Neural cell adhesion molecule 1 is a novel autoantigen in membranous lupus nephritis
Authors: Tiffany N. Caza, Samar I. Hassen, Michael Kuperman, Shree G. Sharma, Zeljko Dvanajscak, John Arthur, Rick Edmondson, Aaron Storey, Christian Herzog, Daniel J. Kenan, Christopher P. Larsen
Journal: Kidney International (2020)

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 complements (C3 and C4 levels) were normal in 6 of 12 patients with available data (50%) and low in 6 of 12 NCAM1-positive patients (50%). Three patients had a positive family history of SLE. Eight patients had evidence of neuropsychiatric disease in addition to nephritis (40%). Neuropsychiatric manifestations in SLE patients with NCAM1-associated MN included seizures (n = 3), cerebritis (n = 3, one with acetylcholine receptor antibodies), serous central retinopathy (n = 1), psychosis (n = 1), and stroke (n = 1) in a young patient suspected to be related to antiphospholipid antibody syndrome. One patient had seizures along with cerebritis. A summary of the clinicopathologic data of NCAM1-associated MN patients is provided in Table 1. There were 2 NCAM1-positive patients without a known history of SLE and showing no autoimmune manifestations or a clinical suspicion for lupus nephritis. One patient was a 54-year-old Caucasian man with a medical history of hypertension, chronic obstructive pulmonary disease, obesity, asthma, obstructive sleep apnea, and bronchiectasis related to repeated episodes of pneumonia due to common variable immunodeficiency. Membranous nephropathy is a common renal manifestation of common variable immunodeficiency.11Caza T.N. Hassen S.I. Larsen C.P. Renal manifestations of common variable immunodeficiency.Kidney360. 2020; 1: 491-500Crossref Google Scholar The second patient was a 40-year-old man with a history of hypertension, obesity, and anxiety. On renal biopsy, both cases lacked tissue ANA staining, TBM staining, or a "full-house" pattern of immunofluorescence. Histopathology was reviewed from all cases of NCAM1-associated MN (n = 20; including 2 cases of primary MN and 18 cases of MLN). NCAM1-positive cases occurred in cases with and without a concurrent proliferative component (n = 5, including ISN/RPS class III or IV) and was not restricted to pure MLN cases (ISN/RPS class V). Four of 20 cases had focal lupus nephritis in addition to MLN (ISN/RPS class III + V), and one case had diffuse lupus nephritis in addition to MLN (ISN/RPS class IV + V). Therefore, 25% of cases had a proliferative component overall, manifested by the presence of endocapillary hypercellularity or crescents. In NCAM1-positive cases, all of which showed NCAM1 positivity in a similar distribution as IgG, 18 of 20 (90%) had diffuse and global IgG staining, whereas 2 of 20 (10%) had an incomplete global to segmental IgG staining pattern. A total of 18 of 20 cases (90%) demonstrated staining for other immune reactants in addition to IgG, kappa, and lambda light chains, with IgA in 13 of 20 cases (65%), IgM in 13 of 19 cases (one case lacked tissue available on sections, 68%), C3 in 17 of 20 cases (85%), and C1q in 11 of 20 cases (55%). Eight of 20 (40%) of the NCAM1-positive cases showed "full house" immunofluorescence with staining for all Ig heavy chains tested (including IgA, IgG, and IgM), as well as both complement components (C1q and C3). There were no cases of monoclonal membranous nephropathy, with all cases showing approximately equal kappa and lambda light chain staining with a distribution similar to IgG. IgG subclasses were performed on 13 cases and did not show a consistent pattern of staining (Supplementary Table S1). However, only 2 of 13 cases were IgG4 dominant or codominant, dissimilar to what occurs with primary membranous nephropathy, for which IgG4 is restricted or codominant within glomerular capillary loop deposits. Extraglomerular staining was present in some NCAM1-associated MN cases, with 11 of 20 cases showing TBM immune deposits (55%), 3 of 20 cases showing vascular immune deposits (15%), and 5 of 20 cases having a tissue ANA pattern (25%). Therefore, NCAM1-associated MN can have varying histopathologic features (Figure 3; Table 2).Table 2Histopathologic features of cases of neural cell adhesion molecule 1-associated membranous nephropathyCase% Global GSIF/TAProliferative componentIgAaGraded from 0 to 3 (0, no staining; 1, mild; 2, moderate; 3, strong).IgGaGraded from 0 to 3 (0, no staining; 1, mild; 2, moderate; 3, strong).IgMaGraded from 0 to 3 (0, no staining; 1, mild; 2, moderate; 3, strong).C3aGraded from 0 to 3 (0, no staining; 1, mild; 2, moderate; 3, strong).C1qaGraded from 0 to 3 (0, no staining; 1, mild; 2, moderate; 3, strong).Full houseMesangial depositsSubendo depositsTBM depositsTissue ANAVascular depositsEM