Mapping Novel Immunogenic Epitopes in IgANephropathy
Sang Hoon Woo,* Tara K. Sigdel,† Van T. Dinh,† Minh-Thien Vu,† Minnie M. Sarwal,† and Richard A. Lafayette‡
AbstractBackground and objectives IgA plays a key role in IgA nephropathy (IgAN) by forming immune complexes anddepositing in the glomeruli, leading to an inflammatory response. However, the antigenic targets and functionalcharacterization of IgA have been incompletely defined in this disease.
Design, setting, participants, & measurements This study was performed in sera from patients who were studiedas part of a prospective, observational study of IgAN. These patients (n=22) all had biopsy-proven IgANwithin 3 years of study initiation, complete clinical data, annual urinary inulin clearance for GFRs, and at least5 years of follow-up. Progression was defined as loss of .5 ml/min per 1.73 m2 per year of inulin clearancemeasured over at least 5 years. A protein microarray was used for detection of IgAN-specific IgA autoantibodiesin blood across approximately 9000 human antigens to specifically identify the most immunogenic proteintargets that drive IgA antibodies in IgAN (n=22), healthy controls (n=10), and non-IgAN glomerular diseases(n=17). Results were validated by ELISA assays in sera and by immunohistochemistry in IgAN kidney biopsies.IgA-specific antibodies were correlated with clinical and histologic variables to assess their effect on diseaseprogression and prognosis.
Results Fifty-four proteins mounted highly significant IgA antibody responses in patients with IgANwith a falsediscovery rate (q value) of#10%; 325 antibodies (P#0.05) were increased overall. Antitissue transglutaminaseIgA was significantly elevated in IgAN (P,0.001, q value of 0%). IgA antibodies to DDX4 (r=20.55, P=0.01)and ZADH2 (r=20.48, P=0.02) were significantly correlated with the decline of renal function. Specific IgA auto-antibodies are elevated in IgAN compared with normal participants and those with other glomerular diseases.
Conclusions In this preliminary study, IgA autoantibodies target novel proteins, highly expressed in the kidneyglomerulus and tubules. These IgA autoantibodies may play important roles in the pathogenesis of IgAN.
Clin J Am Soc Nephrol 10: ccc–ccc, 2014. doi: 10.2215/CJN.02390314
IntroductionIgA nephropathy (IgAN) is the most common GN inthe world and remains a common cause of ESRD (1,2).IgAN is diagnosed by kidney biopsy and defined bythe deposition of IgA in the glomerulus with prolifera-tion of the mesangium (3). The pathogenesis of IgAN isstill poorly understood. There is likely underlying ge-netic predisposition as demonstrated by family, racial,and sex predisposition as well as several candidate ge-netic risk factors (4). Patients with IgAN generally dem-onstrate increased levels of galactose-deficient IgA1 (5).It has been demonstrated that immune complexesformed from IgA or IgG autoantibodies that recog-nize galactose-deficient IgA may precipitate in theglomerulus and cause injury (6–9). The prognosis ofIgAN is also incompletely determined by typicallymeasured clinical and pathologic features. Patientswith greater degrees of proteinuria, renal dysfunction, orpathologic changes (proliferative, necrotic, or sclerotic)have higher risks of progressive disease (10,11).However, greater precision in risk assessment afford-ed by further biomarkers could aid in determining
which patients to observe more closely, treat moreaggressively, or include/stratify in treatment trials.Our group and others previously demonstrated that
autoantibodies may be important in the pathogenesisof IgAN (12,13). However, the specificities of IgA anti-bodies in IgAN have still not been broadly defined.These specificities could help to better understand thepathogenesis of this disease and may provide more re-fined biomarkers to prognosticate and guide therapy.High-density protein microarrays have been used to
screen serum antibodies (14) to .8000 individual hu-man protein targets and have yielded findings of clin-ical interest (15). To characterize IgA and its antigenictargets, we used protein arrays to detect and describeIgA autoantibodies in patients with IgAN. We appliedan integrative genomics approach to cross-map the IgAantibodies that are highly expressed in patients withIgAN with protein targets to assess their specificity tothe kidney as well as other organs. Potential candidatescan then be validated in biopsy tissue by immunohis-tochemistry and in serum samples by ELISA. The over-all study approach is summarized in Figure 1A.
