Article Mapping Novel Immunogenic Epitopes in Iga Nephropathy

CJASN ePress. Published on December 26, 2014 as doi: 10.2215/CJN.02390314 Article

Mapping Novel Immunogenic Epitopes in IgA Nephropathy

Sang Hoon Woo,* Tara K. Sigdel,† Van T. Dinh,† Minh-Thien Vu,† Minnie M. Sarwal,† and Richard A. Lafayette‡

Abstract Background and objectives IgA plays a key role in IgA nephropathy (IgAN) by forming immune complexes and depositing in the glomeruli, leading to an inflammatory response. However, the antigenic targets and functional characterization of IgA have been incompletely defined in this disease. *Division of Hospital Medicine, Department of Design, setting, participants, & measurements This study was performed in sera from patients who were studied Medicine, Thomas as part of a prospective, observational study of IgAN. These patients (n=22) all had biopsy-proven IgAN Jefferson University, within 3 years of study initiation, complete clinical data, annual urinary inulin clearance for GFRs, and at least Philadelphia, Pennsylvania; fi . 2 5 years of follow-up. Progression was de ned as loss of 5 ml/min per 1.73 m per year of inulin clearance †Department of measured over at least 5 years. A protein microarray was used for detection of IgAN-specific IgA autoantibodies Surgery, University of in blood across approximately 9000 human antigens to specifically identify the most immunogenic protein California, San n n Francisco, California; targets that drive IgA antibodies in IgAN ( =22), healthy controls ( =10), and non-IgAN glomerular diseases ‡ (n=17). Results were validated by ELISA assays in sera and by immunohistochemistry in IgAN kidney biopsies. Division of fi Nephrology, IgA-speci c antibodies were correlated with clinical and histologic variables to assess their effect on disease Department of progression and prognosis. Medicine, Stanford University School of Results Fifty-four proteins mounted highly significant IgA antibody responses in patients with IgAN with a false Medicine, Stanford q # P# University, Stanford, discovery rate ( value) of 10%; 325 antibodies ( 0.05) were increased overall. Antitissue transglutaminase California IgA was significantly elevated in IgAN (P,0.001, q value of 0%). IgA antibodies to DDX4 (r=20.55, P=0.01) r 2 P fi fi and ZADH2 ( = 0.48, =0.02) were signi cantly correlated with the decline of renal function. Speci c IgA auto- Correspondence: antibodies are elevated in IgAN compared with normal participants and those with other glomerular diseases. Dr. Richard Lafayette, Department of Conclusions In this preliminary study, IgA autoantibodies target novel proteins, highly expressed in the kidney Medicine, Stanford University, A175, 300 glomerulus and tubules. These IgA autoantibodies may play important roles in the pathogenesis of IgAN. Pasteur Drive, Clin J Am Soc Nephrol 10: ccc–ccc, 2014. doi: 10.2215/CJN.02390314 Stanford, CA 94305; or Dr. Minnie Sarwal, Department of Surgery, University of Introduction which patients to observe more closely, treat more California San IgA nephropathy (IgAN) is the most common GN in aggressively, or include/stratify in treatment trials. Francisco, G893, 513 Parnassus Avenue, San the world and remains a common cause of ESRD (1,2). Our group and others previously demonstrated that fi Francisco, CA 94107. IgAN is diagnosed by kidney biopsy and de ned by autoantibodies may be important in the pathogenesis Email: czar@stanford. the deposition of IgA in the glomerulus with prolifera- of IgAN (12,13). However, the specificities of IgA anti- edu or minnie. tion of the mesangium (3). The pathogenesis of IgAN is bodies in IgAN have still not been broadly defined. sarwal@ucsfmedctr. still poorly understood. There is likely underlying ge- These specificities could help to better understand the org netic predisposition as demonstrated by family, racial, pathogenesis of this disease and may provide more re- and sex predisposition as well as several candidate ge- fined biomarkers to prognosticate and guide therapy. netic risk factors (4). Patients with IgAN generally dem- High-density protein microarrays have been used to onstrate increased levels of galactose-deficient IgA1 (5). screen serum antibodies (14) to .8000 individual hu- It has been demonstrated that immune complexes man protein targets and have yielded findings of clin- formed from IgA or IgG autoantibodies that recog- ical interest (15). To characterize IgA and its antigenic nize galactose-deficient IgA may precipitate in the targets, we used protein arrays to detect and describe glomerulus and cause injury (6–9). The prognosis of IgA autoantibodies in patients with IgAN. We applied IgAN is also incompletely determined by typically an integrative genomics approach to cross-map the IgA measured clinical and pathologic features. Patients antibodies that are highly expressed in patients with with greater degrees of proteinuria, renal dysfunction, or IgAN with protein targets to assess their specificity to pathologic changes (proliferative, necrotic, or sclerotic) the kidney as well as other organs. Potential candidates have higher risks of progressive disease (10,11). can then be validated in biopsy tissue by immunohis- However, greater precision in risk assessment afford- tochemistry and in serum samples by ELISA. The over- ed by further biomarkers could aid in determining all study approach is summarized in Figure 1A. www.cjasn.org Vol 10 March, 2014 Copyright © 2014 by the American Society of Nephrology 1 2 Clinical Journal of the American Society of Nephrology

