Bhave, Gautam

Borza, Dorin-Bogdan

Colon, Selene

Jain, Sanjay

Manral, Pallavi

Zhao, Ming-Hui

English

Digital Commons@Becker

Washington University School of Medicine Digital Commons@Becker Open Access Publications 7-1-2019 Peroxidasin is a novel target of autoantibodies in lupus nephritis Pallavi Manral Selene Colon Gautam Bhave Ming-Hui Zhao Sanjay Jain See next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Authors Pallavi Manral, Selene Colon, Gautam Bhave, Ming-Hui Zhao, Sanjay Jain, and Dorin-Bogdan Borza Peroxidasin Is a Novel Targetof Autoantibodies in Lupus NephritisPallavi Manral1, Selene Colon2, Gautam Bhave2, Ming-Hui Zhao3, Sanjay Jain4 andDorin-Bogdan Borza11Department of Microbiology, Immunology and Physiology, Meharry Medical College, Nashville, Tennessee, USA; 2Division ofNephrology, Vanderbilt University Medical Center, Nashville, Tennessee, USA; 3Renal Division, Peking University First Hos-pital, Beijing, China; and 4Department of Medicine, Division of Nephrology, Washington University School of Medicine, St.Louis, Missouri, USACorrespondence: Dorin-Bogdan Borza, Department of Microbiology, Immunology and Physiology, Meharry Medical College,1005 Dr. D. B. Todd, Jr. Boulevard, Nashville, Tennessee 37208, USA. E-mail: dborza@mmc.eduReceived 20 February 2019; revised 5 April 2019; accepted 8 April 2019; published online 15 April 2019Kidney Int Rep (2019) 4, 1004–1006; https://doi.org/10.1016/j.ekir.2019.04.009ª 2019 International Society of Nephrology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).P eroxidasin is an animal heme peroxidase withbroad tissue distribution, secreted and embeddedin basement membranes and implicated in the homeo-stasis of type IV collagen.1 Peroxidasin uses bromideion (Br) and hydrogen peroxide (H2O2) to generatehypobromous acid (HOBr), which is essential for for-mation of sulfilimine (S¼N) bonds that covalentlycrosslink type IV collagen molecules at the NC1-NC1interface.2 Sulfilimine bonds crosslinking of a345(IV)collagen in the glomerular basement membrane (GBM)confer protection against Goodpasture (GP) disease, anautoimmune kidney disease mediated by autoanti-bodies binding to sites within the NC1 hexamers ofa345(IV) collagen.3 A recent study reports thatapproximately 46% of patients with GP disease haveautoantibodies to peroxidasin, which inhibit HOBrgeneration activity.4 Anti-peroxidasin antibodies,additionally identified in approximately 13% of pa-tients with anti-myeloperoxidase (anti-MPO) ANCA-associated vasculitis (AAV), have been proposed to bea serologic marker of disease in the pulmonary-renalsyndrome spectrum.4 Besides GP disease and AAV,systemic lupus erythematosus (SLE) is another cause ofpulmonary-renal syndrome. We tested the hypothesisthat anti-peroxidasin antibodies are present in lupusnephritis (LN).RESULTSSera from 32 patients with biopsy-confirmed prolif-erative or membranous LN (ISN/RPS class III, IV, or V)were analyzed for peroxidasin immunoreactivity byenzyme-linked immunosorbent assay (ELISA) (seeSupplementary Material). Anti-peroxidasin IgGantibodies were identified in 2 of 25 (8%) patientswith proliferative LN (one each class III and class IV),and also in 3 of 29 (10%) patients with GP disease(Figure 1). The prevalence of anti-peroxidasin anti-bodies in GP disease in our study is lower than thatpreviously reported (46%),4 which may be due todemographic differences in the patient population.Alternatively, our immunoassay may be less sensitiveowing to technical differences between the assays, inwhich case the true prevalence of anti-peroxidasin an-tibodies in LN may be underestimated. Anti-a3NC1 oranti-MPO IgG autoantibodies, which invariably accom-panied previously identified anti-peroxidasin antibodies,are sometimes found in patients with LN,5,6 promptingus to investigate whether these autoantibodies alsooccurred together with anti-peroxidasin antibodies inLN. However, in our LN cohort, none of the seracontained antibodies binding to purified MPO(Suppolementary Figure S1). Anti-a3NC1 antibodieswere also absent in LN, except for q case that did notcontain anti-peroxidasin antibodies (SupplementaryFigure S2). These findings imply that there are anti-peroxidasin antibodies in a subpopulation of patientswith proliferative LN, in the absence of serologicmarkers of anti-MPO AAV or GP disease.We further characterized anti-peroxidasin anti-bodies from LN compared with GP disease. Theirability to bind native peroxidasin was verified bycompetition ELISA using soluble antigen. Soluble per-oxidasin inhibited antibody binding to immobilizedperoxidasin in