*Division of HospitalMedicine,Department ofMedicine, ThomasJefferson University,Philadelphia,Pennsylvania;†Department ofSurgery, University ofCalifornia, SanFrancisco, California;‡Division ofNephrology,Department ofMedicine, StanfordUniversity School ofMedicine, StanfordUniversity, Stanford,California
Correspondence:Dr. Richard Lafayette,Department ofMedicine, StanfordUniversity, A175, 300Pasteur Drive,Stanford, CA 94305;or Dr. Minnie Sarwal,Department ofSurgery, University ofCalifornia SanFrancisco, G893, 513Parnassus Avenue, SanFrancisco, CA 94107.Email: [email protected] or [email protected]
. Published on December 26, 2014 as doi: 10.2215/CJN.02390314CJASN ePress
Materials and MethodsPatientsEntry criteria for this study was biopsy-confirmed IgAN,
and each enrolled patient with IgAN had to have at least5 years of follow-up with complete clinical data, annual seraand urine sampling, and annually measured GFRs forabsolute quantification of renal function. Given these strin-gent selection criteria, we could enroll 22 patients with IgANthat met all entry criteria.A total of 49 participants were enrolled for this study: the 22
patients with biopsy-confirmed IgANwhomet all above studyentry criteria, 17 patientswith biopsy-proven non-IgA causes ofrenal disease, and 10 age- and sex-matched healthy controls.
Within the cohort of IgAN participants, the rate of declineof measured GFR over the 5 years of follow-up was used tosubclassify them into two groups. Patients with IgAN wereclassified as progressors (n=7) if the rate of measured GFRdecline (DGFR) was .5.0 ml/min per 1.73m2 per year. Pa-tients with a DGFR of ,5.0 ml/min per 1.73 m2 per yearwere classified as nonprogressors. Blood (5 ml serum) andurine (50 ml) samples were collected annually over the5-year follow-up from each patient with IgAN. GFR wasmeasured using the urinary clearance of inulin, as previ-ously described (16). The demographics of these patientsare shown in Tables 1 and 2. The characteristics of sevenpatients 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 IgAautoantibodies with a cut-off threshold .1.65-fold increase in IgAN with P value ,0.05 shows the comparison between IgAN and controlparticipants. 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.
2 Clinical Journal of the American Society of Nephrology
male sex (n=4), female sex (n=3), mean age (50.7 years), race(white, 43%; Hispanic, 43%; and Asian, 14%), serum creati-nine (0.88 mg/dl), proteinuria (7.6 g/d). The characteristicsof 10 patients with FSGS include male sex (n=7), female sex(n=3), mean age (50.7 years), race (white, 50%; Hispanic,40%; and black, 10%), serum creatinine (2.0 mg/dl), andproteinuria (4.8 g/d). None of our participants were treatedwith immunosuppression (steroids, cytotoxics) at the time oftheir blood draws.
Immune Response Measurement Using Protein MicroarraysProtoArray Human Protein Microarray v5.0 (Invitrogen,
Carlsbad, CA) was used to characterize the specificity ofIgA-specific autoantibody response in IgAN. The relativefluorescence intensity (in relative fluorescence units [RFU])was measured using GenePix pro 6.0 software (MolecularDevices, Sunnyvale, CA). Data normalization was done usingProtoArray Prospector 5.2. We used the quantile normaliza-tion method because there were no positive controls for IgA toapply Robust Linear Model for normalization (17). Fluores-cence signal values were measured for each protein after ad-justing for background correction so that Z factors for all ofthe corrected intensities of the human protein features couldbe calculated. The differentially increased antibody signal inIgAN was analyzed after based on our previous quality con-trol experience (14,18). The minimum signal threshold was setat 500 RFU and signal difference required between IgAN andhealthy controls for any antibody was at least 200 RFU, and aZ score value of .3.0 was used as a parameter to identifysignificant antibody signals. The IgA signal was normalizedusing quantile normalization. Pearson’s correlation coeffi-cients between selected antibodies and the rate of renal func-tion decline (D inulin GFR, in milliliters per year) werecalculated after transforming antibody signal intensitieswith the use of base-2 logarithms. The list of autoantibodieshighly expressed in both IgAN and non-IgAN groups werecross-mapped based on statistical significance (P,0.05).
