调查表明，足细胞会吸收泄漏的蛋白质，这一过程可能不利于其整体健康状况。该建议与 NIDDK促进基本肾脏生物学研究的目标，包括在肾脏的细胞和分子生物学（蛋白质运输和细胞极性）中以及肾脏疾病病理生物学（肾小球细胞正常生物学和蛋白尿）中的应用。