Abstract
The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy (MN). However, the pathogenic role of anti-PLA2R1 autoantibodies is unclear. Our aim was to evaluate the in vitro cytotoxicity of anti-PLA2R1 antibodies mediated by complement. Forty-eight patients with PLA2R1-related MN from the prospective cohort SOURIS were included. Anti-PLA2R1 titer, epitope profile, and anti-PLA2R1 IgG subclasses were characterized by ELISA. Cell cytotoxicity was evaluated by immunofluorescence in HEK293 cells overexpressing PLA2R1 incubated with patient or healthy donor sera in the presence or absence of rabbit complement or complement inhibitors. Mean cytotoxicity of anti-PLA2R1 sera for HEK293 cells overexpressing PLA2R1 was 2 ± 2%, which increased to 24 ± 6% after addition of rabbit complement (p < 0.001) (n = 48). GVB-EDTA, which inhibits all complement activation pathways, completely blocked cell cytotoxicity, whereas Mg-EGTA, which only inhibits the classical and lectin pathways, highly decreased suggesting a limited role of the alternative pathway. A higher diversity of IgG subclasses beyond IgG4 and high titer of total IgG anti-PLA2R1 were associated with increased cytotoxicity (p = 0.01 and p = 0.03 respectively). In a cohort of 37 patients treated with rituximab, high level of complement-mediated cytotoxicity was associated with less and delayed remission at month 6 after rituximab therapy (5/12 vs. 20/25 (p = 0.03) in 8.5 months ± 4.4 vs. 4.8 ± 4.0 (p = 0.02)). Kaplan-Meier analysis demonstrated that high level of cytotoxicity (≥40%) (p = 0.005), epitope spreading (defined by immunization beyond the immunodominant CysR domain) (p = 0.002), and high titer of anti-PLA2R1 total IgG (p = 0.01) were factors of poor renal prognosis. Anti-PLA2R1 antibodies containing sera can induce in vitro cytotoxicity mediated by complement activation, and the level of cytotoxicity increases with the diversity and the titer of anti-PLA2R1 IgG subclasses. These patients with high level of complement-mediated cytotoxicity could benefit from adjuvant therapy using complement inhibitor associated with rituximab to induce earlier remission and less podocyte injury.
Highlights
Membranous nephropathy (MN) is an autoimmune disease and a major cause of nephrotic syndrome in adults [1]
IgG1, IgG2, IgG3, and IgG4 anti-PLA2R1 antibodies were found in most samples at the first course of the disease, while at relapse, IgG4 anti-PLA2R1 was the predominant subclass (p = 0:02) (Figure 1(c))
Our results demonstrate that anti-PLA2R1 antibodies induce in vitro cytotoxicity mediated by complement activation as suggested by Kistler et al who analyzed TRPC6 protective role from complement-mediated podocyte injury [33]
Summary
Membranous nephropathy (MN) is an autoimmune disease and a major cause of nephrotic syndrome in adults [1]. It is defined by the presence of subepithelial immune complex deposits with alteration of the glomerular membrane and podocyte injury [2]. Kidney Disease Improving Global Outcomes (KDIGO) guidelines recommend a supportive symptomatic treatment (Renine angiotensine system blockers and diuretics) in all patients and immunosuppressive therapy only in the case of persistent nephrotic syndrome or renal function deterioration [9]. Immunosuppressive treatments are often started only after significant and potentially irreversible complications. New KDIGO guidelines will probably modify this recommendation using new markers to start immunosuppressive therapy [10]
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