The analysis was also done with SAS software (version9.2, enterprise guide 4.2). The pathway analysis wasperformed using Ingenuity Pathway Analysis (http://www.ingenuity.com).
Validation of IgA Antibodies Based on ELISAValidation of IgAN-specific IgA autoantibodies identi-
fied by protein arrays was performed using theMSD ELISAplatform (Meso Scale Discovery, Gaithersburg, MD). Receiveroperating characteristic (ROC) analysis was conducted on acombined panel of antibodies to predict IgAN with fittedlogistic regression models using log-transformed ELISAlevels of antibodies against SIX homeobox 2 (SIX2), mem-brane protein, palmitoylated 1 (MPP1), TEA domain familymember 4 (TEAD4), transglutaminase 2 (T-TG), zinc bindingalcohol dehydrogenase domain containing 2 (ZADH2), glu-tamate receptor, ionotropic, N-methyl-D-aspartate-like 1A(GRINL1A), and ariadne homolog 2 (ARIH2). For ROCanalyses, we generated all corresponding sensitivities andspecificities for each of cut-off point (theta).
ImmunohistochemistryTo evaluate the presence of DEAD (Asp-Glu-Ala-Asp) box
polypeptide 4 (DDX4), ZADH2, and GRINL1A in kidneytissue, we performed immunohistochemical staining onformalin-fixed, paraffin-embedded kidney biopsy tissueusing corresponding antibodies. We used a new set of IgANkidney biopsy samples, new normal kidney control samples(obtained from the normal kidney region obtained fromnephrectomy samples for renal tumor), and FSGS and MGNtissue to evaluate their expression in kidneys.
ResultsIgA Autoantibody Profiling in IgANUsing an innovative IgA profiling method that allowed
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
Mean systolic BP (mmHg) 131.569.8 127.161 0.30Mean diastolic BP(mmHg) 79.0613.2 73.665.9 0.16Serum creatinine (mg/dl) 1.060.31 0.9460.18 0.20GFR (ml/min per 1.73 m2) 72.0621.7 88.2612.6 0.10Biopsy featuresVolume fraction of mesangium 0.2260.07 — —Globally sclerotic glomeruli (%) 15.8 (0–36) — —Fractional interstitial area (%) 25.166.7 — —
Values are presented as themean6SD unless otherwise indicated. Pathologic values as reported previously. Median and range normalvalues in our laboratory are volume fraction ofmesangium 0.1460.04, globally sclerotic glomeruli 0, (0–15)%, fractional interstitial area14.9%64.8% (26). IgAN, IgA nephropathy.
Clin J Am Soc Nephrol 10: ccc–ccc, March, 2014 Biomarkers for IgA Nephropathy, Woo et al. 3
human antigens, we were able to detect IgA autoantibodiesin human serum samples. We identified 325 (P#0.05) IgAautoantibodies that were increased in the sera collectedfrom patients with IgAN compared with healthy controls,of which 54 met stricter statistical criteria, because they
were increased with a false discover rate (q value) of#10%. The 20 IgA autoantibody targets of highest signif-icance are listed in Table 3. Principal component analysisof these top 20 antibodies compared with non-IgAN con-trols demonstrated a clear difference in the pattern of
Table 2. Demographics of IgAN progressors versus IgAN nonprogressors
Demographic IgAN Progressors IgAN Nonprogressors P Value
Age (yr) 39.767.3 37.6611.5 0.67Sex (men/women) 5/2 7/8 0.30Mean systolic BP (mmHg) 131.569.8 130.3612.4 0.82Mean diastolic BP (mmHg) 82.269.1 77.9614.8 0.49Serum creatinine (mg/dl) 1.3860.22 1.0560.28 0.01GFR (ml/min per 1.73 m2) 57613.7 79621.4 0.02DGFR (ml/min per 1.73 m2 per yr) 216.4615.3 0.1964.7 ,0.001Proteinuria (g/d) 3.8762.63 1.2860.93 0.04
Table 3. Top 20 most significant antigenic targets for autoantibodies in IgAN
No. Protein Name GeneSymbol
P Value forIgAN
QValue
FoldIncrease
Expressed inGlomerulus
Expressed inTubules
1 Transglutaminase 2 TGM2 ,0.01 0.0 2.3 Y Y2 TEA domain family
participants with IgAN versus controls (Figure 1B). The sig-nificant antigens were subjected to pathway analysis usingIngenuity Pathway Analysis (http://www.ingenuity.com;Ingenuity Systems) which identified cell-mediated immuneresponse, cellular development, cellular function, and main-tenance as the main biologic processes of these proteins. Weused the GO-Elite Pathway Analysis Tool (http://www.genmapp.org/go_elite/go_elite.html) to look for enrichedpathways based on the immunogenic epitopes for IgA re-activity. The most significantly enriched pathways includedthe G13 signaling pathway (P=0.001), DNA damage re-sponse (P=0.001), the MAPK signaling pathway (P=0.002),and the WNT signaling pathway (P=0.003).
