Pathogenicity of Human Anti-PLA 2 R1 Antibodies in Minipigs: A Pilot Study

Abstract

Significance statement: Membranous nephropathy (MN) is an autoimmune kidney disease characterized by immune deposits in the glomerular basement membrane. Circulating anti-phospholipase A 2 receptor 1 (PLA 2 R1) antibodies are detectable in 70%-80% of patients with MN, but experimental evidence of pathogenicity has been lacking. This study demonstrates the pathogenicity of human anti-PLA 2 R1 antibodies in minipigs, a model for MN that intrinsically expresses PLA 2 R1 on podocytes. After passive transfer of human anti-PLA 2 R1 antibody-containing plasma from patients with PLA 2 R1-associated MN to minipigs, antibodies were detected in the minipig glomeruli, but not in response to plasma from healthy controls. The minipigs developed histomorphological characteristics of MN, local complement activation in the glomeruli, and low-level proteinuria within 7 days, showing that human anti-PLA 2 R1 antibodies are pathogenic.

Background: Primary membranous nephropathy (MN) is an autoimmune kidney disease in which immune complexes are deposited beneath the epithelium in the glomeruli. The condition introduces a high risk for end-stage kidney disease. Seventy percent to 80% of patients with MN have circulating antibodies against phospholipase A 2 receptor 1 (PLA 2 R1), and levels correlate with treatment response and prognosis. However, experimental evidence that human anti-PLA 2 R1 antibodies induce MN has been elusive.

Methods: In passive transfer experiments, minipigs received plasma or purified IgG from patients with PLA 2 R1-associated MN or from healthy controls. Anti-PLA 2 R1 antibodies and proteinuria were monitored using Western blot, ELISA, and Coomassie staining. Kidney tissues were analyzed using immunohistochemistry, immunofluorescence, electron microscopy, and proteomic analyses.

Results: Minipigs, like humans, express PLA 2 R1 on podocytes. Human anti-PLA 2 R1 antibodies bound to minipig PLA 2 R1 in vitro and in vivo . Passive transfer of human anti-PLA 2 R1 antibodies from patients with PLA 2 R1-associated MN to minipigs led to histological characteristics of human early-stage MN, activation of components of the complement cascade, and low levels of proteinuria. We observed development of an autologous, later phase of disease.

Conclusions: A translational approach from humans to minipigs showed that human anti-PLA 2 R1 antibodies are pathogenic in MN, although in the heterologous phase of disease only low-level proteinuria developed.

Graphical abstract

Figure 1

Serologic and morphologic analyses after passive transfer of human plasma from anti-PLA₂R1 antibody–positive patients into minipigs. (A) Characterization of minipig A serum samples by Western blot analyses. Reactivity of minipig A serum samples (1:100) used as primary antibody with hPLA₂R1 (top; 4 µg HGE/lane) and pPLA₂R1 (bottom; 4 µg PGE/lane) immediately before serum transfer (pre), 5 minutes after serum transfer (post), or at the specific day of blood collection. *A longer exposure time was chosen to make the band depicting the anti-pPLA₂R1 antibody at “day 29, post” more clear. (B) Detection of hIgG and pAlb in minipig A serum samples (4 µl of 1:50 serum dilution per lane) loaded as antigens in Western blot analysis. (C) Immunofluorescence staining for human IgG (hIgG) and porcine C3 (pC3) (both in green), collagen type IV (in red) and nuclei (in blue) in minipig A and the control minipig B (both day 42). The scale bars indicate 5 µm. (D) Electron microscopy depicting subepithelial electron dense immune deposits (white arrows) in the glomeruli of minipig A, but not in the control minipig B. (E) Proteomic analysis of glomeruli showed an increase of proteins of the complement system as determined by mass spectrometry. Fold changes of label-free quantitation data (minipig A versus control minipig B) were log₂-transformed and z-score–transformed. (F) Reactivity of hIgG4 eluted from minipig A glomeruli as primary antibody (1:2) with recombinant hrPLA₂R1 as antigen (3.7 µg hrPLA₂R1-expressing human embryonic kidney cell lysate per lane) in Western blot analysis. As a positive control, serum from a patient with PLA₂R1-associated MN (1:100) was used. No reactivity to PLA₂R1 was seen when IgG from glomeruli of the control minipig B was eluted. (G) Development of proteinuria in minipig A, but not in the control minipig B during the course of the experiment. Analyzes of all urine samples from minipig A and minipig B using Coomassie blue staining showed dominant albumin and IgG bands, starting already at day 7, in the urine of minipig A, but not in the urine of minipig B. CL, capillary lumen; HGE, human glomerular extract; hrPLA₂R1, human recombinant PLA₂R1; pAlb, minipig albumin; PGE, minipig glomerular extract; PLA₂R1, phospholipase A₂ receptor 1; UR, urinary space.