Materials and Methods Within the cohort of IgAN participants, the rate of decline Patients of measured GFR over the 5 years of follow-up was used to Entry criteria for this study was biopsy-confirmed IgAN, subclassify them into two groups. Patients with IgAN were and each enrolled patient with IgAN had to have at least classified as progressors (n=7) if the rate of measured GFR 5 years of follow-up with complete clinical data, annual sera decline (DGFR) was .5.0 ml/min per 1.73m2 per year. Pa- and urine sampling, and annually measured GFRs for tients with a DGFR of ,5.0 ml/min per 1.73 m2 per year absolute quantification of renal function. Given these strin- were classified as nonprogressors. Blood (5 ml serum) and gent selection criteria, we could enroll 22 patients with IgAN urine (50 ml) samples were collected annually over the that met all entry criteria. 5-year follow-up from each patient with IgAN. GFR was A total of 49 participants were enrolled for this study: the 22 measured using the urinary clearance of inulin, as previ- patients with biopsy-confirmed IgAN who met all above study ously described (16). The demographics of these patients entry criteria, 17 patients with biopsy-proven non-IgA causes of are shown in Tables 1 and 2. The characteristics of seven renal disease, and 10 age- and sex-matched healthy controls. patients with membranous nephropathy (MGN) include

Figure 1. | Work summary and PCA of top 20 IgA autoantibodies. (A) Work ﬂow of IgA autoantibody discovery in IgAN. (B) PCA of top 20 IgA autoantibodies with a cut-off threshold .1.65-fold increase in IgAN with P value ,0.05 shows the comparison between IgAN and control participants. The axes represent principle components (PC1, PC2, and PC3). autoAb, autoantibody; Ctrl, control; IgAN, IgA nephropathy; MGN, membranous nephropathy; PCA, principal component analysis; PC, principle component. Clin J Am Soc Nephrol 10: ccc–ccc, March, 2014 Biomarkers for IgA Nephropathy, Woo et al. 3

male sex (n=4), female sex (n=3), mean age (50.7 years), race The analysis was also done with SAS software (version (white, 43%; Hispanic, 43%; and Asian, 14%), serum creati- 9.2, enterprise guide 4.2). The pathway analysis was nine (0.88 mg/dl), proteinuria (7.6 g/d). The characteristics performed using Ingenuity Pathway Analysis (http://www. of 10 patients with FSGS include male sex (n=7), female sex ingenuity.com). (n=3), mean age (50.7 years), race (white, 50%; Hispanic, 40%; and black, 10%), serum creatinine (2.0 mg/dl), and Validation of IgA Antibodies Based on ELISA proteinuria (4.8 g/d). None of our participants were treated Validation of IgAN-specific IgA autoantibodies identi- with immunosuppression (steroids, cytotoxics) at the time of fied by protein arrays was performed using the MSD ELISA their blood draws. platform (Meso Scale Discovery, Gaithersburg, MD). Receiver operating characteristic (ROC) analysis was conducted on a Immune Response Measurement Using Protein Microarrays combined panel of antibodies to predict IgAN with fitted ProtoArray Human Protein Microarray v5.0 (Invitrogen, logistic regression models using log-transformed ELISA Carlsbad, CA) was used to characterize the specificity of levels of antibodies against SIX homeobox 2 (SIX2), mem- IgA-specific autoantibody response in IgAN. The relative brane protein, palmitoylated 1 (MPP1), TEA domain family fluorescence intensity (in relative fluorescence units [RFU]) member 4 (TEAD4), transglutaminase 2 (T-TG), zinc binding was measured using GenePix pro 6.0 software (Molecular alcohol dehydrogenase domain containing 2 (ZADH2), glu- Devices, Sunnyvale, CA). Data normalization was done using tamate receptor, ionotropic, N-methyl-D-aspartate-like 1A ProtoArray Prospector 5.2. We used the quantile normaliza- (GRINL1A), and ariadne homolog 2 (ARIH2). For ROC tion method because there were no positive controls for IgA to analyses, we generated all corresponding sensitivities and apply Robust Linear Model for normalization (17). Fluores- specificities for each of cut-off point (theta). cence signal values were measured for each protein after ad- Z justing for background correction so that factors for all of Immunohistochemistry the corrected intensities of the human protein features could To evaluate the presence of DEAD (Asp-Glu-Ala-Asp) box be calculated. The differentially increased antibody signal in polypeptide 4 (DDX4), ZADH2, and GRINL1A in kidney IgAN was analyzed after based on our previous quality con- tissue, we performed immunohistochemical staining on trol experience (14,18). The minimum signal threshold was set formalin-fixed, paraffin-embedded kidney biopsy tissue at 500 RFU and signal difference required between IgAN and using corresponding antibodies. We used a new set of IgAN healthy controls for any antibody was at least 200 RFU, and a kidney biopsy samples, new normal kidney control samples Z . score value of 3.0 was used as a parameter to identify (obtained from the normal kidney region obtained from fi signi cant antibody signals. The IgA signal was normalized nephrectomy samples for renal tumor), and FSGS and MGN ’ fi using quantile normalization. Pearson s correlation coef - tissue to evaluate their expression in kidneys. cients between selected antibodies and the rate of renal function decline (D inulin GFR, in milliliters per year) were calculated after transforming antibody signal intensities Results with the use of base-2 logarithms. The list of autoantibodies IgA Autoantibody Profiling in IgAN highly expressed in both IgAN and non-IgAN groups were Using an innovative IgA profiling method that allowed cross-mapped based on statistical significance (P,0.05). us to profile IgA antibodies against approximately 9000

