Autoantibodies targeting glomerular annexin A2 identify patients with proliferative lupus nephritis

Abstract

Purpose: Patients with systemic lupus erythematosus (SLE) frequently develop lupus nephritis (LN), a complication frequently leading to end stage kidney disease. Immune complex deposition in the glomerulus is central to the development of LN. Using a targeted proteomic approach, we tested the hypothesis that autoantibodies targeting glomerular antigens contribute to the development of LN.

Experimental design: Human podocyte and glomerular proteins were separated by SDS-PAGE and immunoblotted with sera from SLE patients with and without LN. The regions of those gels corresponding to reactive bands observed with sera from LN patients were analyzed using LC-MS/MS.

Results: LN reactive bands were seen at approximately 50 kDa in podocyte extracts and between 36 and 50 kDa in glomerular extracts. Those bands were analyzed by LC-MS/MS and 102 overlapping proteins were identified. Bioinformatic analysis determined that 36 of those proteins were membrane associated, including a protein previously suggested to contribute to glomerulonephritis and LN, annexin A2. By ELISA, patients with proliferative LN demonstrated significantly increased antibodies against annexin A2.

Conclusion and clinical relevance: Proteomic approaches identified multiple candidate antigens for autoantibodies in patients with LN. Serum antibodies against annexin A2 were significantly elevated in subjects with proliferative LN, validating those antibodies as potential biomarkers.

Keywords: Autoantibodies; Glomerulonephritis; Lupus nephritis; Systemic lupus erythematosus; Target antigens.

Figure 1. Sera from LN subjects demonstrate selective reactivity to glomerular and podocyte proteins

a. Representative immunoblots of individual lupus subjects with human glomerular proteins. Sera at a dilution of 1:200 from individual lupus subjects were immunoblotted against 30 μg of human glomerular proteins. Immunoblots from a lupus subject without nephritis (LC), a lupus subject with proliferative nephritis (PLN), and a lupus subject with membranous nephritis (MLN) are shown. b. Patterns of sera reactivity to human glomerular proteins among subjects with SLE. Graph demonstrating patterns sera reactivity of individual subjects against human glomerular extracts. 10 subjects with SLE without nephritis (LC), 10 subjects with proliferative lupus nephritis (PLN), and 15 subjects with membranous lupus nephritis (MLN) were compared. The percentage of subjects with reactive bands in each region is displayed. c. Immunoblots of individual lupus subjects with human glomerular proteins highlighting the region of interest from 36-50 kDa. Sera from individual lupus subjects were immunoblotted against 30 μg of human glomerular proteins at 1:200 dilution. Immunoblots from 10 lupus subjects without nephritis (C1-C10), 10 subjects with proliferative lupus nephritis (P1-P10), and 15 subjects with membranous lupus nephritis (M1-M15) are shown. d. Human podocyte immunoblot demonstrating separation of membrane and cytosolic fractions. Podocyte plasma membrane (M) and cytosol (C) fractions were probed with antibodies against plasma membrane proteins PLA₂R and NAK-α1, and cytosolic WT-1. e. Immunoblot of pooled sera with human podocyte membrane proteins. Pooled sera from 10 lupus subjects without nephritis (LC), 5 subjects with MLN, and 10 subjects with PLN were immunoblotted against 20 μg of podocyte membrane proteins at 1:10,000 dilution. A reactive band at approximately 50 kDa was seen in the MLN and PLN subjects, but not in the LC subjects.

Figure 2. Annexin A2 ELISA

Sera from 10 proliferative lupus nephritis (PLN), 10 membranous lupus nephritis (MLN), 10 lupus without nephritis-lupus control (LC), and 10 normal control (NC) subjects were analyzed in duplicate at a 1:200 dilution. PLN subjects demonstrated a mean OD value of 0.204, MLN subjects demonstrated a mean OD value of 0.018, LC subjects demonstrated a mean OD value of 0.059, and NC subjects demonstrated a mean OD value of 0.056. One way analysis of variance (ANOVA) comparing individual anti-annexin A2 levels demonstrated a p-value of 1.25 X 10⁻¹⁰ and Tukey analysis verified statistical difference among groups comparing PLN to MLN, LC, and NC subjects (P < 0.01). Error bars demonstrate standard error of the mean.