IgA Immune Response Profiling in Patients with ProgressiveIgAN versus Nonprogressive IgANWhen we compared the IgA autoantibody profiles of
patients with IgAN with progressive kidney disease versusthose who did not have progressive disease but stable renalfunction, 109 autoantibodies were increased (P,0.05) andthe top 20 most significant antigens are shown in Table 4.We performed a correlation analysis of each of these anti-body levels with the change in GFR over a 5-year duration,as determined by annual measurements of inulin GFR. IgAantibodies to DDX4 showed a significant correlation to thedecline of renal function (r=20.55, P=0.01). DDX4 is a pu-tative RNA helicase, with different spliced forms, and isinvolved in the alteration of RNA secondary structure, andmitochondrial splicing (19). IgA antibodies to GRINL1A andZADH 2 also showed a significant correlation with the declineof renal function (GRINL1A DGFR, r=20.44, P=0.04; ZADH2DGFR, r=20.48, P=0.02) (Figure 2). GRINL1A is the centralcomponent of the basal RNA polymerase II transcription ma-chinery and has multiple alternatively spliced variants (20).
ZADH2 also has four alternatively spliced mRNAs andplays a role in the redox reactions of cells, localizing primarilyin the mitochondria. It is associated with a congenital au-ral atresia phenotype in individuals with the 18q deletionsyndrome (21).
Specificity of IgA AutoantibodiesTo ascertain whether the relative specificity of the IgA
autoantibody profile in patients with IgAN was specific tothis etiology and not just to glomerular injury, 23 addi-tional protein arrays were conducted on sera samples on anadditional group of participants with glomerular diseases(10 with FSGS and seven with MGN) and were evaluatedtogether with six additional normal controls. The autoanti-body panel increased in IgA was found to be largely distinctfrom these other glomerular diseases. Among 93 IgA auto-antibodies that were significantly increased in IgAN(P,0.01), only five autoantibodies were also increased inFSGS and one autoantibody was also increased in MGN.The five autoantibodies increased in both IgAN and FSGSincluded CREB regulated transcription coactivator 2(CRTC2), polyadenylate-binding protein-interactingprotein 2, ARIH2, ankyrin repeat family A 2 (ANKRA2),and ninjurin 2 (NINJ2). Possibly of interest, ANKRA2 wasincreased in all three diseases. ANKRA2 is a protein thatinteracts with the cytoplasmic tail of megalin, which is amultiligand scavenger protein in proximal renal tubules (22).