Table 1. Demographics of patients with IgAN and healthy controls

Demographic Patients with IgAN Controls (Non-IgAN) P Value

Age (yr) 38.0610.2 29.9610.4 0.05 Sex (men/women) 12/10 5/5 0.82 Race (%) White 59 56 Asian 32 33 Hispanic 9 Black 11 Mean systolic BP (mmHg) 131.569.8 127.1610.30 Mean diastolic BP(mmHg) 79.0613.2 73.665.9 0.16 Serum creatinine (mg/dl) 1.060.31 0.9460.18 0.20 GFR (ml/min per 1.73 m2)72.0621.7 88.2612.6 0.10 Biopsy features Volume fraction of mesangium 0.2260.07 —— Globally sclerotic glomeruli (%) 15.8 (0–36) —— Fractional interstitial area (%) 25.166.7 ——

Values are presented as the mean6SD unless otherwise indicated. Pathologic values as reported previously. Median and range normal values in our laboratory are volume fraction of mesangium 0.1460.04, globally sclerotic glomeruli 0, (0–15)%, fractional interstitial area 14.9%64.8% (26). IgAN, IgA nephropathy. 4 Clinical Journal of the American Society of Nephrology

Table 2. Demographics of IgAN progressors versus IgAN nonprogressors

Demographic IgAN Progressors IgAN Nonprogressors P Value

Age (yr) 39.767.3 37.6611.5 0.67 Sex (men/women) 5/2 7/8 0.30 Mean systolic BP (mmHg) 131.569.8 130.3612.4 0.82 Mean diastolic BP (mmHg) 82.269.1 77.9614.8 0.49 Serum creatinine (mg/dl) 1.3860.22 1.0560.28 0.01 GFR (ml/min per 1.73 m2)57613.7 79621.4 0.02 DGFR (ml/min per 1.73 m2 per yr) 216.4615.3 0.1964.7 ,0.001 Proteinuria (g/d) 3.8762.63 1.2860.93 0.04 human antigens, we were able to detect IgA autoantibodies were increased with a false discover rate (q value) of in human serum samples. We identiﬁed 325 (P#0.05) IgA #10%. The 20 IgA autoantibody targets of highest signif- autoantibodies that were increased in the sera collected icance are listed in Table 3. Principal component analysis from patients with IgAN compared with healthy controls, of these top 20 antibodies compared with non-IgAN con- of which 54 met stricter statistical criteria, because they trols demonstrated a clear difference in the pattern of

Table 3. Top 20 most signiﬁcant antigenic targets for autoantibodies in IgAN

Gene P Value for Q Fold Expressed in Expressed in No. Protein Name Symbol IgAN Value Increase Glomerulus Tubules

