特发性膜性肾病中抗足细胞M型磷脂酶A2受体抗体介导足细胞骨架结构改变的相关研究

Circulating autoantibodies against the M-type phospholipase A2 receptor (M-PLA2R) were recently identified in the majority of patients with idiopathic membranous nephropathy. Titer of the antibody reveal a strong correlation with clinical disease activity, and predict treatment response, which indicating it play an important role in the pathogenesis of idiopathic membranous nephropathy. Previous studies found redistribution of cytoskeleton protein and loss of normal structure of slit diaphragm of podocyte in patients with membranous nephropathy. We assume that antibodies against M-PLA2R in patients with idiopathic membranous nephropathy cause the redistribution of cytoskeleton in podocyte via the M-PLA2R pathway, which eventually lead to proteinuria and initiate the disease. We had successfully recombinated M-PLA2R protein which could be used to identify the anti M-PLA2R antibodies in sera, and we had mature thechniques of podocyte culture, to identify the presentation of M-PLA2R on podocyte, and the distribution of cytoskeleton protein in podocyte. The present study aim to evaluate the titer of autoantibody against M-PLA2R in sera from patients with idiopathic membranous nephropathy, to identify the presentation of M-PLA2R on podocyte and the distribution of cytoskeleton such as actin and nephrin in podocyte of patients, both on renal biopsy tissue and in vitro cultrued podocyte which stimulated by sera from patients with idiopathic membranous nephropathy. Furthermore, we will block the binding of antibodies from patients' sera to podocyte with recombinated anti-M-PLA2R antibody, and overexpression M-PLA2R on podocyte, in order to demonstrate that the changes of podocyte caused by anti-M-PLA2R antibody is via the M-PLA2R pathway. The present study will probe into the pathogenesis of idiopathic membranous nephropathy in point of view of redistribution of cytoskeleton proteins of podocyte which were caused by autoantibodies against M-PLA2R on podocyte, and lead to a new angle of view of treatment to this disease.

特发性膜性肾病患者血清中存在针对足细胞膜上M型磷脂酶A2受体(M-PLA2R)的自身抗体。该抗体与疾病活动度、治疗反应密切相关，提示该抗体在膜性肾病发病机制中起重要作用。既往研究发现特发性膜性肾病患者足细胞骨架结构发生改变，导致裂孔膜结构破坏，导致蛋白尿。本研究推测特发性膜性肾病患者血清中抗M-PLA2R抗体通过足细胞膜表面受体导致足细胞骨架结构改变，进而导致蛋白尿的发生而致病。我科前期已重组M-PLA2R蛋白，具备检测抗体滴度能力，同时具备足细胞体外培养及细胞骨架结构荧光染色技术。本课题拟在测定膜性肾病患者血清抗体滴度、肾小球足细胞M-PLA2R表达及细胞骨架结构分布的基础上，利用患者血清刺激体外培养足细胞观察其细胞骨架结构变化情况，进一步通过阻断和过表达足细胞表面M-PLA2R的方法，阐明其通过M-PLA2R途径介导足细胞骨架结构改变这一结论，从而进一步揭示特发性膜性肾病发病机制。

本研究围绕特发性膜性肾病（MN）抗磷脂酶A2受体（PLA2R）抗体，研究其对足细胞细胞骨架结构影响，以及在特殊病理表型中抗PLA2R抗体表达情况。体外培养肾小球足细胞，对细胞骨架结构podocin和actin4在正常足细胞进行染色。使用抗PLA2R抗体阳性患者血浆置换液及商品化的抗PLA2R抗体与体外培养足细胞共孵育后可均观察到细胞骨架改变。该部分研究提示PLA2R抗体可能通过影响细胞骨架结构影响足细胞功能，导致蛋白尿发生。该部分结果尚需进一步验证。调整研究方向后探讨两种特发性MN的特殊病理类型：MN合并局灶节段肾小球硬化病变（FSGS）；以及MN合并新月体。（1）以往研究结果对特发性MN合并FSGS样病变的患者存在争议。可溶性尿激酶受体（Soluble urokinase-type plasminogen activator receptor, suPAR）在特发性FSGS患者中血清水平高于特发性膜性肾病。本研究选取20例MN合并FSGS样病变患者，及29例原发性MN患者和27例原发性FSGS患者为对照，比较各组间临床、病理、抗PLA2R抗体、血浆及尿中suPAR水平。结果示MN合并FSGS样病变患者与特发性MN患者临床病理相似，80%患者抗PLA2R抗体阳性，68.4%肾脏以IgG4沉积为主，但肾小管间质病变偏重；另一方面，与原发性FSGS相比，合并FSGS样病变的MN患者尿suPAR水平明显降低，与特发性MN接近。推测MN合并FSGS样病变与特发性MN存在相似发病机制。该部分已撰写SCI文章审稿中。（2）特发性MN患者少部分肾脏合并新月体形成，其发病机制、临床及病理特点尚存争议。本研究回顾分析401例特发性MN患者，其中28例合并新月体形成，新月体累及肾小球比例平均为4.9%，79.7%合并PLA2R抗体。与特发性MN相比，合并新月体者肾小管萎缩、肾间质淋巴细胞浸润更重；治疗后缓解率更低。合并新月体是肾病综合征不缓解及肾功能恶化的危险因素。该文章已被《Medicine》（IF 4.867）接收。本研究进一步阐释了抗PLA2R抗体在特发性膜性肾病中的作用，无论是否合并特殊的病理表现（如FSGS样病变或形成新月体），其中部分与影响足细胞骨架结构有关。