Customized ELISA Verification of IgAN-Specific AntibodiesTo confirm the findings of the protein array analysis, we
selected seven autoantibodies for further ELISA validationbased on both the confirmed expression of the correspondingprotein (antigen) in the kidney (as previously demonstratedby immunohistochemistry) and strong statistical significance
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 ,0.012 Glial fibrillary acidic protein (GFAP) ,0.013 Ubiquitin-conjugating enzyme E2E 2 (UBC4/5 homolog, yeast) (UBE2E2) ,0.014 Regulator of G-protein signaling 3 (RGS3), transcript variant 4 ,0.015 MAGUK p55 subfamily member 5 ,0.016 Membrane protein, palmitoylated 5 (MAGUK p55 subfamily member 5) (MPP5) ,0.017 Piccolo (presynaptic cytomatrix protein) (PCLO) ,0.018 DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4)a ,0.019 Putative uncharacterized protein C14orf177 ,0.0110 RWD domain containing 2B (RWDD2B) ,0.0111 DEAD (Asp-Glu-Ala-Asp) box polypeptide 17 (DDX17), transcript variant 2 0.0112 Rho GTPase-activating protein 15 0.0113 Glutamate receptor, N-methyl D-aspartate-like 1A (GRINL1A)a 0.0114 Tropomodulin 4 (muscle) (TMOD4) 0.0115 p21(CDKN1A)-activated kinase 4 (PAK4), transcript variant 1 0.0116 Branched chain ketoacid dehydrogenase kinase (BCKDK) 0.0117 TAF6 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 80kDa
(TAF6), transcript variant 10.01
18 Kinesin family member 6 (KIF6) 0.0119 Tropomodulin 1 (TMOD1) 0.0120 Zinc binding alcohol dehydrogenase, domain containing 2 (ZADH2)a 0.01
aAutoantibodies of antigens correlated with renal function decline (Figure 2).
Clin J Am Soc Nephrol 10: ccc–ccc, March, 2014 Biomarkers for IgA Nephropathy, Woo et al. 5
(measured by the fold increase and q value ,10%). Theseautoantibodies included IgA against SIX2, membrane pro-tein, MPP1, ZADH2, GRINL1A, TEAD4, T-TG (also knownas TGM2), and ARIH2. As demonstrated in Figure 3, each ofthese autoantibodies was confirmed to be significantly in-creased in IgAN compared with controls (P,0.01). The dif-ference in these IgA antibodies remained significant whennormalized against total IgA to test for possible effect ofvarying IgA levels in these samples: MPP1 (P=0.001),TEAD4 (P=0.01), T-TG (P=0.01), ZADH2 (P=0.03), GRIN1LA(P=0.03), and ARIH2 (P=0.01).
Prediction of IgAN by AutoantibodiesThe diagnosis of IgANwas strongly associated with seven
autoantibodies by ROC curve analysis. The individual areaunder the curve (AUC) values for each of the autoantibodieswere as follows: 0.84 forMPP1, 0.79 for T-TG, 0.79 for ARIH2,0.78 for SIX2, 0.77 for GRIN1LA, 0.76 for TEAD4, and 0.74 forZADH2. The significance of IgAN-specific antibodies wasdemonstrated by comparative ROC analyses that was per-formed on four IgAN-specific IgG antibodies against MATN2,UBE2W, DDX17, and PRKD1 (13) and six IgAN-specific IgAantibodies against GRINL1A, ZADH2, TEAD4, MPP1,TGM2, and SIX2. As evident from AUC from the two ROCanalyses, combined performance of six IgAN-specific IgA an-tibodies was more discriminatory (AUC=1) compared withIgAN-specific IgG antibodies (AUC=0.86) as described previ-ously (13). Of note, anti–T-TG IgA antibody has been used forthe diagnosis of celiac disease (23), wherein there appears tobe an increased risk of IgAN (24).
ImmunohistochemistryFor those IgA autoantibodies that significantly associated
with decline of renal function in IgAN (ZADH2, GRINL1A,and DDX4), we performed immunohistochemistry for theircorresponding proteins/antigens on kidney biopsy samplesfrom patients with the histologic diagnosis of IgAN, non-IgAN glomerular diseases (FSGS, membranous), and normalcontrol kidney tissue (from microdissected kidney biopsysamples of normal kidney from resected renal tumor speci-mens). As demonstrated in Figure 4, immune staining forthese antigens in the podocytes and tubular epithelium ofparticipants with IgAN was present or relatively increasedcompared with normal control tissue or other glomerular
diseases. This may suggest that increased or altered tissueexpression of the corresponding antigen may underlie theincreased autoantibody response to these renal proteins inIgAN.