1 Transglutaminase 2 TGM2 ,0.01 0.0 2.3 Y Y 2 TEA domain family TEAD4 ,0.01 0.0 3.1 NA NA member 4 3 Four and a half LIM FHL3 ,0.01 4.8 1.8 Y N domains protein 3 4 Homeobox C8 HOXC8 ,0.01 4.8 1.9 Y Y 5 Membrane protein, MPP1 ,0.01 4.8 2.8 N Y palmitoylated 1 6Polyadenylate- PAIP2 ,0.01 4.8 2.5 Y Y interacting protein 2 7 Ariadne homolog 2 ARIH2 ,0.01 4.8 1.7 NA NA 8 LUC7-like 2 LUC7L2 0.02 4.8 1.9 NA NA 9 Tandem C2 MTAC2D1 0.02 4.8 1.7 N Y domains, nuclear 10 Protein PPP2R5C 0.01 5.9 2.0 Y Y phosphatase 2 11 Pescadillo homolog 1 PES1 ,0.01 7.6 2.6 NA NA 12 Leucine rich repeat LRRC48 ,0.0 7.6 1.9 Y Y containing 48 13 Developmental DPPA4 ,0.01 7.6 2.1 Y Y pluripotency- protein 14 ATM/ATR- ASCIZ 0.01 9.1 3.4 NA NA Substrate Chk2- Interacting Zn2 +-ﬁnger protein 15 Nuclear respiratory NRF1 ,0.01 7.6 2.2 Y Y factor 1 16 Hypothetical protein FLJ23356 0.01 7.6 1.9 NA NA FLJ23356 17 G protein-coupled GIT2 0.01 7.6 2.2 Y Y receptor kinase 18 TANK-binding TBK1 0.01 7.6 1.9 NA NA kinase 1 19 Golgi SNAP receptor GOSR1 0.01 7.6 1.9 N Y complex 20 Protein MYLE DEXI 0.02 7.6 2.4 Y Y

Source: www.proteinatlas.org. Y, yes; NA, data not available; N, no. Clin J Am Soc Nephrol 10: ccc–ccc, March, 2014 Biomarkers for IgA Nephropathy, Woo et al. 5

Table 4. Top 20 most signiﬁcant antigenic targets for autoantibodies in progressive IgAN

No. Protein Name P Value

1 LIM and senescent cell antigen-like-containing domain protein 1 ,0.01 2 Glial ﬁbrillary acidic protein (GFAP) ,0.01 3 Ubiquitin-conjugating enzyme E2E 2 (UBC4/5 homolog, yeast) (UBE2E2) ,0.01 4 Regulator of G-protein signaling 3 (RGS3), transcript variant 4 ,0.01 5 MAGUK p55 subfamily member 5 ,0.01 6 Membrane protein, palmitoylated 5 (MAGUK p55 subfamily member 5) (MPP5) ,0.01 7 Piccolo (presynaptic cytomatrix protein) (PCLO) ,0.01 8 DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4)a ,0.01 9 Putative uncharacterized protein C14orf177 ,0.01 10 RWD domain containing 2B (RWDD2B) ,0.01 11 DEAD (Asp-Glu-Ala-Asp) box polypeptide 17 (DDX17), transcript variant 2 0.01 12 Rho GTPase-activating protein 15 0.01 13 Glutamate receptor, N-methyl D-aspartate-like 1A (GRINL1A)a 0.01 14 Tropomodulin 4 (muscle) (TMOD4) 0.01 15 p21(CDKN1A)-activated kinase 4 (PAK4), transcript variant 1 0.01 16 Branched chain ketoacid dehydrogenase kinase (BCKDK) 0.01 17 TAF6 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 80kDa 0.01 (TAF6), transcript variant 1 18 Kinesin family member 6 (KIF6) 0.01 19 Tropomodulin 1 (TMOD1) 0.01 20 Zinc binding alcohol dehydrogenase, domain containing 2 (ZADH2)a 0.01

aAutoantibodies of antigens correlated with renal function decline (Figure 2).

