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. 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. There are an estimated 121,000 to 402,000 patients living with systemic lupus erythematosus (SLE) in the United States, which disproportionately affects women and African Americans.1Stojan G. Petri M. Epidemiology of systemic lupus erythematosus: an update.Curr Opin Rheumatol. 2018; 30: 144-150Crossref PubMed Scopus (87) Google Scholar SLE can involve multiple organ systems, and lupus nephritis is a serious complication that affects up to half of lupus patients.2Almaani S. Meara A. Rovin B.H. Update on lupus nephritis.Clin J Am Soc Nephrol. 2017; 5: 825-835Crossref Scopus (310) Google Scholar Lupus nephritis causes significant morbidity in SLE patients, with 11% progressing to end-stage kidney disease and a requirement for renal replacement therapies.3Tektonidou M.G. Dasgupta A. Ward M.M. Risk of end-stage renal disease in patients with lupus nephritis, 1971-2015: a systematic review and Bayesian meta-analysis.Arthritis Rheumatol. 2016; 68: 1432-1441Crossref PubMed Scopus (153) Google Scholar Of lupus patients with biopsy-proven nephritis, 10%–20% have membranous lupus nephritis (MLN), which can occur with or without a concurrent proliferative component .4Huong D.L. Papo T. Beaufils H. et al.Renal involvement in systemic lupus erythematosus. A study of 180 patients from a single center.Medicine. 1999; 78: 148-166Crossref PubMed Scopus (187) Google Scholar MLN is the leading cause of nephrotic syndrome in patients with SLE. It is characterized by the presence of subepithelial and intramembranous immune deposits, which are caused by circulating immune complexes and/or in situ immune complex formation within glomeruli. In the majority of cases of MLN, the causative autoantigen is unknown. Recently, the exostosin (EXT) 1/2 protein complex was identified in the glomeruli of 34.6% of cases of SLE-associated membranous nephropathy (MN).5Sethi S. Madden B.J. Debiec H. et al.Exostosin 1/exostosin 2-associated membranous nephropathy.J Am Soc Nephrol. 2019; 30: 1123-1136Crossref PubMed Scopus (81) Google Scholar However, to date, circulating autoantibodies to EXT1 or EXT2 have not been identified. Serum testing in idiopathic membranous nephropathy has proven useful to monitor disease and inform treatment. Additionally, it has been shown that serologic remission can be detected months before resolution of proteinuria and evidence of clinical remission. For phospholipase A2 receptor (PLA2R)–positive membranous nephropathy, the causative autoantigen in the majority of idiopathic MN cases,6Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1332) Google Scholar antibody testing can serve as a biomarker of disease activity and response to treatment, as measured by indirect immunofluorescence and/or enzyme-linked immunosorbent assay–based testing.7Hofstra J.M. Beck Jr., L.H. Beck D.M. et al.Anti-phospholipase A(2) receptor antibodies correlate with clinical status in idiopathic membranous nephropathy.Clin J Am Soc Nephrol. 2011; 6: 1286-1291Crossref PubMed Scopus (263) Google Scholar Thrombospondin–type 1 domain containing 7a, the second identified autoantigen in MN,8Godel M. Grahammer F. Huber T.B. Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy.N Engl J Med. 2015; 372: 1073-1075Crossref PubMed Scopus (21) Google Scholar,9Tomas N.M. Beck Jr., L.H. Meyer-Schwesinger C. et al.Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy.N Engl J Med. 2014; 371: 2277-2287Crossref PubMed Scopus (446) Google Scholar also can be monitored by serum testing through indirect immunofluorescence-based assays.10Hoxha E. Beck Jr., L.H. Wiech T. et al.An indirect immunofluorescence method facilitates detection of thrombospondin type 1 domain-containing 7a-specific antibodies in membranous nephropathy.J Am Soc Nephrol. 