Multi-Autoantibody Signature and Clinical Outcome in Membranous Nephropathy

Abstract

Background and objectives: Patients with membranous nephropathy can have circulating autoantibodies against membrane-bound (phospholipase A2 receptor 1 [PLA2R1] and thrombospondin type-1 domain containing 7A [THSD7A]) and intracellular (aldose reductase, SOD2, and α-enolase) podocyte autoantigens. We studied their combined association with clinical outcomes.

Design, setting, participants, & measurements: Serum levels of anti-PLA2R1, anti-THSD7A, anti-aldose reductase, anti-SOD2, and anti-α-enolase autoantibodies were determined in 285 patients at diagnosis and during follow-up using standardized and homemade assays. An eGFR>60 ml/min per 1.73 m² and remission of proteinuria (<0.3/<3.5 g per d) after 12 months were the outcomes of interest.

Results: At diagnosis, 182 (64%), eight (3%), and 95 (33%) patients were anti-PLA2R1⁺, anti-THSD7A⁺, and double negative, respectively. The prevalence of a detectable antibody to at least one intracellular antigen was similarly distributed in patients who were anti-PLA2R1⁺ (n=118, 65%) and double negative (n=64, 67%). Positivity for anti-PLA2R1, anti-SOD2, and anti-α-enolase antibodies and higher titers at diagnosis were associated with poor clinical outcome independently to each other. Combined positivity for anti-PLA2R1, anti-SOD2, and anti-α-enolase was associated with highest risk of poor outcome (odds ratio, 5.5; 95% confidence interval, 1.2 to 24; P=0.01). In Kaplan-Meier analysis, patients who were anti-PLA2R1⁺/anti-SOD2⁺ or anti-PLA2R1⁺/anti-α-enolase⁺ had lower eGFR at 12 months compared with patients who were anti-PLA2R1⁺/anti-SOD2^- or anti-α-enolase^-. Predictive tests (net reclassification index and area under the curve-receiver-operating characteristic analysis) showed that combined assessment of antibodies improved classification of outcome in 22%-34% of cases for partial remission of proteinuria and maintenance of normal eGFR. For patients with nephrotic syndrome at diagnosis, anti-SOD2 positivity and high anti-PLA2R1 titer were associated with a lack of complete remission. Patients who were anti-PLA2R1^-/anti-intracellular antigens^- had the lowest proteinuria and the highest eGFR at diagnosis and the lowest risk of lower eGFR at 12 months. Epitope spreading was present in 81% of patients who were anti-PLA2R1⁺ and was associated with increased positivity for intracellular antigens and poor eGFR at diagnosis and 12 months.

Conclusions: Combined serological analysis of autoantibodies targeting membrane-bound and intracellular autoantigens identifies patients with poor clinical outcomes.

Keywords: autoantibodies; glomerular disease; glomerulonephritis; membranous nephropathy.

Graphical abstract

Figure 1.

Detection of circulating autoantibodies against the membrane-bound autoantigens (phospholipase A2 receptor 1 [PLA2R1] and thrombospondin type-1 domain containing 7A [THSD7A]) and against the intracellular autoantigens (aldose reductase [AR], superoxide dismutase 2 [SOD2], and α-enolase [αENO]) allows the stratification of patients in different groups.(A) Patients were first divided according to the presence of circulating autoantibodies targeting the membrane-bound autoantigens PLA2R1 or THSD7A. The three groups of patients (PLA2R1⁺, THSD7A⁺, and PLA2R1⁻/THSD7A⁻) were further divided according to positivity for autoantibodies targeting any of the three intracellular podocyte autoantigens (AR, SOD2, αENO). (B) Stratification of PLA2R1⁺ patients according to anti-PLA2R1 titers below and above the median. *The percentage of patients positive for intracellular autoantibodies was higher in those with high anti-PLA2R1 titers (P=0.02, Fisher’s exact test). MN, membranous nephropathy.

Figure 2.

Positivity for anti-SOD2 and anti-αENO has an additive effect with positivity for anti-PLA2R1 for decreased eGFR at 12 months. Kaplan–Meier curve showing the proportion of patients in the different groups who reached the endpoint of reduction of kidney function (eGFR <60 ml/min per 1.73 m²) after 12 months of follow-up based on the concomitant positivity or not for anti-PLA2R1 and anti-SOD2 (A) or anti-αENO (B). The Kaplan–Meier curve of the difference was calculated with the log-rank (Mantel-Cox) test and the hazard ratios with confidence intervals for the different groups.

Figure 3.

Patients with single or multiple positivity for autoantibodies have different clinical outcome of proteinuria and eGFR.(A) Proteinuria and (B) eGFR values (data are presented as median with interquartile range [IQR]) at diagnosis and after 12 months of follow-up for patients divided in four groups based on the concomitant positivity or not for anti-PLA2R1 (PLA2R1⁺ or PLA2R1⁻) and anti-intracellular antigen antibodies (intracellular⁺ or intracellular⁻). Proteinuria (A) and eGFR (B) values are presented at diagnosis (open bars) and after 12 months (gray bars) for all patients (overall), for patients with nephrotic syndrome (NS) or not (non NS) at diagnosis and for patients who had been treated with an immunosuppressive treatment (IS-treated with cyclophosphamide, cyclosporine A, or rituximab) after 12 months of follow-up (black bars).

Figure 4.

Patients with positivity for multiple autoantibodies have worse clinical outcome (complete remission and eGFR) than those all negative. (A) Kaplan–Meier curve showing the proportion of patients in the different groups, based on the concomitant positivity or not for anti-PLA2R1 (P⁺/P⁻) and anti-intracellular antibodies (IC⁺/IC⁻) who reached the endpoint of complete remission (proteinuria <0.3 g/d) after 12 months of follow-up. The Kaplan–Meier curve of the difference was calculated with the log-rank (Mantel-Cox) test and the hazard ratios with confidence intervals for the different groups. (B) Kaplan–Meier curve showing the proportion of patients in the different groups, based on the concomitant negativity for anti-PLA2R1 (P⁻) and anti-intracellular antigen antibody negativity or positivity (IC⁺/IC⁻) who reached the endpoint of reduction of kidney function (eGFR<60 ml/min per 1.73 m²) after 12 months of follow-up. (C) Odds ratios and confidence intervals were calculated for the association between the different groups and the risk of not reducing kidney function (eGFR<60 ml/min per 1.73 m²) after 12 months of follow-up.

Figure 4.

Patients with positivity for multiple autoantibodies have worse clinical outcome (complete remission and eGFR) than those all negative. (A) Kaplan–Meier curve showing the proportion of patients in the different groups, based on the concomitant positivity or not for anti-PLA2R1 (P⁺/P⁻) and anti-intracellular antibodies (IC⁺/IC⁻) who reached the endpoint of complete remission (proteinuria <0.3 g/d) after 12 months of follow-up. The Kaplan–Meier curve of the difference was calculated with the log-rank (Mantel-Cox) test and the hazard ratios with confidence intervals for the different groups. (B) Kaplan–Meier curve showing the proportion of patients in the different groups, based on the concomitant negativity for anti-PLA2R1 (P⁻) and anti-intracellular antigen antibody negativity or positivity (IC⁺/IC⁻) who reached the endpoint of reduction of kidney function (eGFR<60 ml/min per 1.73 m²) after 12 months of follow-up. (C) Odds ratios and confidence intervals were calculated for the association between the different groups and the risk of not reducing kidney function (eGFR<60 ml/min per 1.73 m²) after 12 months of follow-up.