participants with IgAN versus controls (Figure 1B). The sig- ZADH2 also has four alternatively spliced mRNAs and nificant antigens were subjected to pathway analysis using plays a role in the redox reactions of cells, localizing primarily Ingenuity Pathway Analysis (http://www.ingenuity.com; in the mitochondria. It is associated with a congenital au- Ingenuity Systems) which identified cell-mediated immune ral atresia phenotype in individuals with the 18q deletion response, cellular development, cellular function, and main- syndrome (21). tenance as the main biologic processes of these proteins. We used the GO-Elite Pathway Analysis Tool (http://www. Specificity of IgA Autoantibodies genmapp.org/go_elite/go_elite.html) to look for enriched To ascertain whether the relative specificity of the IgA pathways based on the immunogenic epitopes for IgA re- autoantibody profile in patients with IgAN was specificto activity. The most significantly enriched pathways included this etiology and not just to glomerular injury, 23 addi- the G13 signaling pathway (P=0.001), DNA damage re- tional protein arrays were conducted on sera samples on an sponse (P=0.001), the MAPK signaling pathway (P=0.002), additional group of participants with glomerular diseases and the WNT signaling pathway (P=0.003). (10 with FSGS and seven with MGN) and were evaluated together with six additional normal controls. The autoanti- IgA Immune Response Profiling in Patients with Progressive body panel increased in IgA was found to be largely distinct IgAN versus Nonprogressive IgAN from these other glomerular diseases. Among 93 IgA auto- fi When we compared the IgA autoantibody profiles of antibodies that were signi cantly increased in IgAN P, fi patients with IgAN with progressive kidney disease versus ( 0.01), only ve autoantibodies were also increased in those who did not have progressive disease but stable renal FSGS and one autoantibody was also increased in MGN. fi function, 109 autoantibodies were increased (P,0.05) and The ve autoantibodies increased in both IgAN and FSGS the top 20 most significant antigens are shown in Table 4. included CREB regulated transcription coactivator 2 We performed a correlation analysis of each of these anti- (CRTC2), polyadenylate-binding protein-interacting body levels with the change in GFR over a 5-year duration, protein2,ARIH2,ankyrinrepeatfamilyA2(ANKRA2), as determined by annual measurements of inulin GFR. IgA and ninjurin 2 (NINJ2). Possibly of interest, ANKRA2 was antibodies to DDX4 showed a significant correlation to the increased in all three diseases. ANKRA2 is a protein that decline of renal function (r=20.55, P=0.01). DDX4 is a pu- interacts with the cytoplasmic tail of megalin, which is a tative RNA helicase, with different spliced forms, and is multiligand scavenger protein in proximal renal tubules (22). involved in the alteration of RNA secondary structure, and mitochondrial splicing (19). IgA antibodies to GRINL1A and Customized ELISA Verification of IgAN-Specific Antibodies ZADH 2 also showed a significant correlation with the decline To confirm the findings of the protein array analysis, we of renal function (GRINL1A DGFR, r=20.44, P=0.04; ZADH2 selected seven autoantibodies for further ELISA validation DGFR, r=20.48, P=0.02) (Figure 2). GRINL1A is the central based on both the confirmed expression of the corresponding componentofthebasalRNApolymeraseIItranscriptionma- protein (antigen) in the kidney (as previously demonstrated chinery and has multiple alternatively spliced variants (20). by immunohistochemistry) and strong statistical significance 6 Clinical Journal of the American Society of Nephrology

Figure 2. | Correlation of DDX4, ZADH2, and GRINL1A with renal function decline. DDX4 (A), ZADH2 (B), and GRINL1A (C) showed signiﬁcant correlations with the decline of renal function (DGFR). Log[2] RFU, relative ﬂuorescent units.