2017; 28: 520-531Crossref PubMed Scopus (94) Google Scholar Identification of serum biomarkers in MLN could enable disease monitoring similar to what is now possible with PLA2R-positive primary membranous nephropathy, and is the focus of these investigations. MS of renal biopsies from patients with MLN was performed for autoantigen discovery. Mass spectra of laser-capture microdissected (LCMD) glomeruli from 13 cases of MN of unknown antigen type, including both lupus and non-lupus patients, were compared with 12 cases of known etiology (including 8 PLA2R and 4 EXT-associated MN) to identify proteins present uniquely in the unknown cases (Supplementary Figure S1). Three cases showed the presence of NCAM1 peptides uniquely in the unknown MN glomeruli with a total of 18 unique peptides identified (Supplementary Figure S2). All 3 of these patients with NCAM1 detected had SLE. The mass spectrometry (MS) profile of these LCMD glomeruli from 3 cases with abundant neural cell adhesion molecule 1 (NCAM1, also known as CD56) peptides were compared with the 12 cases of MN of known type to identify the protein with the greatest fold change. The protein differential abundance for each type of MN was compared against the remaining 2 groups using normalized intensity-based absolute quantification (iBAQ) values produced by MaxQuant (Max Planck Institute of Biochemistry, Planegg, Germany). Statistical analysis was performed using Welch's t test, and the results were depicted on a volcano plot (Figure 1). To determine the ability of this approach to identify glomerular antigens, we first compared samples from patients with known membranous antigens, PLA2R and EXT1, to cases that did not stain for these antigens. PLA2R was the protein with the strongest fold increase in cases of PLA2R-associated MN (Figure 1a). EXT1 displayed the strongest fold changes in EXT-associated MN (Figure 1c). These results demonstrate that analysis of the laser-capture microdissected glomeruli MS data in this way can identify proteins significant to the pathogenesis of disease in MN. When this same statistical analysis was used on the samples with NCAM1 identified, NCAM1 was identified as the protein with the highest fold increase (Figure 1e), and NCAM1 peptides were not present in the other samples. This confirms that NCAM1 is uniquely present in the glomeruli of a subset of patients with membranous nephropathy of unknown antigen type. As a further independent discovery method, protein G co-immunoprecipitation was performed on fresh-frozen tissue from a total of 11 kidney biopsies to identify proteins uniquely bound to Ig within the glomeruli of MN biopsies. This included 4 PLA2R MN, 4 EXT MN, and the 3 cases predicted as NCAM1 MN by LCMD-MS. All 3 patients predicted as NCAM1 MN had a history of SLE with MLN diagnosed on kidney biopsy. A total of 1448 distinct proteins were identified in these samples. A similar analysis was performed as with the LCMD samples in that each group was evaluated for proteins with the greatest fold change when compared with MN of other types. The protein differential abundance for each type of MN was compared against the remaining 2 groups using normalized iBAQ values. Statistical analysis was performed using Welch's t test, and the results were depicted on volcano plots (Figure 1). PLA2R was the protein with the strongest fold increase in cases of PLA2R-associated MN (Figure 1, a and b). EXT1 and EXT2 displayed the strongest fold changes in EXT-associated MN (Figure 1, c and d). NCAM1 was identified as the protein with the highest fold increase in the NCAM1-associated cases (Figure 1, e and f). NCAM1 was present in all 3 of the NCAM1-associated samples but none of the PLA2R- or EXT-associated samples. These immunoprecipitations from frozen tissue confirm that IgG is bound to NCAM1 in the samples with increased glomerular NCAM1 detected by MS. A rabbit polyclonal antibody against NCAM1 showed strong positive staining along the glomerular capillary loops of cases with NCAM-1-positive MN with essentially complete colocalization with IgG by confocal microscopy. Colocalization of NCAM1 with IgG was 96.1% ± 2.3% in NCAM1-associated MN cases (Figure 2) and 19.6% ± 5.8% of control PLA2R-positive MN cases (Figure 2). This background staining in PLA2R-positive MN cases is artifactual, likely due to serum trapping within glomeruli in formalin-fixed paraffin-embedded tissue. Colocalization with NCAM1 and IgG was significantly increased in NCAM1-associated MN