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Mechanisms for the generation of anti-dsDNA autoantibodies are still not completely elucidated. One theory states that dsDNA interacts for mimicry with antibodies raised versus other antigens but molecular features for mimicry are unknown. Here we show that, at physiological acid-base balance, anti-Annexin A1 binds IgG2 dsDNA in a competitive and dose-dependent way with Annexin A1 and that the competition between the two molecules is null at pH 9. On the other hand, these findings also show that dsDNA and Annexin A1 interact with their respective antibodies on a strictly pH-dependent basis: in both cases, the binding was minimal at pH 4 and maximal at pH9-10. The anionic charge of dsDNA is mainly conferred by the numerous phosphatidic residues. The epitope binding site of Annexin A1 for anti-Annexin A1 IgG2 was here characterized as a string of 34 amino acids at the NH2 terminus, 10 of which are anionic. Circulating levels of anti-dsDNA and anti-Annexin A1 IgG2 antibodies were strongly correlated in patients with systemic lupus erythematosus (n 496) and lupus nephritis (n 425) stratified for age, sex, etc. These results show that dsDNA competes with Annexin A1 for the binding with anti-Annexin A1 IgG2 on a dose and charged mediated base, being able to display an inhibition up to 75%. This study provides the first demonstration that dsDNA may interact with antibodies raised versus other anionic molecules (anti-Annexin A1 IgG2) because of charge mimicry and this interaction may contribute to anti-dsDNA antibodies generation.

The podocyte injury, and consequent proteinuria, that characterize the pathology of idiopathic membranous nephropathy (IMN) is mediated by an autoimmune reaction against podocyte antigens. In particular, the activation of pathways leading to abundant renal deposits of complement is likely to involve the binding of mannose-binding lectin (MBL) to aberrant glycans on immunoglobulins. To obtain a landscape of circulatory IgG Fc glycosylation characterizing this disease, we conducted a systematic N-glycan profiling study of IgG1, 2, and 4 by mass spectrometry. The cohort included 57 IMN patients, a pathological control group with nephrotic syndrome (PN) (n = 20), and 88 healthy control subjects. The effect of sex and age was assessed in all groups and controlled by rigorous matching. Several IgG Fc glycan traits were found to be associated with IMN. Interestingly, among them, only IgG4-related results were specific for IMN and not for PN. Hypo-galactosylation of IgG4, already shown for IMN, was observed to occur in the absence of core fucose, in line with a probable increase of pro-inflammatory IgG. In addition, elevated levels of fucosylated IgG4, along with low levels of hybrid-type glycans, were detected. Some of these IgG4 alterations are likely to be more pronounced in high PLA2R (phospholipase A2 receptor) patients. IgG Fc glycosylation patterns associated with IMN warrant further studies of their role in disease mechanisms and may eventually enrich the diagnostic spectrum regarding patient stratification.

Background: Belimumab (Benlysta) is currently approved for the treatment of active Lupus despite standard therapy. Few data are available on the efficacy of this drug in lupus nephritis (LN). Methods: 17 LN female followed in two Nephrology Italian Unit received belimumab for a median period of 36 months (range 6–42 months). The indications were: arthralgia in 3 patients, cutaneous manifestations in 2, residual proteinuria in 8, and the need to reduce steroids for severe side effects in 4. Of interest, 1 patient started therapy during Peritoneal Dialysis and continued after kidney transplantation due to non-responsive arthralgias. Results: Arthralgia and skin manifestations resolved in all patients. Proteinuria normalized in three patients and stabilized in all but one of the others. Steroids were indefinitely stopped in six patients (35%) and reduced to around 40% of the basal dosage in the other patients. During belimumab therapy, three extrarenal and one renal SLE flares were diagnosed accounting for a rate of renal flares of 0.02/patient/year. No major adverse events leading to therapy withdrawal occurred. Clinical case: Arthralgia resolved, immunological parameters improved and prednisone could be reduced within few months in the patient who started belimumab during peritoneal dialysis. After kidney transplantation belimumab was stopped but due to arthralgias unresponsive to standard immunosuppressive therapy it was restarted with success. Conclusions: Belimumab allows the achievement of complete response together with the withdrawal or the reduction of corticosteroids in almost all our patients. Of interest its satisfactory use in a patient in peritoneal dialysis and after kidney transplantation.