stage10AbsentNo23020.5NoYesNoAbsentAbsentAbsent220MildYes13113YesYesYesPresentAbsentAbsent332.5AbsentNo03010NoNoNoAbsentAbsentAbsent2435SevereNo03100NoYesNoAbsentAbsentAbsent351AbsentNo0.53121YesYesNoPresentPresentAbsent2616MildNo0.5322YesYesNoPresentAbsentAbsent1733ModerateNo0300.50NoYesNoAbsentAbsentAbsent382MildNo23030NoYesNoAbsentAbsentAbsent393AbsentNo03020NoYesNoAbsentPresentAbsent21018ModerateNo1210.50NoYesYesAbsentAbsentAbsent31118ModerateYes0320.50NoYesNoPresentAbsentPresent3123ModerateYes1.5312.52.5YesYesNoPresentPresentAbsent2137MildNo030.50.50.5NoYesNoAbsentAbsentPresent11442ModerateYes13000NoYesN/AAbsentPresentAbsentN/A1511SevereYes0220.51NoYesYesPresentAbsentAbsent21623SevereNo23223YesYesYesPresentAbsentPresent3170AbsentNo2221.51.5YesYesNoPresentPresentAbsent31833ModerateNo110.500NoYesNoPresentAbsentAbsent3190AbsentNo23220.5YesYesNoPresentAbsentAbsent3200ModerateNo32.5320.5YesYesNoPresentAbsentAbsent3ANA, antinuclear antibodies; C1q/C3, complement proteins; EM, electron microscopy; GS, glomerulosclerosis; IF/TA, interstitial fibrosis and tubular atrophy; N/A, not available; TBM, tubular basement membrane.a Graded from 0 to 3 (0, no staining; 1, mild; 2, moderate; 3, strong). Open table in a new tab ANA, antinuclear antibodies; C1q/C3, complement proteins; EM, electron microscopy; GS, glomerulosclerosis; IF/TA, interstitial fibrosis and tubular atrophy; N/A, not available; TBM, tubular basement membrane. Electron microscopy was available for 19 of the 20 NCAM1-positive cases. Mesangial electron-dense deposits were present in glomeruli in 18 of 19 cases (95%), and subendothelial electron-dense deposits were present in 4 of 19 cases (21%). Subepithelial electron-dense deposits were present in all cases and had variable stages of maturation present by the Ehrenreich and Churg classification12Ehrenreich T. Churg J. Pathology of membranous nephropathy.Pathol Annu. 1968; 2: 145-186Google Scholar (stage 1 to stage 3). Serum from NCAM1 patients reacted with recombinant NCAM1 120-kDa protein in 2 of 2 patients with NCAM1-associated MN on non-reducing Western blots (Figure 4) and not under reducing conditions (Supplementary Figure S6). There was no reactivity from serum from patients with PLA2R MN (0 of 14) or EXT MN (0 of 4) (Figure 4; Supplementary Figure S7). These serum reactivity studies confirmed that circulating anti-NCAM1 antibodies exist in a subset of lupus patients. Loss of seroreactivity under reducing conditions suggests that the epitope may be dependent on internal disulfide bonds. Additionally, indirect immunofluorescence assays show seroreactivity against NCAM1-expressing human embryonic kidney (HEK)-293 cells, but not within vector controls within NCAM1-associated MN. PLA2R-positive MN controls lacked seroreactivity (Supplementary Figure S8). This newly identified target antigen in MLN, NCAM1, is a member of the Ig superfamily of proteins. In adults, NCAM1 expression is seen at high levels within the central nervous system (including the cerebral cortex, cerebellum, and hippocampus), peripheral nerves, thyroid gland, adrenal gland, heart, stomach (within gastric chief cells), and cells within the immune system.13Uhlen M. Fagerberg L. Hallstrom B.M. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (5470) Google Scholar Immune cells expressing NCAM1 include natural killer (NK) cells, γΔ T cells, CD8+ T cells that have an activated phenotype, and dendritic cells. NCAM1 is expressed in the kidney during development within the metanephrogenic mesenchyme, and with maturity, NCAM1 expression decreases. In adults, NCAM1 expression is mostly restricted to interstitial cells that are in proximity to the corticomedullary junction,14Markovic-Lipkovski J. Muller C.A. Klein G. et al.Neural cell adhesion molecule expression on renal interstitial cells.Nephrol Dial Transplant. 2007; 22: 1558-1566Crossref PubMed Scopus (20) Google Scholar but also occurs within podocytes.15Rinschen M.M. Gödel M. Grahammer F. et al.A multi-layered quantitative in vivo expression atlas of the podocyte unravels kidney disease candidate genes.Cell Rep. 2018; 23: 2495-2508Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Increased NCAM1 expression within interstitial cells has been associated with interstitial fibrosis in glomerulonephritis, including lupus nephritis.16Markovic-Lipkovski J. Zivotic M. Muller C.A. et al.Variable expression of neural cell adhesion molecule isoforms in renal tissue: possible role in incipient renal fibrosis.PloS One. 2015; 10e0137028Crossref PubMed Scopus (10) Google Scholar However, increased interstitial fibrosis was not observed in the NCAM1-associated MLN cases that we evaluated. NCAM1 is also present in urinary exosomes,17Prunotto M. Farina A. Lane L. et al.Proteomic analysis of podocyte exosome-enriched fraction from normal human urine.J Proteomics. 