cases, compared to PLA2R-positive MN controls (P < 0.0001, 1-way analysis of variance). NCAM1 did not show significant colocalization with IgG in the glomeruli from control PLA2R-positive MN cases (additional images provided in Supplementary Figure S3). There are rare foci of NCAM1 positivity along tubular basement membranes (TBMs) with colocalization with IgG; however, the majority of TBM deposits are NCAM1-negative (Supplementary Figure S4). Additionally, NCAM1 was not found to be positive in TBM deposits in every case of NCAM1 MN that contained IgG TBM deposits, so this finding is unlikely to be significant. No PLA2R-associated MN cases had TBM deposits for evaluation. To further establish specificity of NCAM1 detection in NCAM1-associated MN, NCAM1 staining was also performed on 20 proliferative lupus nephritis biopsies (International Society of Nephrology and the Renal Pathology Society [ISN/RPS] class III or IV) and 10 EXT-associated MN biopsies. Zero of 20 proliferative lupus nephritis biopsies and 0 of 10 EXT-associated MN biopsies were NCAM1-positive (Supplementary Figure S5). Additionally, no proliferative lupus nephritis or EXT-associated MN cases showed NCAM1 staining within TBM deposits (Supplementary Figure S5). To confirm antibody specificity, a second commercial antibody (Invitrogen anti-CD56, PA5-83479, Thermo Fisher Scientific - Invitrogen, Waltham, MA) demonstrated a similar pattern of staining within glomeruli in NCAM1-associated MN cases (data not shown). A series of consecutive MLN biopsies were identified over a 6-month period that could be tested for the prevalence of NCAM1-associated MN. A total of 216 consecutive cases of MLN with or without proliferative changes by light microscopy were diagnosed in our laboratory during the study time period. Among these, there were 212 cases with sufficient tissue remaining to stain for the type of MLN present. All cases were stained for NCAM1 by immunofluorescence. A total of 14 of 212 of MLN cases were NCAM1-positive (6.6%). EXT-staining was also performed on this cohort (of which 209 cases had residual tissue for staining), and 33 of 209 cases were EXT2-positive (15.8% of MLN cases). NCAM1-associated MN was rarely positive in a separate analysis of 101 cases of idiopathic MN stained for NCAM1 (2 of 101 cases, 2.0%). Twenty-four patients in this cohort were EXT2-positive; however, our biorepository frequency is over-representative of the true frequency, as the data are not from consecutive patients and there was increased recruitment of EXT-positive patients into our biorepository. Nineteen of 20 cases were mutually exclusive, with one MLN case identified with dual EXT and NCAM1 positivity. Clinical details for patients with NCAM1-associated MN are shown in Table 1. The patients were on average 34 years of age (±12.1 years) and were predominantly female (14 of 20 patients; 70%). The mean serum creatinine level was 1.35 ± 0.88 mg/dl. Quantitative proteinuria was on average 6.7 ± 9.5 g/d, determined by either urine protein-to-creatinine ratio or 24-hour urinary protein levels. The mean serum albumin level was depressed (2.2 ± 0.78 g/dl).Table 1Clinical and serologic features of cases of neural cell adhesion molecule 1–associated membranous nephropathyCaseDiagnosisAge (y)SexCr (mg/dl)Albumin (g/dl)Proteinuria (g/d)Serologies (positive)SLEDxNeuropsychiatric symptomsTreatment1Lupus V42F3.21.4ANA+, dsDNA+, Smith+YesSeizuresHydroxychloroquine2Lupus IV + V36M2.73.1YesHydroxychloroquine, mycophenolateprednisone3Membranous40M0.80.9No4Lupus V44M1.52.94ANA+, dsDNA+YesStrokePrednisone, azathioprine5Lupus V23F0.53.8YesSeizures, lupus cerebritisMonthly IV cyclophosphamide, prednisone, hydroxychloroquine6Lupus V38F2.1240ANA+, dsDNA+YesPsychiatric disorderHydroxychloroquine, methylprednisolone7Lupus V28F1.51.56.8ANA+YesSeizuresPrednisone, hydroxychloroquine, mycophenolate8Membranous54M15NoHydroxychloroquine, mycophenolate, ACEI9Lupus V24F0.72.30ANA+, Smith+YesACTHar, ACEI10Lupus V33F0.91.8Yes11Lupus III + V60M0.72YesCyclophosphamide, prednisone12Lupus III + V27F0.61.98.2ANA+, dsDNA+YesTocilizumab, ARB, prednisone, mycophenolate13Lupus V23F0.82.23YesMycophenolate, prednisone14Lupus