DiscussionThis study was carried out to further the understanding
of IgAN by examining the antigen specificity of IgA amongpatients with IgAN. Utilizing protoarray technology, wewere able to analyze each individual’s repertoire of auto-antibodies to .9000 human antigens. We found a substan-tial difference in the levels of many antigen-specific IgAantibodies in participants with IgAN compared with healthycontrols. When compared by principal component analysis,the patterns were highly distinct between controls and pa-tients with disease. The results of the protoarray analysiswere subsequently confirmed by ELISA for each of the auto-antibodies that were tested, selected for the magnitude ofdifference from controls and for their possible biologic rele-vance. Because our patients had been observed and charac-terized for an average of 5 years, autoantibody levels couldbe compared in a group of patients with progression towarddialysis versus those without progression. Several of the au-toantibodies were significantly higher among the progressorgroup, and a group of these antibodies correlated signifi-cantly with the loss of renal function, defined by the declinein measured GFR observed during the study. Comparison ofautoantibody levels in the patients with IgAN to anothercontrol group of normal participants and patients with otherprogressive, proteinuric renal diseases suggests that the au-toantibody patterns are distinct among different glomerulardiseases with very little overlap. Finally, in an analysis of thedescribed functions of the antigens targeted by IgA, potentialmechanisms of disease are revealed, such as apoptosis, fibro-sis, and inflammation. For example, we find potential foractivation of the G13 pathway, which has been associatedwith podocyte collagen activation, proteinuria, and glo-merulosclerosis (25). Likewise, the antibodies interactedwith proteins that interact with the WNT signaling path-way and there has been evidence that changes in WNTsignaling may be important in IgAN (26). These findingssuggest that IgA could potentially be pathogenic in thisdisease through stimulating these mechanisms. If confirmedin larger cohorts, these IgA autoantibodies could serve as a
Figure 2. | Correlation of DDX4, ZADH2, and GRINL1A with renal function decline. DDX4 (A), ZADH2 (B), and GRINL1A (C) showedsignificant correlations with the decline of renal function (DGFR). Log[2] RFU, relative fluorescent units.
6 Clinical Journal of the American Society of Nephrology
biomarker or prognostic measure and may be proven toplay a role in the progression of renal injury.The pathogenesis of IgAN continues to emerge. Over recent
years, there has been substantial focus on the role of systemicIgA in subsequent renal injury. Many studies have document-ed increased levels of galactose-deficient IgA among patientswith IgAN (27). However, levels do not necessarily predict
disease or disease severity among individuals. Further workhas established the existence of autoantibodies to galactose-deficient IgA1. The levels of IgG and IgA specific to galactose-deficient IgA1 seem to be more predictive of disease and ofdisease severity (28). However, given that these autoantibod-ies are also found in healthy individuals, and that some pa-tients with relatively low levels can manifest severe disease,
Figure 3. | ELISA verification of IgA antibodies. Seven antibodies were subjected for verification by ELISA assay. This included IgA against SIXhomeobox 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).
