Department of Nephrology and Renal Division, Peking University First Hospital, Beijing, China.
Kintor Pharmaceutical Limited, Suzhou, China.
Ren Fail. 2022 Dec;44(1):304-313. doi: 10.1080/0886022X.2022.2039705.
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 = 104.3 ± 19.2 vs. 36.7 ± 7.6, = 0.004). The increase of calcium concentration in podocytes (MFI = 3309.3 ± 363.6 vs. 1776.3 ± 212.7, = 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, = 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, = 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, = 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.
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，其中大多数为 M 型磷脂酶 A2 受体（PLA2R），并伴有严重的足细胞功能障碍。抗 PLA2R 抗体对足细胞的直接影响尚不清楚。
使用与重组人 PLA2R 蛋白偶联的柱亲和纯化 PLA2R 相关 MN 患者的抗 PLA2R 抗体。通过细胞凋亡检测、细胞内钙检测、划痕愈合试验和免疫荧光染色评估其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 PLA2R 抗体的刺激可诱导足细胞早期凋亡（Annexin 的 MFI = 104.3 ± 19.2 比 36.7 ± 7.6，= 0.004）。足细胞内钙浓度的增加（MFI = 3309.3 ± 363.6 比 1776.3 ± 212.7，= 0.015）可能归因于内质网钙流出。钙/钙调蛋白依赖性蛋白激酶 IV（CaMK4）的表达也增加（MFI = 134.4 ± 9.8 比 105.3 ± 10.1，= 0.011）。蛋白质组学结果表明，抗 PLA2R 抗体治疗导致细胞结构损伤，并对蛋白质结合、肌动蛋白丝结合和微管马达活性产生功能障碍。足突上 F-肌动蛋白的染色减少（MFI = 27.3 ± 2.8 比 47.5 ± 1.0，= 0.001），与抗 PLA2R 抗体孵育后足细胞的迁移和粘附能力降低（迁移细胞数= 44.7 ± 3.1 比 53.3 ± 4.9，= 0.001）。
这些数据表明，抗 PLA2R 抗体可能独立于补体系统直接诱导足细胞损伤，这扩展了抗 PLA2R 抗体在 MN 中的作用机制。