III + V22F2.32.72.1ANA+YesLupus cerebritisHydroxychloroquine, mycophenolate,prednisone15Lupus III + V26F22.54.6ANA+YesBelimumab, prednisone16Lupus V47F3.71.811ANA+YesMethylprednisolone17Lupus V12F0.32.13+ UA, no quantitationANA+, ENA+YesHydroxychloroquine, mycophenolate18Lupus V44F1.63.22.5ANA+, SS-A+, SS-B+YesHydroxychloroquine, mycophenolate,prednisone19Lupus V24M0.82.161.3ANA+, Smith+, RNP+YesCentral serous retinopathyPrednisone, mycophenolate20Lupus V33F1.2160.5ANA+, SS-A+YesInflammatory cerebritis, positive AchR antibodiesPrednisone, hydroxychloroquineACEI, angiotensin-converting enzyme inhibitor; ANA, antinuclear antibody; ARB, angiotensin receptor blocker; Cr, creatinine; Dx, diagnosis; ENA, extractable nucleic acid (antibody); SLE, systemic lupus erythematosus; RNP, ribonucleoprotein; SS-A/B, Sjogren's syndrome-A/B; UA, urinalysis. Open table in a new tab ACEI, angiotensin-converting enzyme inhibitor; ANA, antinuclear antibody; ARB, angiotensin receptor blocker; Cr, creatinine; Dx, diagnosis; ENA, extractable nucleic acid (antibody); SLE, systemic lupus erythematosus; RNP, ribonucleoprotein; SS-A/B, Sjogren's syndrome-A/B; UA, urinalysis. Serologic studies showed that 13 of 13 patients with serologic data available at the time of biopsy had a positive antinuclear antibody (ANA) test (100%); 4 of 13 had a positive double-stranded DNA antibody test (30%), and 3 of 13 had anti-Smith antibodies (23%). Two patients had SS-A antibodies, and one patient had anti-ribonucleoprotein antibodies. Serum and were in of 12 patients with available data and in of 12 NCAM1-positive patients Three patients had a positive history of SLE. patients had evidence of disease in to nephritis in SLE patients with NCAM1-associated MN included one with receptor serous and in a patient to be to antibody patient had along with A of the data of NCAM1-associated MN patients is provided in Table 1. There were 2 NCAM1-positive patients without a known history of SLE and no or a clinical for lupus nephritis. patient was a with a history of and to of due to Membranous nephropathy is a renal of Renal of Google Scholar The second patient was a with a history of and renal both cases tissue TBM or a pattern of immunofluorescence. was from all cases of NCAM1-associated MN including 2 cases of primary MN and 18 cases of NCAM1-positive cases in cases with and without a concurrent proliferative component including class III or IV) and was not to MLN cases class of 20 cases had lupus nephritis in to MLN class III + and one case had lupus nephritis in to MLN class IV + of cases had a proliferative component by the presence of or In NCAM1-positive cases, all of which showed NCAM1 positivity in a similar as 18 of 20 had and IgG 2 of 20 had an to IgG staining A total of 18 of 20 cases demonstrated staining for other immune in to and light with in 13 of 20 cases in 13 of cases case tissue available on in of 20 cases and in 11 of 20 cases of 20 of the NCAM1-positive cases showed immunofluorescence with staining for all Ig tested (including and as as both and There were no cases of membranous nephropathy, with all cases and light staining with a similar to IgG were performed on 13 cases and did not show a pattern of staining (Supplementary Table S1). However, 2 of 13 cases were or to what with primary membranous nephropathy, for which is or within glomerular capillary staining was present in some NCAM1-associated MN cases, with 11 of 20 cases TBM immune deposits 3 of 20 cases immune deposits and of 20 cases a tissue pattern NCAM1-associated MN can have features (Figure Table features of cases of neural cell adhesion molecule membranous from 0 to 3 no 1, from 0 to 3 no 1, from 0 to 3 no 1, from 0 to 3 no 1, from 0 to 3 no 1, antinuclear and tubular not tubular basement from 0 to 3 no 1, Open table in a new tab ANA, antinuclear and tubular not tubular basement microscopy was available for of the 20 NCAM1-positive cases. deposits were present in glomeruli in 18 of cases and deposits were present in 4 of cases deposits were present in all cases and had of present by the and T. Pathology of membranous Scholar 1 to Serum from NCAM1 patients with recombinant NCAM1 protein