a dose-dependent manner, with similarhalf-maximal inhibitory concentration for LN and GPsera (Figure 2a). To determine whether the autoanti-bodies recognize conformational epitopes, we compared1004 Kidney International Reports (2019) 4, 1004–1018RESEARCH LETTERSIgG binding with native versus misfolded peroxidasin.Decreased binding to misfolded peroxidasin, indicativeof conformational epitopes, was observed for anti-peroxidasin IgG from LN but not GP sera (Figure 2b).DISCUSSIONOur results identify peroxidasin as a novel target ofautoantibodies in LN. In contrast to most autoantigensimplicated in SLE, peroxidasin is an intrinsic compo-nent of basement membranes that provides a target fordirect autoantibody binding. Immunofluorescencestaining of the renal biopsy from the patient with LNwith higher titer of anti-peroxidasin showed strongIgG staining (3þ) accompanied by IgA, C1q, C3, kappa,and lambda (2–3þ each) in glomeruli, tubules, inter-stitium, and vessels, with IgM (2þ) additionally foundin glomeruli and tubules. Antibodies binding to per-oxidasin in the GBM may accelerate or exacerbateglomerular immune complex formation, potentiallyincreasing disease activity or severity. Interestingly,the patient with LN with higher titer of anti-peroxidasin antibodies showed declining renal func-tion (estimated glomerular filtration rate decreased from96 ml/min per 1.73 m2 at serum collection to 52 ml/minper 1.73 m2 at follow-up), but a larger sample size willbe required to analyze clinical associations.Altered crosslinking of type IV collagen has beenproposed as a major pathogenic mechanism for anti-peroxidasin autoantibodies from GP sera, based ontheir ability to inhibit in vitro HOBr generation byperoxidasin by 70%.4 Reduced crosslinking of a345(IV)collagen in the GBM is essential for the pathogenesis ofGP disease because: (i) GP autoantibodies bind only to asmall subset of a345(IV)NC1 hexamers that are not sta-bilized by crosslinks,7 and (ii) sulfilimine bonds lock theepitopes preventing the in vivo binding of GP autoanti-bodies to the GBM.8 Consistent with a role in promotingGP disease, anti-peroxidasin immunoreactivity has beendetected before the onset of clinical disease in approxi-mately half of the prediagnostic cases of GP disease.4In analogous manner, continued presence of anti-Figure 1. Anti-peroxidasin (anti-Pxdn) IgG autoantibodies are pre-sent in sera of lupus nephritis. Immunoreactivity toward peroxidasinwas evaluated by enzyme-linked immunosorbent assay in sera fromcontrols (n ¼ 13) and patients with proliferative lupus nephritis(class III or class IV), membranous lupus nephritis (class V), andGoodpasture disease (GP). Sera diluted 1:100 were incubated inwells coated with 200 ng purified recombinant peroxidasin, then IgGbinding was measured. The positivity threshold (indicated by a grayline) was set at 3 SD above the mean of controls (dotted line). OD,optical density.Figure 2. Characterization of anti-peroxidasin (anti-Pxdn) antibodies.(a) The specificity of antibodies for native peroxidasin was verifiedby competition enzyme-linked immunosorbent assay using solubleantigen. Lupus nephritis (LN) or Goodpasture disease (GP) seradiluted 1:1000 were preincubated for 1 hour at room temperaturewith the shown concentrations of native peroxidasin (Pxdn), thenadded to wells coated with 50 ng of recombinant peroxidasin. Foreach serum, IgG binding was calculated relative to binding in theabsence of an inhibitor, which was considered 100% (dotted refer-ence line). Means and SD from 2 independent experiments areshown. (b) Anti-Pxdn antibodies from LN sera recognizeconformation-dependent epitopes. A 1:100 dilution of sera containinganti-Pxdn antibodies was applied to wells coated with 200 ng ofperoxidasin in phosphate-buffered saline (native Pxdn) or in 3Mguanidinium chloride containing 20 mM tris(2-carboxyethyl)phos-phine as a reducing agent (misfolded Pxdn). The mean  SD of therelative decrease in IgG binding to misfolded Pxdn was 34%  5%for LN sera compared with 3%  5% for GP sera (P ¼ 0.012, 2-tailedunpaired t test). OD, optical density.RESEARCH LETTERSKidney International Reports (2019) 4, 1004–1018 1005peroxidasin antibodies over a period of months(commensurate with the rate of collagen IV turnover)may impair the crosslinking of a345(IV) collagen in LN.An association between LN and GP disease has beenreported in 1 retrospective study, in which 9% (14/157)of patients with SLE had detectable levels of anti-GBM(anti-a3NC1) autoantibodies along with antinuclear an-tibodies, and some of these patients presented withrapid progressive