抗PLA2R抗体糖基化异常在原发性膜性肾病发病机制中的作用研究

Although the pathogenesis of idiopathic membranous nephropathy has not been fully delineated, an increasing number of studies have confirmed that M-type phospholipase A2 receptor expressed on podocytes as the targeting antigens, especially the interaction between IgG4 anti-PLA2R and complement system potentially leading to the podocytes injury has been paid much more attention. There exists N-glycosyltion site in the Cγ2 domain of IgG4 molecule. Recently, there is finding that the purified anti-PLA2R IgG4 autoantibodies from MN patients have a high ratio of GalNAC to Gal in a terminal position，indicating that aberrant N-glycosylation IgG4 may induce the complement system activation through MBL pathway. However, the aberrant N-glycosylation form and the role it played in MBL pathway were not well illustrated. This study will focus on the IgG4 anti-PLA2R antibody and attempt to delineate the potential aberrant N-glycosylation form of IgG4 and the mechanism of podocytes injury through the interaction between IgG4 and MBL pathway by molecular immunology, mass spectrometry and podocytes culture, which has great significance for the pathogenesis and treatment strategies of membranous nephropathy.

原发性膜性肾病（IMN）发病机制的研究越来越关注于足细胞上膜抗原磷脂酶A2受体（PLA2R），近来抗PLA2R抗体与补体之间相互作用导致足细胞损伤已成为该领域的研究热点。然而抗PLA2R抗体通过何种机制激活补体系统导致IMN足细胞损伤并不清楚。IgG4型抗PLA2R抗体重链恒定区γ2区（Cγ2）存在N-糖基化修饰位点，其对IgG4分子Fc段属性具有重要意义。近年来研究发现，IMN患者IgG4型抗PLA2R抗体N-糖基化末端存在N-乙酰葡萄糖胺（GlcNAc）与半乳糖（Galactose）比例异常，提示IgG4分子有可能通过MBL途径激活补体系统。本研究以IgG4型抗PLA2R抗体为核心，运用分子免疫学技术、质谱分析、体外细胞培养等技术探讨其N-糖基化异常的作用及其激活MBL途径介导膜性肾病足细胞损伤的机制，此研究将为IMN发病机制及临床诊疗策略提供新的理论依据。

大约70%以上膜性肾病患者肾组织局部有补体C3沉积，提示补体系统的活化参与膜性肾病肾损伤。大部分研究发现补体经典途径、MBL途径可能是参与膜性肾病发病发病的主要途径。然而并未有明确的体内、体外实验证实抗PLA2R抗体与补体途径相互作用可能损伤足细胞。本研究首先利用本中心膜性肾病随访患者研究MBL在肾组织沉积情况，进而分析其与患者蛋白尿、肾功能关系，发现MBL沉积患者倾向于蛋白尿缓解，而与肾功能进展关系未明确（此部分文章在修稿中）。为进一步明确抗PLA2R抗体是否通过MBL途径损伤足细胞，我们纯化了含PLA2R抗体IgG，构建抗PLA2R抗体与MBL共刺激足细胞体系，发现二者共刺激组较单成分刺激组，足细胞损伤更明确，表现为足细胞膜上攻膜复合物C5b-9表达增多、足细胞骨架蛋白podocin、neprhin表达下降，足细胞骨架紊乱、凋亡增多。进而我们初步探讨了抗PLA2R抗体可能激活补体MBL途径的机制，利用凝集素法检测患者抗PLA2R抗体末端半乳糖的缺失情况，发现原发性膜性肾病患者抗PLA2R抗体末端半乳糖结构缺失更多，推测其结构异常可能参与MBL途径活化，该部分结果有待进一步实验验证。