in 2 of 2 patients with NCAM1-associated MN on Western (Figure and not (Supplementary Figure There was no reactivity from serum from patients with PLA2R MN of or EXT MN of (Figure Supplementary Figure These serum reactivity studies that circulating anti-NCAM1 antibodies in a subset of lupus of that the be on Additionally, indirect immunofluorescence show against kidney but not within controls within NCAM1-associated MN. PLA2R-positive MN controls (Supplementary Figure This identified target antigen in MLN, is a of the Ig of In NCAM1 is at levels within the (including the and and within the immune M. et of the 2015; PubMed Scopus Google Scholar NCAM1 that have an and NCAM1 is in the kidney during within the and with NCAM1 In NCAM1 is to that are in to the G. et cell adhesion molecule on renal PubMed Scopus Google Scholar but also within M.M. M. Grahammer F. et in of the kidney disease 2018; PubMed Scopus Google Scholar NCAM1 within has been with in including lupus M. et of neural cell adhesion molecule in renal possible in renal 2015; PubMed Scopus Google Scholar However, increased was not in the NCAM1-associated MLN cases that we NCAM1 is also present in urinary M. A. et analysis of from PubMed Scopus Google Scholar of which the is is to determine the of NCAM1-associated MN. NCAM1 within we not a staining pattern within kidney biopsies or membranous cases. NCAM1-associated MN cases were within lupus patients, we the that it could also serve as a primary There are no studies to that NCAM1 in However, increased NCAM1 within urine has been shown in a subset of lupus patients with proliferative lupus nephritis with to disease et detection of urinary adhesion for determination of lupus nephritis 2018; PubMed Scopus Google Scholar When lupus were against renal glomerular NCAM1 was significantly This is to be due to NCAM1 with the Lupus serum glomerular cell adhesion in association with of and adhesion It is possible that in some lupus nephritis patients, NCAM1 could be an autoantigen that that some patients had concurrent with nephritis, and NCAM1 is at levels within the and it is possible that NCAM1 a subset of patients at for both and nephritis. Of NCAM1 has been as a biomarker for within for patients with et of a neural cell adhesion molecule found in as a biomarker for PubMed Scopus Google Scholar and it increased within T. T. et in from in the PubMed Scopus Google Scholar our frequency of was in SLE patients it is unknown whether it is increased within lupus patients with MLN, as at this there are no data in the on the frequency of MLN and Additionally, that our cohort is it be to to determine clinical of lupus and NCAM1-associated MN. Membranous nephropathy can be the first of SLE in a subset of lupus patients, and other clinical and serologic not be but can at a In the patients with membranous nephropathy are NCAM1 these patients to determine lupus be to determine NCAM1 positivity or anti-NCAM1 antibodies could serve as a of SLE. There are of this The study is of a cohort of NCAM1-associated MN patients, with a 20 we are to determine there are features that could NCAM1-associated MN cases from other cases of MLN, as the and on the kidney biopsies in this cohort had a could the when to stain membranous biopsies with S. Madden B.J. Debiec H. et al.Exostosin 1/exostosin 2-associated membranous nephropathy.J Am Soc Nephrol. 2019; 30: 1123-1136Crossref PubMed Scopus (81) Google Scholar It is possible that anti-NCAM1 antibodies could be used as biomarkers to the of remission and monitor response to but we have data at this time for that anti-NCAM1 antibodies in serum correlate with proteinuria is as we had 2 serum samples from NCAM1-positive patients available for without this it is also unknown NCAM1-associated MN cases have differential response to used in the of MN, as cyclophosphamide, or NCAM1-associated membranous nephropathy is in patients with systemic lupus has a female and in patients with PLA2R- or thrombospondin type 1 containing membranous nephropathy. NCAM1-associated MN up of all MLN cases, which have There also be an association with of SLE, but is for

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