glomerulonephritis or were clinicallydiagnosed with GP disease.6 It is plausible that anti-peroxidasin antibodies may be among the factorstriggering GP disease in patients with LN, a rare pre-sentation with worse outcomes.To decrease sulfilimine bond formation, given thatalready formed bonds are not affected, anti-peroxidasinantibodies must be present when nascent collagen IVmolecules assemble within basement membranes. Theirimpact would be maximal during periods of rapidturnover of basement membranes, such as during fetaldevelopment or the formation of feto-maternal inter-face. Therefore, the potentially harmful role of anti-peroxidasin autoantibodies in lupus pregnancieswarrants further investigation. As precedent, autoan-tibodies to phospholipids or laminin-111 (a componentof basement membranes in placenta and developingtissues) may occur in lupus and are associated withadverse pregnancy outcomes, whereas maternal anti-Ro/SS-A and anti-La/SS-B autoantibodies have beenimplicated in neonatal lupus.9Among limitations, this was an exploratory study ina relatively small number of patients. Our findingsshould be validated in a larger study, also investigatingclinical associations. Due to the small amounts of seraavailable, we could not isolate sufficient IgG to testwhether anti-peroxidasin antibodies inhibit HOBrgeneration. Definitive proof that anti-peroxidasin an-tibodies are pathogenic will require future studies inanimal models yet to be developed.DISCLOSUREAll the authors declared no competing interests.ACKNOWLEDGMENTSWe thank Mary Hoffmann and Diane Salamon for spec-imen and data procurement. This work was supported by apilot project (to Dorin-Bogdan Borza) from the MeharryTranslational Research Center grant U54 MD0007593 fromthe National Institute on Minority Heath and Health Dis-parities. The Kidney Translational Research Center atWashington University School of Medicine was partlysupported by the George O’Brien Center for Kidney Dis-ease Research (grant P30 DK079333) and by the Division ofNephrology at Washington University School of Medicine.Additional support was from the Paul Teschan ResearchFund grant from Dialysis Clinic Inc, R01 DK-116964 fromNational Institute of Diabetes and Digestive and KidneyDiseases, and the Burroughs Wellcome Fund CareerAward for Medical Scientists (13030995).SUPPLEMENTARY MATERIALFigure S1. Absence of anti-MPO antibodies in sera of pa-tients with LN.Figure S2. Analysis of anti-a3NC1 antibodies in sera ofpatients with LN.Supplementary Methods.Supplementary material is linked to the online version ofthe paper at www.kireports.org.REFERENCES1. Bhave G, Cummings CF, Vanacore RM, et al. Peroxidasinforms sulfilimine chemical bonds using hypohalous acids intissue genesis. Nat Chem Biol. 2012;8:784–790.2. McCall AS, Cummings CF, Bhave G, et al. Bromine is anessential trace element for assembly of collagen IV scaffolds intissue development and architecture. Cell. 2014;157:1380–1392.3. Pedchenko V, Bondar O, Fogo AB, et al. Molecular architectureof the Goodpasture autoantigen in anti-GBM nephritis. N EnglJ Med. 2010;363:343–354.4. McCall AS, Bhave G, Pedchenko V, et al. Inhibitory anti-peroxidasin antibodies in pulmonary-renal syndromes. J AmSoc Nephrol. 2018;29:2619–2625.5. Turner-Stokes T, Wilson HR, Morreale M, et al. Positive anti-neutrophil cytoplasmic antibody serology in patients withlupus nephritis is associated with distinct histopathologicfeatures on renal biopsy. Kidney Int. 2017;92:1223–1231.6. Li CH, Li YC, Xu PS, et al. Clinical significance of anti-glomerular basement membrane antibodies in a cohort ofChinese patients with lupus nephritis. Scand J Rheumatol.2006;35:201–208.7. Borza DB, Bondar O, Colon S, et al. Goodpasture autoanti-bodies unmask cryptic epitopes by selectively dissociatingautoantigen complexes lacking structural reinforcement: novelmechanisms for immune privilege and autoimmune patho-genesis. J Biol Chem. 2005;280:27147–27154.8. Luo W, Wang XP, Kashtan CE, et al. Alport alloantibodies butnot Goodpasture autoantibodies induce murine glomerulone-phritis: protection by quinary crosslinks locking crypticalpha3(IV) collagen autoepitopes in vivo. J Immunol. 2010;185:3520–3528.9. Shoenfeld Y, Blank M. Autoantibodies associated with repro-ductive failure. Lupus. 2004;13:643–648.RESEARCH LETTERS1006 Kidney International Reports (2019) 4, 1004–1018

Peroxidasin is a novel target of autoantibodies in lupus nephritis

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Peroxidasin is a novel target of autoantibodies in lupus nephritis

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