2013; 82: 193-229Crossref PubMed Scopus (98) Google Scholar of which the significance is unclear. Further work is required to determine the pathophysiology of NCAM1-associated MN. Despite NCAM1 expression occurring within podocytes, we do not observe a podocyte staining pattern within normal kidney biopsies or non-NCAM1 membranous cases. Although NCAM1-associated MN cases were primarily within lupus patients, we cannot exclude the possibility that it could also serve as a primary podocyte antigen. There are no studies to date that explore NCAM1 in MLN. However, increased NCAM1 within urine has been shown in a subset of lupus patients with proliferative lupus nephritis with correlation to disease activity.18Wang Y. Tao Y. Liu Y. et al.Rapid detection of urinary soluble intercellular adhesion molecule-1 for determination of lupus nephritis activity.Medicine. 2018; 97: e11287Crossref PubMed Scopus (11) Google Scholar When lupus sera were reacted against human renal glomerular endothelial cells, NCAM1 expression was significantly reduced. This effect is thought to be due to soluble NCAM1 competing with the membrane-bound form (Russell D, Markiewicz M, Oates JC. Lupus serum induces glomerular endothelial cell neutrophil adhesion in association with soluble mediators of chemotaxis and adhesion [abstract]. Arthritis Rheumat. 2017;69. Abstract 2642). It is possible that in some lupus nephritis patients, membrane-bound NCAM1 could be an autoantigen that drives disease. Given that some patients had neuropsychiatric manifestations concurrent with nephritis, and NCAM1 is expressed at high levels within the central and peripheral nervous system, it is possible that NCAM1 autoreactivity may indicate a subset of patients at risk for both neuropsychiatric manifestations and nephritis. Of note, NCAM1 has been implicated as a biomarker for epilepsy within cerebrospinal fluid for patients with seizures,19Wang W. Wang L. Luo J. et al.Role of a neural cell adhesion molecule found in cerebrospinal fluid as a potential biomarker for epilepsy.Neurochem Res. 2012; 37: 819-825Crossref PubMed Scopus (19) Google Scholar and it shows increased expression within reactive astrocytes.20Nomura T. Yabe T. Rosenthal E.S. et al.PSA-NCAM distinguishes reactive astrocytes in 6-OHDA-lesioned substantia nigra from those in the striatal terminal fields.J Neurosci Res. 2000; 61: 588-596Crossref PubMed Scopus (45) Google Scholar Although our frequency of neuropsychiatric manifestations was higher than reported in SLE patients overall, it is unknown whether it is increased within lupus patients with MLN, as at this time, there are no data in the literature on the frequency of MLN and neuropsychiatric disease. Additionally, given that our cohort is relatively small, it may be premature to try to determine clinical associations of lupus manifestations and NCAM1-associated MN. Membranous nephropathy can be the first presentation of SLE in a subset of lupus patients, and other clinical and serologic sequelae may not be seen but can arise at a later time. In the patients with "idiopathic" membranous nephropathy who are NCAM1 positive, following these patients to determine if they later develop lupus would be important to determine if NCAM1 positivity or anti-NCAM1 antibodies could serve as a harbinger of SLE. There are several limitations of this study. The study is of a small cohort of NCAM1-associated MN patients, with a relatively small sample size (n = 20 patients). Currently, we are unable to determine if there are histopathologic features that could distinguish NCAM1-associated MN cases from other cases of MLN, as the histopathologic and immunologic patterns on the kidney biopsies in this small cohort had variable appearances. Determining a distinguishing factor could help the nephropathologist decide when to stain membranous biopsies with NCAM1.5Sethi 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 It is possible that anti-NCAM1 antibodies could be used as biomarkers to predict the likelihood of remission and monitor response to therapy, but we have insufficient data at this time for that conclusion. Whether anti-NCAM1 antibodies in serum correlate with proteinuria is currently unknown, as we had only 2 serum samples from NCAM1-positive patients available for evaluation, without serial sampling. At this time, it is also unknown if NCAM1-associated MN cases have differential response to common therapies used in the treatment of MN, such as rituximab, cyclophosphamide, or calcineurin inhibitors. NCAM1-associated membranous nephropathy is enriched in patients with systemic lupus erythematosus, has a female predominance, and presents in younger patients than those seen with PLA2R- or thrombospondin type 1 containing 7A–associated membranous nephropathy. NCAM1-associated MN makes up 7.1% of all MLN cases, which have variable histopathologic patterns. There may also be an association with neuropsychiatric manifestations of SLE, but more work is required for confirmation.