Anti-phospholipase A2 receptor antibodies directly induced podocyte damage in vitro.
Anti-phospholipase A2 receptor antibodies directly induced podocyte damage in vitro.
复制标题
抗磷脂酶A2受体抗体直接诱导体外足细胞损伤
DOI：
10.1080/0886022x.2022.2039705
复制
发表时间：
2022-12
期刊：
影响因子：
3
通讯作者：
Zhao MH
中科院分区：
文献类型：
--
作者：
Li Y;Yu J;Wang M;Cui Z;Zhao MH
原发性膜性肾病（MN）的发病机制涉及针对足细胞表面抗原的抗体，其中大部分是M型磷脂酶A2受体（PLA 2 R），以及足细胞功能障碍。抗PLA 2 R抗体直接作用于足细胞的效果尚不清楚。使用与重组人PLA 2 R蛋白偶联的柱亲和纯化来自PLA 2 R相关MN患者的抗PLA 2 R抗体。通过凋亡测定、细胞钙离子检测、伤口愈合测定和免疫荧光染色评估它们对条件永生化人足细胞的影响。通过LC-MS/MS和PANTHER数据库进行蛋白质组学分析。抗PLA 2 R抗体的刺激可诱导足细胞的早期凋亡（膜联蛋白V的MFI = 104.3 ± 19.2对36.7 ± 7.6，p = 0.004）。足细胞内钙离子浓度增加（MFI = 3309.3 ± 363.6 vs.1776.3 ± 212.7，p = 0.015）可能与内质网钙外流有关。钙/钙调蛋白依赖性蛋白激酶IV（CaMK 4）的表达也增加（MFI = 134.4 ± 9.8 vs. 105.3 ± 10.1，p = 0.011）。蛋白质组学结果表明，抗PLA 2 R抗体处理导致细胞结构损伤，并产生蛋白质结合、肌动蛋白丝结合和微管运动活性的功能障碍。与抗PLA 2 R抗体孵育后，足突上F-肌动蛋白的染色减少（MFI = 27.3 ± 2.8 vs.47.5 ± 1.0，p = 0.001），足细胞的运动性和粘附能力降低（迁移细胞数= 44.7 ± 3.1 vs.53.3 ± 4.9，p = 0.001）。这些数据表明，抗PLA 2 R抗体可以直接诱导足细胞损伤，而不依赖于补体系统，这扩展了抗PLA 2 R抗体对MN的机制。
The pathogenesis of primary membranous nephropathy (MN) involves the antibodies against antigens on the cell surface of podocytes, with the majority of M-type phospholipase A2 receptor (PLA2R), and a profound podocyte dysfunction. The effects of anti-PLA2R antibodies directly to the podocytes remain unclear. Anti-PLA2R antibodies from patients with PLA2R-associated MN were affinity-purified using a column coupled with recombinant human PLA2R protein. Their effects on conditionally immortalized human podocytes were assessed by apoptosis assays, cellular calcium detection, wound healing assay, and immunofluorescent staining. Proteomics analysis was performed by LC-MS/MS and on PANTHER database. The stimulation by anti-PLA2R antibodies could induce early-stage apoptosis of podocytes (MFI of Annexin V = 104.3 ± 19.2 vs. 36.7 ± 7.6, p = 0.004). The increase of calcium concentration in podocytes (MFI = 3309.3 ± 363.6 vs. 1776.3 ± 212.7, p = 0.015) might attribute to the endoplasmic reticulum calcium efflux. The expression of calcium/calmodulin-dependent protein kinase IV (CaMK4) was also increased (MFI = 134.4 ± 9.8 vs. 105.3 ± 10.1, p = 0.011). Proteomics results suggested that anti-PLA2R antibody treatment led to damage on cellular structure, and produced functional disorders on protein binding, actin filament binding, and microtubule motor activity. The staining of F-actin on foot process was reduced (MFI = 27.3 ± 2.8 vs. 47.5 ± 1.0, p = 0.001) and the motility and adherence capacity of podocytes were reduced (number of migrated cells = 44.7 ± 3.1 vs. 53.3 ± 4.9, p = 0.001) after incubation with anti-PLA2R antibodies. These data indicate that anti-PLA2R antibodies may directly induce podocyte damage independent of the complement system, which expands the mechanism of anti-PLA2R antibodies on MN.