Neural cell adhesion molecule 1 is a novel autoantigen in membranous lupus nephritis

Abstract

Membranous lupus nephritis is a frequent cause of nephrotic syndrome in patients with systemic lupus erythematosus. It has been shown in phospholipase A2 receptor positive membranous nephropathy that known antibodies can be detected within sera, determination of the target autoantigen can have diagnostic significance, inform prognosis, and enable non-invasive monitoring of disease activity. Here we utilized mass spectrometry for antigen discovery in laser captured microdissected glomeruli from formalin-fixed paraffin embedded tissue and tissue protein G immunoprecipitation studies to interrogate immune complexes from frozen kidney biopsy tissue. We identified neural cell adhesion molecule 1 (NCAM1) to be a target antigen in some cases of membranous lupus nephritis and within rare cases of primary membranous nephropathy. The prevalence of NCAM1 association was 6.6% of cases of membranous lupus nephritis and in 2.0% of primary membranous nephropathy cases. NCAM1 was found to colocalize with IgG within glomerular immune deposits by confocal microscopy. Additionally, serum from patients with NCAM1-associated membranous nephropathy showed reactivity to NCAM1 recombinant protein on Western blotting and by indirect immunofluorescence assay, demonstrating the presence of circulating antibodies. Thus, we propose that NCAM1 is a target autoantigen in a subset of patients with membranous lupus nephritis. Future studies are needed to determine whether anti-NCAM1 antibody levels correlate with disease activity or response to therapy.

Keywords: glomerular disease; immune complex; lupus nephritis; membranous nephropathy; serum testing; systemic lupus erythematosus.

Figure 1.

NCAM1 is significantly enriched in glomeruli from a subset of membranous lupus nephritis patients. Normalized iBAQ values from MaxQuant were used for statistical analysis. Figures on the left (A, C, E) compare protein abundance in LCMD glomeruli and figures on the right (B, D, E) compare abundance of proteins obtained by immunoglobulin captured from kidney tissue. Dashed lines depict cutoff for p value=0.05. (A) Volcano plot comparing MS of laser capture microdissected glomeruli from eight PLA2R-positive MN cases compared with seven membranous nephropathy samples of other types identifies PLA2R as the protein with the highest fold change. (B) Volcano plot comparing MS of immunoglobulin capture from four PLA2R frozen kidney biopsy samples compared with seven membranous nephropathy samples of other types identifies identifies PLA2R as the protein with the highest fold change. (C) Volcano plot comparing MS of laser capture microdissected glomeruli from four EXT-positive membranous cases compared with eleven membranous nephropathy samples of other types identifies EXT1 as the protein with the highest fold change. (D) Volcano plot comparing MS of immunoglobulin capture from four frozen kidney biopsy samples that show EXT-positive MN compared with seven membranous nephropathy samples of other types identifies EXT1 and EXT2 as the proteins with the highest fold change. (E) Volcano plot comparing MS of laser capture microdissected glomeruli from three NCAM1-positive membranous cases with twelve membranous nephropathy samples of other types identifies NCAM1 as the protein with the highest fold change. (F) Volcano plot comparing MS of immunoglobulin capture from three frozen kidney biopsy samples that show NCAM1-positive MN with eight membranous nephropathy samples of other types identifies NCAM1 as the protein with the highest fold change. Each dot on the volcano blot represents a protein detected by mass spectrometry.

Figure 2.

Co-localization of NCAM1 with IgG in cases of NCAM1-associated MN, but not PLA2R controls. A) IgG staining of a predicted NCAM1-positive case; B) NCAM1 staining of a predicted NCAM1-positive case; C) Overlay of IgG and NCAM1 of a predicted NCAM1 positive case; D) IgG staining of a PLA2R MN case; E) NCAM1 staining of a PLA2R MN case; F) Overlay of IgG and NCAM1 in a PLA2R MN case.

Figure 3.

Histopathology of NCAM1-associated MN. (A) NCAM1 positivity in a granular capillary loop distribution within glomeruli (NCAM1 labeled with rhodamine red X; original magnification × 200, scale bar = 20 μm) (B) Glomerulus with endocapillary hypercellularity (Periodic acid Schiff; original magnification ×400, scale bar = 20 μm) (c), Spikes and holes seen along the glomerular capillary loops by Jones methenamine silver staining (original magnification ×400, scale bar = 20 μm). (D and E) IgG immunofluorescence shows (D) global granular glomerular capillary loops staining (original magnification ×400, scale bar = 20 μm) and (E) granular tubular basement membrane staining (original magnification ×200, scale bar = 50 μm). (F), Subepithelial electron dense deposits by electron microscopy (original magnification ×5000).

Figure 4.

Serum reactivity against NCAM1 identified in patients with NCAM1-associated MN. Western blotting shows immunoreactivity against NCAM1 recombinant protein in NCAM1-associated MN but not PLA2R-associated MN controls. A band of 120 kDa was identified, corresponding to that obtained after incubation with an anti-NCAM1 primary antibody.