The Dual Role of the Neonatal Fc Receptor (FcRn) in Podocytes

 描述（由申请人提供）：在正常肾脏中，每天通过肾小球滤过屏障过滤大量血液（180 L）。即使是最保守的估计，每天也有1 - 3 g白蛋白通过过滤屏障。足细胞是GFB的关键部分，足细胞中蛋白质运输的分子基础尚不清楚。在初步研究中，我们已经发现足细胞主动转胞吞两种主要的血清蛋白，白蛋白和免疫球蛋白G（IgG），并且转胞吞作用是处理这些蛋白质的主要途径。我们还发现IgG的运输比白蛋白的运输更复杂，并且足细胞根据IgG是否与抗原偶联而不同地指导IgG的运输。在其他细胞类型如肾小管细胞和内皮细胞中，新生儿Fc受体（FcRn）是白蛋白和IgG转胞吞作用所必需的。在树突状细胞中，FcRn是抗原/IgG（免疫复合物）运输至溶酶体以在MHC II类上呈递抗原所必需的。基于我们的初步研究和已知的FcRn在其他细胞类型中的作用，我们提出了FcRn在足细胞中的双重作用。具体而言，我们假设（1）FcRn是通过足细胞转胞吞白蛋白和IgG所必需的，（2）FcRn是通过将抗原/IgG复合物导向溶酶体以在足细胞MHC II中加工和呈递而在足细胞中呈递免疫复合物所必需的。在本提案中，我们将使用从野生型和FcRn敲除小鼠中分离的原代足细胞来确定FcRn是否是白蛋白和IgG转胞吞作用所必需的。我们还将确定白蛋白和IgG的肾小球筛选系数是否在野生型与FcRn敲除小鼠中显著更低，以及体内足细胞特异性敲除FcRn是否导致肾小球内蛋白质蓄积和肾小球硬化。我们将检查是否需要FcRn来将抗原/IgG复合物引导至溶酶体以降解并在足细胞MHC II类上呈递抗原，并确定缺乏FcRn是否会消除足细胞中的抗原/IgG呈递。最后，我们将确定足细胞特异性敲除FcRn是否能改善免疫介导的肾炎小鼠模型的疾病。我们还将使用活体多光子显微镜，一种动态的先进成像技术，以检查是否足细胞特异性敲除FcRn减少T细胞在诱导免疫介导的肾炎后肾小球内的滞留时间。在这个建议中概述的工作是重要的，因为它是在确切地了解血清蛋白是如何通过足细胞运输的第一步。足细胞中的蛋白质积累已被证明对足细胞具有毒性，并与足细胞损失和肾小球硬化相关。足细胞如何处理蛋白质的分子水平的知识将形成合理的治疗设计的基础，以防止蛋白质在足细胞中的积累。理解FcRn在足细胞中的双重作用（转胞吞作用和抗原呈递）对于设计安全有效的治疗免疫介导的肾小球疾病的疗法也至关重要。

 DESCRIPTION (provided by applicant): In the normal kidney, an enormous volume of blood (180 L) is filtered each day through the glomerular filtration barrier. By even the most conservative estimates, between 1 - 3 g of albumin a day make it through the filtration barrier. Podocytes are a key part of the GFB and the molecular basis of protein trafficking in podocytes is unknown. In preliminary studies, we have found that podocytes actively transcytose two major serum proteins, albumin and immunoglobulin G (IgG), and that transcytosis is the major pathway for handling these proteins. We have also found that trafficking of IgG is more complicated than that of albumin and that podocytes direct trafficking of IgG differently depending on whether IgG is coupled to antigen. In other cell types such as renal tubular cells and endothelial cells, the neonatal Fc receptor (FcRn) is required for transcytosis of albumin and IgG. In dendritic cells, FcRn is required for antigen/IgG (immune complex) trafficking to the lysosome for antigen presentation on MHC Class II. Based on our preliminary studies and the known role of FcRn in other cell types we propose a dual role for FcRn in podocytes. Specifically, we hypothesize that (1) FcRn is required for transcytosis of albumin and IgG through the podocyte and (2) FcRn is required for immune complex presentation in podocytes by directing antigen/IgG complexes to the lysosome for processing and presentation in podocyte MHC II. In this proposal, we will use primary podocytes isolated from wild type and FcRn knockout mice to determine whether FcRn is required for transcytosis of albumin and IgG. We will also determine whether the glomerular sieving coefficient for albumin and IgG is significantly less in wild type versus FcRn knockout mice and whether podocyte-specific knockout of FcRn in vivo leads to protein accumulation within the glomerulus and glomerulosclerosis. We will examine whether FcRn is required to direct antigen/IgG complexes to the lysosome for degradation and antigen presentation on podocyte MHC Class II and determine whether lack of FcRn abrogates antigen/IgG presentation in podocytes. Finally, we will determine whether podocyte-specific knockout of FcRn ameliorates disease in a mouse model of immune-mediated nephritis. We will also use intravital multiphoton microscopy, a dynamic advanced imaging technique, to examine whether podocyte-specific knockout of FcRn reduces T retention times within the glomerulus after induction of immune-mediated nephritis. The work outlined in this proposal is significant because it is the initial step in understanding exactly how serum proteins are trafficked through the podocyte. Protein accumulation in podocytes has been shown to be toxic to podocytes and correlates with podocyte loss and glomerulosclerosis. Knowledge at the molecular level of how proteins are handled by the podocyte will form the basis for the design of rational therapies to prevent protein accumulation in podocytes. An understanding of the dual role of FcRn in podocytes (both in transcytosis and antigen presentation) is also critical to the design of safe and effective therapies to treat immune-mediated glomerular diseases.