(measured by the fold increase and q value ,10%). These diseases. This may suggest that increased or altered tissue autoantibodies included IgA against SIX2, membrane pro- expression of the corresponding antigen may underlie the tein, MPP1, ZADH2, GRINL1A, TEAD4, T-TG (also known increased autoantibody response to these renal proteins in as TGM2), and ARIH2. As demonstrated in Figure 3, each of IgAN. these autoantibodies was confirmed to be significantly increased in IgAN compared with controls (P,0.01). The difference in these IgA antibodies remained significant when Discussion normalized against total IgA to test for possible effect of This study was carried out to further the understanding fi varying IgA levels in these samples: MPP1 (P=0.001), of IgAN by examining the antigen speci city of IgA among TEAD4 (P=0.01), T-TG (P=0.01), ZADH2 (P=0.03), GRIN1LA patients with IgAN. Utilizing protoarray technology, we ’ (P=0.03), and ARIH2 (P=0.01). were able to analyze each individual s repertoire of autoantibodies to .9000 human antigens. We found a substantial difference in the levels of many antigen-specific IgA Prediction of IgAN by Autoantibodies antibodies in participants with IgAN compared with healthy The diagnosis of IgAN was strongly associated with seven controls. When compared by principal component analysis, autoantibodies by ROC curve analysis. The individual area the patterns were highly distinct between controls and pa- under the curve (AUC) values for each of the autoantibodies tients with disease. The results of the protoarray analysis were as follows: 0.84 for MPP1, 0.79 for T-TG, 0.79 for ARIH2, were subsequently confirmed by ELISA for each of the auto- 0.78 for SIX2, 0.77 for GRIN1LA, 0.76 for TEAD4, and 0.74 for fi fi antibodies that were tested, selected for the magnitude of ZADH2. The signi cance of IgAN-speci c antibodies was difference from controls and for their possible biologic rele- demonstrated by comparative ROC analyses that was per- vance. Because our patients had been observed and charac- formed on four IgAN-specific IgG antibodies against MATN2, fi terized for an average of 5 years, autoantibody levels could UBE2W, DDX17, and PRKD1 (13) and six IgAN-speci cIgA be compared in a group of patients with progression toward antibodies against GRINL1A, ZADH2, TEAD4, MPP1, dialysis versus those without progression. Several of the au- TGM2, and SIX2. As evident from AUC from the two ROC fi fi toantibodies were signi cantly higher among the progressor analyses, combined performance of six IgAN-speci cIgAan- group, and a group of these antibodies correlated signifi- tibodies was more discriminatory (AUC=1) compared with cantly with the loss of renal function, defined by the decline IgAN-specific IgG antibodies (AUC=0.86) as described previ- – in measured GFR observed during the study. Comparison of ously (13). Of note, anti T-TG IgA antibody has been used for autoantibody levels in the patients with IgAN to another the diagnosis of celiac disease (23), wherein there appears to control group of normal participants and patients with other be an increased risk of IgAN (24). progressive, proteinuric renal diseases suggests that the autoantibody patterns are distinct among different glomerular Immunohistochemistry diseases with very little overlap. Finally, in an analysis of the For those IgA autoantibodies that significantly associated described functions of the antigens targeted by IgA, potential with decline of renal function in IgAN (ZADH2, GRINL1A, mechanisms of disease are revealed, such as apoptosis, fibro- and DDX4), we performed immunohistochemistry for their sis, and inflammation. For example, we find potential for corresponding proteins/antigens on kidney biopsy samples activation of the G13 pathway, which has been associated from patients with the histologic diagnosis of IgAN, non- with podocyte collagen activation, proteinuria, and glo- IgAN glomerular diseases (FSGS, membranous), and normal merulosclerosis (25). Likewise, the antibodies interacted control kidney tissue (from microdissected kidney biopsy with proteins that interact with the WNT signaling path- samples of normal kidney from resected renal tumor speci- way and there has been evidence that changes in WNT mens). As demonstrated in Figure 4, immune staining for signaling may be important in IgAN (26). These findings these antigens in the podocytes and tubular epithelium of suggest that IgA could potentially be pathogenic in this participants with IgAN was present or relatively increased disease through stimulating these mechanisms. If confirmed compared with normal control tissue or other glomerular in larger cohorts, these IgA autoantibodies could serve as a Clin J Am Soc Nephrol 10: ccc–ccc, March, 2014 Biomarkers for IgA Nephropathy, Woo et al. 7

Figure 3. | ELISA verification of IgA antibodies. Seven antibodies were subjected for verification by ELISA assay. This included IgA against SIX homeobox 2 (SIX2) (A), membrane protein, palmitoylated 1 (MPP1) (B), zinc binding alcohol dehydrogenase domain containing 2 (ZADH2) (C), glutamate receptor, ionotropic, N-methyl D-aspartate-like 1A (GRINL-1A) (D), TEA domain family member 4 (TEAD4) (E), Trans- glutaminase 2 (T-TG) (F), and ariadne homolog 2 (ARIH2) (G). biomarker or prognostic measure and may be proven to disease or disease severity among individuals. Further work play a role in the progression of renal injury. has established the existence of autoantibodies to galactose- The pathogenesis of IgAN continues to emerge. Over recent deficient IgA1. The levels of IgG and IgA specifictogalactose- years, there has been substantial focus on the role of systemic deficient IgA1 seem to be more predictive of disease and of IgA in subsequent renal injury. Many studies have document- disease severity (28). However, given that these autoantibod- ed increased levels of galactose-deficient IgA among patients ies are also found in healthy individuals, and that some pa- with IgAN (27). However, levels do not necessarily predict tients with relatively low levels can manifest severe disease, 8 Clinical Journal of the American Society of Nephrology