ObjectiveTo investigate second kidney biopsy as predictor of end-stage kidney disease (ESKD) in active lupus nephritis (LN).MethodsPatients with biopsy-proven LN (International Society of Nephrology/Renal Pathology Society 2003) who had undergone a second kidney biopsy between January 1990 and December 2018 were included. Clinical and histological findings at first and at second biopsy were analysed with Cox proportional hazard models to predict ESKD, defined as start of kidney replacement therapy. Survival curves were calculated with Kaplan-Meier method.ResultsNinety-two patients with LN were included, 87% females, mean follow-up 17.9±10.1 years. Reasons for second kidney biopsy encompassed nephritic flares (n=28, 30.4%), proteinuric flares (n=46, 50%) or lack of renal response (n=18, 19.5%). Class switch from first biopsy occurred in 50.5% of cases, mainly from non-proliferative towards proliferative classes. Class IV remained stable in over 50% of cases. Twenty-five patients (27.2%) developed ESKD, mostly belonging to the nephritic flare group (17/28, 60.7%). Independent predictors of ESKD at second biopsy were activity index (AI; (HR 95% CI) 1.20 (1.03 to 1.41), p=0.022), chronicity index (CI; 1.41 (1.09 to 1.82), p=0.008) and 24h-proteinuria (1.22 (1.04 to 1.42), p=0.013). AI≥2 (log-rank p=0.031), CI >4 (log-rank p=0.001) or proteinuria ≥3.5 g/day (log-rank=0.009) identified thresholds for higher ESKD risk. In a subgroup analysis, glomerular activity and tubular chronicity mostly accounted for AI and CI association with ESKD. No histological or laboratory predictors emerged at first biopsy (95% CI): AI: 0.88 to 1.19; CI: 0.66 to 1.20; proteinuria 0.85 to 1.08.ConclusionsFindings at second but not at first kidney biopsy in patients with persistently active or relapsing LN inform about ESKD development in a long-term follow-up.

Objectives Serum anti-dsDNA and anti-nucleosome IgGs have been proposed as signatures for SLE and LN in limited numbers of patients. We sought to show higher sensitivity and specificity of the same antibodies with the IgG2 isotype and included IgG2 antibodies vs specific intracellular antigens in the analysis. Methods A total of 1052 SLE patients with (n = 479) and without (n = 573) LN, recruited at different times from the beginning of symptoms, were included in the study. Patients with primary APS (PAPS, n = 24), RA (RA, n = 24) and UCTD (UCTD, n = 96) were analysed for comparison. Anti-nucleosome (dsDNA, Histone2A, Histone3), anti-intracellular antigens (ENO1), anti-annexin A1 and anti-C1q IgG2 were determined by non-commercial techniques. Results The presence in the serum of the IgG2 panel was highly discriminatory for SLE/LN vs healthy subjects. Serum levels of anti-dsDNA and anti-C1q IgG2 were more sensitive than those of IgGs (Farr radioimmunoassay/commercial assays) in identifying SLE patients at low–medium increments. Of more importance, serum positivity for anti-ENO1 and anti-H2A IgG2 discriminated between LN and SLE (ROC T0–12 months), and high levels at T0–1 month were detected in 63% and 67%, respectively, of LN, vs 3% and 3%, respectively, of SLE patients; serum positivity for each of these was correlated with high SLEDAI values. Minor differences existed between LN/SLE and the other rheumatologic conditions. Conclusion Nephritogenic IgG2 antibodies represent a specific signature of SLE/LN, with a few overlaps with other rheumatologic conditions. High levels of anti-ENO1 and anti-H2A IgG2 correlated with SLE activity indexes and were discriminatory between SLE patients limited to the renal complication and other SLE patients. Trial registration The Zeus study was registered at https://clinicaltrials.gov, NCT02403115.

Objectives Circulating anti-ENO1 and anti-H2A IgG2 have been identified as specific signatures of LN in a cross-over approach. We sought to show whether the same antibodies identify selected population of patients with LN with potentially different clinical outcomes. Methods Here we report the prospective analysis over 36 months of circulating IgG2 levels in patients with newly diagnosed LN (n=91) and SLE (n=31) and in other patients with SLE recruited within 2 years from diagnosis (n=99). Anti-podocyte (ENO1), anti-nucleosome (DNA, histone 2 A, histone 3) and anti-circulating proteins (C1q, AnnexinA1-ANXA1) IgG2 antibodies were determined by home-made techniques. Results LN patients were the main focus of the study. Anti-ENO1, anti-H2A and anti-ANXA1 IgG2 decreased in parallel to proteinuria and normalized within 12 months in the majority of patients while anti-dsDNA IgG2 remained high over the 36 months. Anti-ENO1 and anti-H2A had the highest association with proteinuria (Heat Map) and identified the highest number of patients with high proteinuria (68% and 71% respectively) and/or with reduced estimated glomerula filtration rate (eGFR) (58% for both antibodies) compared with 23% and 17% of anti-dsDNA (agreement analysis). Anti-ENO1 positive LN patients had higher proteinuria than negative patients at T0 and presented the maximal decrement within 12 months. Conclusions Anti-ENO1, anti-H2A and anti-ANXA1 antibodies were associated with high proteinuria in LN patients and Anti-ENO1 also presented the maximal reduction within 12 months that paralleled the decrease of proteinuria. Anti-dsDNA were not associated with renal outcome parameters. New IgG2 antibody signatures should be utilized as tracers of personalized therapies in LN. Trial registration The Zeus study was registered at https://clinicaltrials.gov (study number: NCT02403115).

