Podocyte-specific human PLA2R transgenic mouse model of membranous nephropathy

足细胞特异性人PLA2R转基因小鼠膜性肾病模型

• 批准号: 7976290
• 负责人: DAVID J SALANT
• 金额: $ 25.35万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-02 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7976290
• 关键词: Animal Testing; Antibodies; Antigen Targeting; Antigens; Autoantibodies; Binding; Biopsy; Cavia; Cell Aging; Chinchilla (genus); Clinical; Complement; Deposition; Development; Disease; Eicosanoid Production; Epitopes; Future; Goals; Human; Idiopathic Membranous Nephropathy; IgG1; IgG4; Immune; Immunoglobulin G; Injury; Kidney; Laboratory Animals; Ligand Binding; Location; Mediating; Membranous Glomerulonephritis; Modeling; Mus; Oryctolagus cuniculus; Pathogenicity; Patients; Phospholipase; Play; Positioning Attribute; Proteinuria; Rattus; Role; Serum; Specificity; System; Techniques; Testing; Transgenic Mice; Transgenic Organisms; complement pathway; in vivo; model development; mouse model; nephrin; podocyte; promoter; public health relevance; receptor; tool

DESCRIPTION (provided by applicant): The primary goal of this exploratory proposal is to develop a tool to determine if circulating autoantibodies directed at the M-type phospholipase receptor (PLA2R) from patients with idiopathic membranous nephropathy (MN) are pathogenic. We have established that about 75% of patients with idiopathic MN have circulating anti-PLA2R autoantibodies whereas none of the disease or normal controls has tested positive, thus indicating a high degree of specificity. The anti-PLA2R antibodies are IgG4, the subclass that predominates in idiopathic but not secondary MN, the antigen PLA2R is expressed on podocytes and colocalizes with IgG4 in the glomerular immune deposits of patients with MN, and the antibodies can be eluted from the glomerular immune deposits of MN renal biopsies. Moreover, the presence of circulating anti-PLA2R antibodies correlates with disease activity in MN. Although these findings strongly suggest that anti-PLA2R antibodies are responsible for, or at least contribute to disease development, proof of pathogenicity has yet to be established. Whereas simple transfer of the disease to laboratory animals with human anti-PLA2R would readily answer this question, human anti-PLA2R does not recognize PLA2R in the glomeruli of any small laboratory animal tested to date. Therefore, we plan to take advantage of both the lack of immunoreactive PLA2R in mouse glomeruli and the refinement of podocyte- specific transgenic techniques to develop a mouse model in which the human PLA2R is expressed in podocytes. To that end we propose the following specific aims: Specific Aim 1: Produce a transgenic mouse utilizing the NPHS1 (nephrin) promoter to drive expression of human PLA2R exclusively in mouse podocytes. Specific Aim 2: Verify that human PLA2R is expressed on mouse podocytes in vivo. Specific Aim 3: a) Determine if injected human anti-PLA2R autoantibodies bind to human PLA2R and form immune deposits typical of MN in the transgenic mouse glomeruli. b) Establish if injected human anti-PLA2R antibodies are able to cause proteinuria in the human PLA2R transgenic mouse. Specific Aim 4: Determine if the human PLA2R transgenic mice develop anti-PLA2R antibodies and MN when actively immunized with human PLA2R. Successful development of this model will pave the way for studies to both establish the pathogenic role of human anti-PLA2R autoantibodies and define the mechanisms of injury. PUBLIC HEALTH RELEVANCE: Having demonstrated that the M-type phospholipase receptor (PLA2R) is a specific target antigen of circulating autoantibodies in a high proportion of patients with idiopathic membranous nephropathy, we propose to develop a mouse model to determine if the antibodies are responsible for the clinical (proteinuria) and pathological manifestations of the disease. To that end, we will produce transgenic mice in which the human PLA2R is expressed on mouse podocytes, the location of the antigen in human kidneys.

描述(由申请人提供)：这项探索性提案的主要目标是开发一种工具，以确定特发性膜性肾病(MN)患者针对M型磷脂酶受体(PLA2R)的循环自身抗体是否具有致病性。我们已经确定，大约75%的特发性MN患者有循环中的抗PLA2R自身抗体，而该病或正常对照组均未检测出阳性，因此显示出高度的特异性。抗PLA2R抗体为IgG4，在特发性MN中占优势，但在继发性MN中不占优势，PLA2R抗原表达于足细胞，并与IgG4共存于MN患者的肾小球免疫沉积物中，抗体可从MN肾活检的肾小球免疫沉积物中洗脱。此外，循环中抗PLA2R抗体的存在与MN的疾病活动性有关。尽管这些发现强烈表明抗PLA2R抗体是疾病发展的原因，或者至少有助于疾病的发展，但致病性的证据尚未建立。虽然简单地将疾病转移到带有人抗PLA2R的实验动物身上可以很容易地回答这个问题，但到目前为止，人类抗PLA2R不识别任何小型实验动物的肾小球中的PLA2R。因此，我们计划利用小鼠肾小球缺乏PLA2R的免疫反应和足细胞特异性转基因技术的改进来发展一种在足细胞中表达人PLA2R的小鼠模型。为此，我们提出了以下特定目标：特定目标1：利用NPHS1(Neparin)启动子来驱动人PLA2R在小鼠足细胞中的表达，从而获得转基因小鼠。特定目的2：验证人PLA2R在体内小鼠足细胞上的表达。具体目的3：a)确定注射的人抗PLA2R自身抗体是否与人PLA2R结合，并在转基因小鼠肾小球中形成典型的MN免疫沉积。B)确定注射的人抗PLA2R抗体是否能够导致人类PLA2R转基因小鼠的蛋白尿。特异性目的4：确定人PLA2R转基因小鼠主动免疫后是否产生抗PLA2R抗体和MN。该模型的成功开发将为确定人类抗PLA2R自身抗体的致病作用和确定损伤机制的研究铺平道路。 公共卫生相关性：在证明M型磷脂酶受体(PLA2R)是高比例特发性膜性肾病患者循环自身抗体的特异性靶抗原后，我们建议建立一个小鼠模型，以确定抗体是否与疾病的临床(蛋白尿)和病理表现有关。为此，我们将生产转基因小鼠，其中人类PLA2R在小鼠足细胞上表达，足细胞是人类肾脏中抗原的位置。