Figure 4. | Immunohistological assessment of IgA specific antigens ZADH2, GRINL1A, and DDX4 in kidneys. (A) ZADH2 staining illustrates increased staining in podocytes in IgAN compared with normal kidney and MGN. (B) GRINL1A staining shows staining in IgAN and MGN, but normal kidneys did not show significant staining. (C) DDX4 staining shows cytoplasmic staining in glom and FSGS shows 1+ staining in cytoplasm. other mechanisms and measures are still being sought. This etiology, in a way similar to proteinuria, irrespective of study could not evaluate IgA autoantibodies to galactose- the initiating pathogenic mechanisms. Because we do not deficient IgA because specific hinge region components are have serial GFR measurements in the non-IgAN cohort, not included in the antigen array. we cannot test this hypothesis. This could be addressed by We previously suggested that there are a wide number of validation of these autoantibodies in prospective studies IgG autoantibodies to various antigens, including many of progressive renal injury. In addition, although the exact found in the kidney, increased among patients with IgAN functions of these autoantibodies are uncertain, some like (13).Inthisstudy,wefind that IgA autoantibodies to various anti T-Tg, found in celiac disease, may point to common antigens, again including many that are found in the kidney pathways of autoimmune injury as a trigger for many re- itself, occur in a distinct pattern in IgAN and correlate with nal diseases. progression. However, many of the autoantibodies most spe- A distinct strength of this study is its highly select sample cific for IgAN diagnosis were not strongly associated with group, with detailed demographics, quantitative renal func- the actual progression of renal disease. First, this observation tion data, and extended follow-up to evaluate the natural may be due to the limited number of participants in this history of the disease with outcomes of disease progression. study who experienced disease progression. Inclusion of a The bioinformatics analyses allowed for robust selection of larger cohort may show that some of these autoantibodies highly significant IgA autoantibodies that segregated with are significant in renal disease progression. Second, the most IgA diagnosis and IgA-related progressive renal injury, significant autoantibodies associated with the progression of which was verified and validated by the customized reverse the renal disease in this study may be nonspecifictodisease ELISA assays. The defined panel of autoantibodies appears to Clin J Am Soc Nephrol 10: ccc–ccc, March, 2014 Biomarkers for IgA Nephropathy, Woo et al. 9

