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Non-HLA Antibodies to Immunogenic Epitopes Predictthe Evolution of Chronic Renal Allograft Injury
Tara K. Sigdel, Li Li, Tim Q. Tran, Purvesh Khatri, Maarten Naesens, Poonam Sansanwal,Hong Dai, Szu-chuan Hsieh, and Minnie M. Sarwal
Department of Pediatrics, Stanford University School of Medicine, Stanford, California
ABSTRACTChronic allograft injury (CAI) results from a humoral response to mismatches in immunogenic epitopesbetween the donor and recipient. Although alloantibodies against HLA antigens contribute to thepathogenesis of CAI, alloantibodies against non-HLA antigens likely contribute as well. Here, we used high-density protein arrays to identify non-HLA antibodies in CAI and subsequently validated a subset in a cohortof 172 serum samples collected serially post-transplantation. There were 38 de novo non-HLA antibodiesthat significantly associated with the development of CAI (P,0.01) on protocol post-transplant biopsies,with enrichment of their corresponding antigens in the renal cortex. Baseline levels of preformed antibodiesto MIG (also called CXCL9), ITAC (also called CXCL11), IFN-g, and glial-derived neurotrophic factor posi-tively correlated with histologic injury at 24 months. Measuring levels of these four antibodies could helpclinicians predict the development of CAI with .80% sensitivity and 100% specificity. In conclusion, pre-transplant serum levels of a defined panel of alloantibodies targeting non-HLA immunogenic antigensassociate with histologic CAI in the post-transplant period. Validation in a larger, prospective transplantcohort may lead to a noninvasive method to predict and monitor for CAI.
J Am Soc Nephrol 23: 750–763, 2012. doi: 10.1681/ASN.2011060596
Despite improvements in short-term graft survivalover the past decades,1 chronic allograft injury(CAI) remains a major challenge in renal and othersolid organ transplants. In addition, the rate of pro-gression of CAI remained relatively unabated overthe last decade, with limited improvements in ex-tending graft survival.2,3 CAI in the graft is the pri-mary reason for accelerated graft loss,4 categorizedby progressive interstitial fibrosis (IF) and tubularatrophy (TA) of the parenchyma,5 that is also asso-ciated with glomerulopathy, fibrointimal hyperpla-sia of arteries, and arteriolar hyalinosis.6 This injuryis believed to be mostly a humoral injury responseto mismatched immunogenic epitopes between thedonor and recipient,7 with current understandingmostly focused on HLA antigens.8 Despite improvedunderstanding of the assessment and monitoring forHLA mismatched epitopes,8 the pathogenicity ofdonor-specific anti-HLA antibodies on CAI remainselusive.9 This is shownwith complete HLAmatchedtransplants exhibiting finite survival due to progres-sive CAI,10 and not infrequently, CAI observed in
HLAmismatched grafts without demonstrable anti-bodies to donor-specific HLA antigens.7 These find-ings strongly suggest that additional pathogenicantibodies drive the humoral axis of injury in CAI,9,11
and may be the final common outcome. The acti-vation or transition of these non-HLA antibodiestoward pathogenicity is likely through the underlyingtriggers of acute rejection, hypoperfusion, ischemiareperfusion, calcineurin toxicity, infection, and re-current diseases.12 Currently, there is no means topredict which transplant patients will develop accel-erated CAI in the allograft. Histologic diagnosis ofCAI is the current gold standard for diagnosing
Received June 21, 2011. Accepted October 28, 2011.
Published online ahead of print. Publication date available atwww.jasn.org.
Correspondence: Dr. Minnie M. Sarwal, Department of Pediat-rics, Stanford University, G306, 300 Pasteur Drive, Stanford, CA94305-5208. Email: [email protected]
Copyright © 2012 by the American Society of Nephrology
750 ISSN : 1046-6673/2304-750 J Am Soc Nephrol 23: 750–763, 2012
CAI; however, this only detects advanced, established, and oftenirreversible injury. The renal biopsy is also an invasive procedurethat suffers from sampling heterogeneity, is associated with var-ious complications,13 and provides a limited prognostic value.Identification of informative, minimally invasive biomarkers iscritically needed to monitor and predict CAI, and remains acritically important unmet need in solid organ transplantation.