ObjectivesShort-term predictive endpoints of chronic kidney disease (CKD) are needed in lupus nephritis (LN). We tested response to therapy at 1 year.MethodsWe considered patients with LN who underwent renal biopsy followed by induction therapy between January 1970 and December 2016. LN was assessed using the International Society of Nephrology/Renal Pathology Society (2003) criteria and the National Institute of Health (NIH) activity and chronicity index. The renal outcome was CKD. Response was defined according to EULAR/European League Against Rheumatism/European Renal Association-European Dialysis and Transplant Association (EULAR/ERA-EDTA) recommendations:complete:proteinuria partial:≥50% proteinuria reduction to subnephrotic levels, (near) normal eGFR; andno response:all the other cases. Logistic regression analysis was employed for 12-month response and Cox regression for CKD prediction.ResultsWe studied 381 patients (90.5% Caucasians). After 12-month therapy, 58%, 26% and 16% of patients achieved complete, partial and no response, respectively, according to EULAR/ERA-EDTA. During a median follow-up of 10.7 (IQR: 4.97–18.80) years, 53 patients developed CKD. At 15 years, CKD-free survival rate was 95.2%, 87.6% and 55.4% in patients with complete, partial and no response at 12 months, respectively (pConclusionsLack of EULAR/ERA-EDTA response at 12 months predicts CKD.

Autoantibodies to M-type phospholipase A2 receptor (PLA2R) are specific markers of idiopathic membranous nephropathy (IMN). They can differentiate IMN from other glomerular diseases and primary from secondary forms of MN. Preliminary data suggest that anti-PLA2R antibody titer correlates with disease activity but more solid evidence is needed.To evaluate the performance of anti-PLA2R antibody for monitoring nephropathy activity, 149 anti-PLA2R antibody measurements were performed during the follow-up of 42 biopsy proven IMN consecutive patients. Patients were enrolled either at time of diagnosis (33 cases, inception cohort) or after diagnosis (9 patients, non-inception cohort).Anti-PLA2R detection was performed using the highly sensitive transfected cell-based indirect immunofluorescence (IIFT).Over the follow-up there was a linear time-trend of decreasing proteinuria (P < 0.001), increasing serum albumin (P < 0.001) and decreasing PLA2R antibody levels (P = 0.002). There was a statistically significant association between changes in PLA2R antibody levels and the clinical course of PLA2R-positive IMN. The positive PLA2R serum antibody status was linearly associated with increasing proteinuria and decreasing serum albumin over time, compared with negative antibody status. Moreover, the strong correlation between the clinical conditions and PLA2R antibody levels allowed the prediction of prevalence distribution of patients with active disease, partial and complete remission. Over the course of the follow-up, the probability of halving proteinuria increased 6.5 times after disappearance of PLA2R antibodies.Our data suggest that the serial evaluation of anti-PLA2R antibodies could help in optimal timing and duration of the immunosuppressive therapy, reducing over(under)-treatment and associated side-effects.