have substantial accuracy for noninvasive detection of IgA 2. Berger J, Hinglais N: [Intercapillary deposits of IgA-IgG]. J Urol disease and prediction of IgA renal progression. Nephrol (Paris) 74: 694–695, 1968 3. Cattran DC, Coppo R, Cook HT, Feehally J, Roberts IS, Troyanov We previously reported that IgG autoantibodies may S, Alpers CE, Amore A, Barratt J, Berthoux F, Bonsib S, Bruijn JA, play a role in IgAN through the recognition of kidney D’Agati V, D’Amico G, Emancipator S, Emma F, Ferrario F, antigens and activation of immune response. The present, Fervenza FC, Florquin S, Fogo A, Geddes CC, Groene HJ, Haas more robust results may suggest that circulating IgA has M, Herzenberg AM, Hill PA, Hogg RJ, Hsu SI, Jennette JC, Joh K, more refined specificities to antigens within the kidney and Julian BA, Kawamura T, Lai FM, Leung CB, Li LS, Li PK, Liu ZH, Mackinnon B, Mezzano S, Schena FP, Tomino Y, Walker PD, elsewhere, which may prove helpful in the diagnosis and Wang H, Weening JJ, Yoshikawa N, Zhang H; Working Group of prognosis of IgAN. It appears possible that some of these the International IgA Nephropathy Network and the Renal Pa- autoantibodies could be mediators of injury and potential thology Society: The Oxford classification of IgA nephropathy: targets of therapy. The possibility that both IgG and IgA Rationale, clinicopathological correlations, and classification. contribute to disease activity is consistent with the hy- Kidney Int 76: 534–545, 2009 4. Kiryluk K, Julian BA, Wyatt RJ, Scolari F, Zhang H, Novak J, pothesis of others, who have demonstrated this for anti- Gharavi AG: Genetic studies of IgA nephropathy: Past, present, bodies to galactose-deficient IgA (28). Although mesangial and future. Pediatr Nephrol 25: 2257–2268, 2010 deposits of IgA immune complex have been known to be a 5. Allen AC, Bailey EM, Brenchley PE, Buck KS, Barratt J, Feehally main pathogenic feature of IgAN, it has also been shown J: Mesangial IgA1 in IgA nephropathy exhibits aberrant that interstitial expansion and podocytopenia are strong O-glycosylation: Observations in three patients. Kidney Int 60: 969–973, 2001 predictors of the progression of IgAN (1,29). This study 6. Galla JH: IgA nephropathy. Kidney Int 47: 377–387, 1995 suggests that autoantibodies to specific antigenic targets 7. Coppo R, Amore A, Hogg R, Emancipator S: Idiopathic ne- in podocytes and tubules may be pathogenic for IgAN as phropathy with IgA deposits. Pediatr Nephrol 15: 139–150, they correlate with disease progression. 2000 fi 8. Coppo R, Troyanov S, Camilla R, Hogg RJ, Cattran DC, Cook HT, Nevertheless, there are signi cant limitations of this Feehally J, Roberts IS, Amore A, Alpers CE, Barratt J, Berthoux F, study. We did not have the ability to measure antibodies to Bonsib S, Bruijn JA, D’Agati V, D’Amico G, Emancipator SN, galactose-deficient IgA antibodies or to measure glycation Emma F, Ferrario F, Fervenza FC, Florquin S, Fogo AB, Geddes status of antigens. Therefore, we cannot comment on the CC, Groene HJ, Haas M, Herzenberg AM, Hill PA, Hsu SI, potential contribution of galactose-deficient IgA antibodies Jennette JC, Joh K, Julian BA, Kawamura T, Lai FM, Li LS, Li PK, Liu fi ZH, Mezzano S, Schena FP, Tomino Y, Walker PD, Wang H, to these ndings. Furthermore, to allow for robust discov- Weening JJ, Yoshikawa N, Zhang H; Working Group of the In- ery across the large number of antigens screened, we used ternational IgA Nephropathy Network and the Renal Pathology very stringent criteria for patient selection to limit false dis- Society: The Oxford IgA nephropathy clinicopathological clas- covery rates, which limited the number of the participants that sification is valid for children as well as adults. Kidney Int 77: were available for inclusion in the discovery set in this study. 921–927, 2010 9. D’Amico G: Natural history of idiopathic IgA nephropathy: role Finally, because of the high cost associated with using high- of clinical and histological prognostic factors. Am J Kidney Dis density protein arrays, we have not been able to test a larger 36: 227–237, 2000 cohort of patients without IgAN. To overcome these limi- 10. Berthoux F, Mohey H, Laurent B, Mariat C, Afiani A, Thibaudin L: tations, the predefined antibody panels that correlated with Predicting the risk for dialysis or death in IgA nephropathy. JAm IgAN diagnosis and progression might be used for indepen- Soc Nephrol 22: 752–761, 2011 11. Roberts IS, Cook HT, Troyanov S, Alpers CE, Amore A, Barratt J, dent validation in larger studies of patients with IgAN and Berthoux F, Bonsib S, Bruijn JA, Cattran DC, Coppo R, D’Agati V, other glomerular diseases, using lower-cost ELISA-based D’Amico G, Emancipator S, Emma F, Feehally J, Ferrario F, assays of the predefined antigenic targets. The validity of our Fervenza FC, Florquin S, Fogo A, Geddes CC, Groene HJ, Haas studies and these conclusions cannot be well established until M, Herzenberg AM, Hill PA, Hogg RJ, Hsu SI, Jennette JC, Joh K, Julian BA, Kawamura T, Lai FM, Li LS, Li PK, Liu ZH, Mackinnon this is completed. B, Mezzano S, Schena FP, Tomino Y, Walker PD, Wang H, Weening JJ, Yoshikawa N, Zhang H; Working Group of the In- Acknowledgments ternational IgA Nephropathy Network and the Renal Pathology The authors thank Dr. Bryan Myers for contributing precious Society: The Oxford classification of IgA nephropathy: Pathology plasma/serum samples and clinical histories on patients, as well as definitions, correlations, and reproducibility. Kidney Int 76: 546– 556, 2009 Dr. Neeraja Kambham for her reviewing and grading of the pa- 12. Matsiota P, Dosquet P, Louzir H, Druet E, Druet P, Avrameas S: thology for immunohistochemistry. The authors also thank the IgA polyspecific autoantibodies in IgA nephropathy. Clin Exp Stanford Functional Genomics Center at Stanford University for the Immunol 79: 361–366, 1990 Axon scanner, Dr. Marianne Delville for her support with the MSD 13. Sigdel TK, Woo SH, Dai H, Khatri P, Li L, Myers B, Sarwal MM, Lafayette RA: Profiling of autoantibodies in IgA nephropathy, an ELISA, and Sarwal laboratory members for their help. integrative antibiomics approach. Clin J Am Soc Nephrol 6: This work was supported by the Sobrato Research Fund (to S.H.W 2775–2784, 2011 and R.A.L.). 14. Li L, Wadia P, Chen R, Kambham N, Naesens M, Sigdel TK, Miklos DB, Sarwal MM, Butte AJ: Identifying compartment- Disclosures specific non-HLA targets after renal transplantation by in- A patent related to theIgAN biomarkerispending.M.S. servesas a tegrating transcriptome and “antibodyome” measures. Proc Natl Acad Sci U S A 106: 4148–4153, 2009 consultant for Bristol Meyers, Immucor, and ISIS. 15. Mattoon D, Michaud G, Merkel J, Schweitzer B: Biomarker discovery using protein microarray technology platforms: Antibody- antigen complex profiling. Expert Rev Proteomics 2: 879–889, References 2005 1. 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