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

 描述(申请人提供)：在正常肾脏，每天有大量的血液(180L)通过肾小球滤过屏障过滤。即使是最保守的估计，每天也有1-3克白蛋白通过过滤屏障。足细胞是GFB的关键部分，足细胞内蛋白质运输的分子基础尚不清楚。在初步研究中，我们发现足细胞主动地转运两种主要的血清蛋白，即白蛋白和免疫球蛋白G(Ig G)，而转运是处理这些蛋白的主要途径。我们还发现，免疫球蛋白的转运比白蛋白的转运更为复杂，足细胞根据免疫球蛋白是否与抗原偶联而以不同的方式引导免疫球蛋白的转运。在其他类型的细胞中，如肾小管细胞和内皮细胞，新生儿Fc受体(FcRN)是白蛋白和免疫球蛋白转胞所必需的。在树突状细胞中，FcRN是抗原/免疫复合物运输到溶酶体以呈递MHC II类抗原所必需的。根据我们的初步研究和FcRN在其他细胞类型中的已知作用，我们提出了FcRN在足细胞中的双重作用。具体地说，我们假设(1)FcRN是白蛋白和免疫球蛋白通过足细胞转运所必需的，(2)FcRN是足细胞中免疫复合物提呈所必需的，FcRN通过引导抗原/免疫球蛋白复合体进入足细胞MHC II进行加工和递送。在本方案中，我们将使用从野生型和FcRN基因敲除小鼠分离的原代足细胞来确定白蛋白和免疫球蛋白的跨细胞转运是否需要FcRN。我们还将确定野生型和FcRN基因敲除小鼠的肾小球白蛋白和免疫球蛋白的筛选系数是否显著低于FcRN基因敲除小鼠，以及体内足细胞特异性FcRN基因敲除是否会导致蛋白在肾小球内积聚和肾小球硬化。我们将研究FcRN是否需要将抗原/IgG复合体定向到溶酶体，以降解足细胞MHC II类的抗原和提呈抗原，并确定缺乏FcRN是否会取消足细胞中的抗原/IgG提呈。最后，我们将确定足细胞特异性FcRN敲除是否可以改善免疫介导性肾炎小鼠的疾病。我们还将使用活体多光子显微镜，一种动态先进的成像技术，来检查足细胞特异性敲除FcRN是否减少了免疫介导性肾炎诱导后T细胞在肾小球内的滞留时间。这项提案中概述的工作意义重大，因为它是准确了解血清蛋白如何通过足细胞运输的第一步。足细胞中蛋白质的积累已被证明对足细胞是有毒的，并与足细胞丢失和肾小球硬化有关。关于足细胞如何处理蛋白质的分子水平的知识将构成设计合理疗法的基础，以防止蛋白质在足细胞中积累。了解FcRN在足细胞中的双重作用(包括跨细胞和抗原提呈)，对于设计安全有效的治疗免疫介导性肾小球疾病的方法也是至关重要的。

 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.