Autoantibody against phospholipase A2 receptor (anti-PLA2R) is a sensitive and specific biomarker of idiopathic membranous nephropathy (iMN), being found in approximately 70% of iMN patients and only occasionally in other glomerular diseases. However, whereas its diagnostic specificity vs. normal controls and other glomerulonephritides (GN) has been firmly established, its specificity vs. membranous nephropathy associated with various diseases (sMN) has given inconsistent results. The aim of our study was to evaluate the prevalence of anti-PLA2R antibodies in iMN in comparison with various control groups, including sMN. A total of 252 consecutive iMN patients, 184 pathological and 43 healthy controls were tested for anti-PLA2R antibody using indirect immunofluorescence (PLA2R IIFT, Euroimmun). Anti-PLA2R autoantibodies were detectable in 178/252 iMN patients, 1/80 primary GN, 0/72 secondary GN, 9/32 sMN and 0/43 healthy controls, with a diagnostic sensitivity of 70.6%. The diagnostic specificity of anti-PLA2R antibody vs. normal and pathological controls was 100 and 94.6% respectively. However, when the diagnostic specificity was calculated only vs. secondary forms of MN, it decreased considerably to 71.9%. Interestingly enough, 9 out of 10 anti-PLA2R positive patients in the disease control groups had membranous nephropathy associated with various diseases (7 cancer, 1 Crohn's disease, 1 scleroderma). In conclusion, anti-PLA2R positivity in a patient with MN, should not be considered sufficient to abstain from seeking a secondary cause, especially in patients with risk factors for neoplasia. The causal relationship between tumors and anti-PLA2R-induced MN remains to be established, as well as the possible mechanisms through which malignancies provoke autoimmunity.

Glomerular planted antigens (histones, DNA, and C1q) are potential targets of autoimmunity in lupus nephritis (LN). However, the characterization of these antigens in human glomeruli in vivo remains inconsistent. We eluted glomerular autoantibodies recognizing planted antigens from laser-microdissected renal biopsy samples of 20 patients with LN. Prevalent antibody isotypes were defined, levels were determined, and glomerular colocalization was investigated. Renal and circulating antibodies were matched, and serum levels were compared in 104 patients with LN, 84 patients with SLE without LN, and 50 patients with rheumatoid arthritis (RA). Autoantibodies against podocyte antigens (anti–α-enolase/antiannexin AI) were also investigated. IgG2 autoantibodies against DNA, histones (H2A, H3, and H4), and C1q were detected in 50%, 55%, and 70% of biopsy samples, respectively. Anti-DNA IgG3 was the unique non-IgG2 anti-DNA deposit, and anti-C1q IgG4 was mainly detected in subepithelial membranous deposits. Anti-H3, anti-DNA, and anti-C1q IgG2 autoantibodies were also prevalent in LN serum, which also contained IgG3 against the antigen panel and anti-C1q IgG4. Serum and glomerular levels of autoantibodies were not strictly associated. High serum levels of all autoantibodies detected, including anti–α-enolase and antiannexin AI, identified LN versus SLE and RA. Anti-H3 and anti–α-enolase IgG2 levels had the most remarkable increase in LN serum and represented a discriminating feature of LN in principal component analysis. The highest levels of these two autoantibodies were also associated with proteinuria>3.5 g/24 hours and creatinine>1.2 mg/dl. Our findings suggest that timely autoantibody characterization might allow outcome prediction and targeted therapies for patients with nephritis.

Rapidly progressive glomerulonephritis (RPGN) is mainly caused by anti-glomerular basement membrane (GBM) antibody-mediated glomerulonephritis, immune-complex or anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides and leads to rapid loss of renal function. Detection of ANCA and autoantibodies (autoAbs) to GBM and dsDNA enables early diagnosis and appropriate treatment of RPGN aiding in preventing end-stage renal disease. Determination of ANCA on neutrophils (ANCA) as well as autoAbs to myeloperoxidase (MPO-ANCA), proteinase 3 (PR3-ANCA), GBM, and dsDNA was performed by the novel multiplex CytoBead technology combining cell- and microbead-based autoAb analyses by automated indirect immunofluorescence (IIF). Forty patients with granulomatosis with polyangiitis (GPA), 48 with microscopic polyangiitis (MPA), 2 with eosinophilic GPA, 42 with systemic lupus erythematosus (SLE), 43 with Goodpasture syndrome (GPS), 57 with infectious diseases (INF), and 55 healthy subjects (HS) were analyzed and findings compared with classical single testing. The CytoBead assay revealed for GPA, MPA, GPS, and SLE the following diagnostic sensitivities and for HS and INF the corresponding specificities: PR3-ANCA, 85.0% and 100.0%; MPO-ANCA, 77.1% and 99.1%; anti-GBM autoAb, 88.4% and 96.4%; anti-dsDNA autoAb, 83.3% and 97.3%; ANCA, 91.1% and 99.1%, respectively. Agreement with classical enzyme-linked immunosorbent assay and IIF was very good for anti-GBM autoAb, MPO-ANCA, PR3-ANCA, and ANCA, respectively. Anti-dsDNA autoAb comparative analysis demonstrated fair agreement only and a significant difference (P = 0.0001). The CytoBead technology provides a unique multiplex reaction environment for simultaneous RPGN-specific autoAb testing. CytoBead RPGN assay is a promising alternative to time-consuming single parameter analysis and, thus, is well suited for emergency situations.