Clin J Am Soc Nephrol 10: ccc–ccc, March, 2014 Biomarkers for IgA Nephropathy, Woo et al. 7
other mechanisms and measures are still being sought. Thisstudy could not evaluate IgA autoantibodies to galactose-deficient IgA because specific hinge region components arenot included in the antigen array.We previously suggested that there are a wide number of
IgG autoantibodies to various antigens, including manyfound in the kidney, increased among patients with IgAN(13). In this study, we find that IgA autoantibodies to variousantigens, again including many that are found in the kidneyitself, occur in a distinct pattern in IgAN and correlate withprogression. However, many of the autoantibodies most spe-cific for IgAN diagnosis were not strongly associated withthe actual progression of renal disease. First, this observationmay be due to the limited number of participants in thisstudy who experienced disease progression. Inclusion of alarger cohort may show that some of these autoantibodiesare significant in renal disease progression. Second, the mostsignificant autoantibodies associated with the progression ofthe renal disease in this study may be nonspecific to disease
etiology, in a way similar to proteinuria, irrespective ofthe initiating pathogenic mechanisms. Because we do nothave serial GFR measurements in the non-IgAN cohort,we cannot test this hypothesis. This could be addressed byvalidation of these autoantibodies in prospective studiesof progressive renal injury. In addition, although the exactfunctions of these autoantibodies are uncertain, some likeanti T-Tg, found in celiac disease, may point to commonpathways of autoimmune injury as a trigger for many re-nal diseases.A distinct strength of this study is its highly select sample
group, with detailed demographics, quantitative renal func-tion data, and extended follow-up to evaluate the naturalhistory of the disease with outcomes of disease progression.The bioinformatics analyses allowed for robust selection ofhighly significant IgA autoantibodies that segregated withIgA diagnosis and IgA-related progressive renal injury,which was verified and validated by the customized reverseELISA assays. The defined panel of autoantibodies appears to
Figure 4. | Immunohistological assessment of IgA specific antigens ZADH2, GRINL1A, and DDX4 in kidneys. (A) ZADH2 staining illustrates in-creased staining in podocytes in IgAN comparedwith normal kidney andMGN. (B) GRINL1A staining shows staining in IgAN andMGN, but normalkidneys did not show significant staining. (C) DDX4 staining shows cytoplasmic staining in glom and FSGS shows 1+ staining in cytoplasm.
8 Clinical Journal of the American Society of Nephrology
have substantial accuracy for noninvasive detection of IgAdisease and prediction of IgA renal progression.We previously reported that IgG autoantibodies may
play a role in IgAN through the recognition of kidneyantigens and activation of immune response. The present,more robust results may suggest that circulating IgA hasmore refined specificities to antigens within the kidney andelsewhere, which may prove helpful in the diagnosis andprognosis of IgAN. It appears possible that some of theseautoantibodies could be mediators of injury and potentialtargets of therapy. The possibility that both IgG and IgAcontribute to disease activity is consistent with the hy-pothesis of others, who have demonstrated this for anti-bodies to galactose-deficient IgA (28). Although mesangialdeposits of IgA immune complex have been known to be amain pathogenic feature of IgAN, it has also been shownthat interstitial expansion and podocytopenia are strongpredictors of the progression of IgAN (1,29). This studysuggests that autoantibodies to specific antigenic targetsin podocytes and tubules may be pathogenic for IgAN asthey correlate with disease progression.Nevertheless, there are significant limitations of this
study. We did not have the ability to measure antibodies togalactose-deficient IgA antibodies or to measure glycationstatus of antigens. Therefore, we cannot comment on thepotential contribution of galactose-deficient IgA antibodiesto these findings. Furthermore, to allow for robust discov-ery across the large number of antigens screened, we usedvery stringent criteria for patient selection to limit false dis-covery rates, which limited the number of the participants thatwere available for inclusion in the discovery set in this study.Finally, because of the high cost associated with using high-density protein arrays, we have not been able to test a largercohort of patients without IgAN. To overcome these limi-tations, the predefined antibody panels that correlated withIgAN diagnosis and progression might be used for indepen-dent validation in larger studies of patients with IgAN andother glomerular diseases, using lower-cost ELISA-basedassays of the predefined antigenic targets. The validity of ourstudies and these conclusions cannot be well established untilthis is completed.
AcknowledgmentsThe authors thank Dr. Bryan Myers for contributing precious
plasma/serum samples and clinical histories on patients, as well asDr. Neeraja Kambham for her reviewing and grading of the pa-thology for immunohistochemistry. The authors also thank theStanford Functional Genomics Center at StanfordUniversity for theAxon scanner, Dr. Marianne Delville for her support with the MSDELISA, and Sarwal laboratory members for their help.Thisworkwas supportedby the SobratoResearchFund (to S.H.W
and R.A.L.).
DisclosuresApatent related to the IgANbiomarker ispending.M.S. servesasa
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Received: April 22, 2014 Accepted: November 10, 2014
S.HW. and T.K.S. contributed equally to this work as first authors.M.M.S. and R.A.L. contributed equally to this work as seniorauthors.
Published online ahead of print. Publication date available at www.cjasn.org.
10 Clinical Journal of the American Society of Nephrology