Summary Membranous nephropathy (MN), the leading cause of nephrotic syndrome in adults, is characterized by the deposition of subepithelial immune deposits that consist mainly of immunoglobulin (Ig)G and complement. Most of the cases are primary or idiopathic (iMN), while only approximately 25% of the cases are secondary to some known disease such as systemic lupus erythematosus, hepatitis B, drugs and malignancies. Most of our knowledge on the pathogenesis of iMN has relied upon old experimental models (i.e. Heymann nephritis) that have shown that immune deposits are formed in situ by the reaction of autoantibodies against the respective podocyte antigen. Recent findings indicate that podocyte proteins also act as an autoantigen in human iMN. The M-type phospholipase A2 receptor (PLA2R) has been identified as the main target antigen, as it can be found in approximately 70% of iMN patients but only rarely in other glomerulonephritides. Podocytes damage in the experimental model of Heymann nephritis is complement-mediated. In humans, the presence of complement within the subepithelial deposits is well established, but IgG4, which does not activate complement by classical or alternative pathways, represents the predominant subclass of IgG anti-PLA2R. Some evidence suggests that IgG4 anti-PLA2R autoantibodies can bind mannan-binding lectin (MBL) and activate the lectin complement pathway. A genetic background for iMN has been demonstrated by genome-wide association studies that have shown highly significant associations of the PLA2R1 and the human leucocyte antigen (HLA)-DQA1 loci with iMN. In addition to their diagnostic value, anti-PLA2R antibodies may be useful to monitor disease activity and predict response to treatment.

Few studies have correlated serum biomarkers with renal histology, the gold standard for renal activity, in lupus nephritis (LN). We tested a panel of autoantibodies and complement at the time of kidney biopsy and after treatment. Anti-dsDNA, anti-nucleosome, anti-ribosome P, and anti-C1q antibodies and C3/C4 were measured in 107 patients with LN at the time of renal biopsy and after 6–12 months and were correlated with clinical/histological parameters. At multivariate analysis, high titers of anti-C1q antibodies or of anti-dsDNA antibodies (P=0.005, OR = 8.67, CI: 2.03–37.3) were the independent predictors that discriminate proliferative from nonproliferative LN. All the immunological parameters, except anti-ribosome, showed a significant correlation with activity index but not with chronicity index. Only anti-C1q showed a significant correlation with the amount of proteinuria (R=0.2,P=0.03). None of the immunological parameters were predictive of remission at 6 and 12 months. We found that anti-C1q alone or in combination with anti-dsDNA emerged as the most reliable test in differentiating proliferative and nonproliferative LN. Anti-C1q was the only test correlated with the clinical presentation of LN. After treatment, the titre of the autoantibodies was significantly reduced, but none was predictive of remission.

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti–α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti–α-enolase/low anti-annexin AI IgG2 and patients with low anti–α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti–α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum.

Objective. We report the first comparison between rituximab (RTX) and either MMF or CYC pulses in the treatment of active LN. Methods. Fifty-four patients with active LN received three methylprednisolone pulses for 3 consecutive days followed by oral prednisone and RTX 1 g at days 3 and 18 (17 patients) or MMF 2-2.5 g/day (17 patients) or six CYC pulses (0.5 g every fortnight) (20 patients). At 4 months MMF, AZA or ciclosporin were associated to prednisone as a consolidation/maintenance therapy in all groups. The outcomes of the three groups were compared at 3 and 12 months. Results. Patients in the RTX group were older, had a longer duration of SLE and LN, had more renal flares, had higher activity and had higher chronicity indexes at renal biopsy than the other two groups. Four patients in each group had acute renal dysfunction and ∼50% had nephrotic syndrome. At 3 months, proteinuria was reduced by 50% in 58.8% of patients on RTX, in 64.7% on MMF and in 63.1% on CYC. At 12 months, complete remission was present in 70.6% of patients on RTX, in 52.9% on MMF, and in 65% on CYC. Partial remission was reached in 29.4% on RTX, 41.2% on MMF, and 25% on CYC. Conclusion. RTX seems to be at least as effective as MMF and CYC pulses in inducing remission. Considering that patients treated with RTX had more negative renal prognostic factors, this drug should be considered a viable alternative for the treatment of active LN. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.