The antibody axis has been implicated in different diseaseconditions, and profiling and measuring the level of IgG an-tibodies against thousands of defined human proteins to iden-tify antibodies against non-HLA antigens have been previouslyattempted in autoimmune diseases14 and CKD.15 In an at-tempt to evaluate clinically relevant de novo, renal-specific,non-HLA antibody responses, we utilized protein arrays andcustomized informatics for studying non-HLA antibody re-sponses in stable grafts16,17 and acute rejection11,18 to inter-rogate previously unidentified non-HLA antibody CAI. Toinvestigate the non-HLA pathogenicity in CAI, we excludedpatients with signs of acute rejection, purely focusing onnon-HLA antibody responses in the absence of any intervalacute rejection episodes. From this cohort, we then excludedpediatric patients with interval episodes of infection, delayedgraft function, or body surface area ,0.75 m2,19 thuscreating a homogenous set of patients without any major con-founders to CAI. This resulted in the removal of 47 patientswith interval acute rejection episodes, 3 patients in intervalinfectious episodes, 3 patients with delayed graft function, aswell as 4 recipients with body surface area ,0.75 m2. Fromthese remaining patients, we carefully selected a subset of 20patients (n=10 CAI versus n=10 without CAI) as the learningset and collected surveillance samples of sera matched to di-agnostic biopsy at 0, 6, and 24 months post-transplantationfrom each patient, thus analyzing 60 serum samples for diag-nosis and prediction of CAI. The increased presence of signif-icant non-HLA antibodies was analyzed for their correlationwith injury progression post-transplant. Finally, from thelarger confounder-controlled cohort, we validated the mostsignificant and relevant antibodies (from discovery) by usingELISAs on 112 unique and independent sera samples from 68patients (Figure 1).
RESULTS
Identification of CAI-Specific Novel Non-HLAAntibodiesCompared with the non-HLA antibody levels before trans-plantation, there was a significant immune responsepost-transplantwith thedevelopmentofCAI.ByusingM-statistics ofthe Prospector Analyzer with the robust linear normalizationmethod published elsewhere,20 we identified a total of 231non-HLA antibodies with statistically significant P values#0.05.Among the 231 changed antibodies, 111 non-HLA antibodiesincreased significantly in the CAI group (P#0.05) (Figure 2Aand Supplemental Table 1). On the basis of a time course
analysis of the most statistically significant (P#0.05) non-HLAantibodies, the CAI-specific non-HLA antibody could be categor-ically divided into an non-HLA showing significant detection ei-ther early (,6months) or late (.6months) post-transplantation.The early responsewas observed to be against non-HLAproteinsinvolved in pathways such as cell-mediated immune response,connective tissue development and function, and EGF signaling.Some of these selected antigens are shown in Table 1. The lateresponding non-HLA proteins were noted to immunologicallyreactive proteins involved in pathways such as cell death, cell-to-cell signaling and interaction, and cellular movement (Figure 2Band Supplemental Figure 1).
We analyzed the antibody response for 12 HLA antigens,including 2HLAclass Imolecules (HLA-B andHLA-C) and 10HLA class II molecules (HLA-DOA, HLA-DOB, HLA-DPA1,HLA-DPB1,HLA-DQA1,HLA-DQB1,HLA-DRA,HLA-DRB1,HLA-DRB3, and HLA-DRB5) in the CAI and non-CAI groups.The signal intensity of antibody detection to these targets is verylow (average ,500 RFU signal intensity). Thus, it seems thatHLA antibody levelsmay be less influenced in the course of CAI,in the absence of interval acute rejection.
Cross-Mapping CAI-Specific Non-HLA Antibodies toExpression of the Target Proteins in SubcellularCompartments of the KidneyTo triage the selection of non-HLA antibodies to further pursue,we chose to assign physiologic and potentially pathologicrelevance to the significant CAI-specific antibodies, by selectingantibodies with reactivities to kidney-specific antigens. Theassumption is that these de novo antibody responses after kidneytransplantation are more likely against the antigens in the newlytransplanted kidney. To enable this analysis, we performed cross-mapping of kidney and kidney-compartment–specific genes ob-tained frommicrodissected compartments of normal kidney byprofiling these tissues on cDNA microarrays with protoarrayprotein targets, using our published approach of integrated anti-biomics.18,21,22 This analysis revealed that there was an enrich-ment for antigens expressed in the renal cortex (P=0.029). Aspreviously shown, the renal pelvis antigens are highly immuno-genic, and enrichment for pelvis-specific antigens was alsonoted. These antigen lists are shown in Table 2.
CAI-Specific Antibodies Track Injury ProgressionWe took the antibodies that were increased with CAI andperformed Spearman correlation analyses with the ChronicAllograft Damage Index (CADI) and IF/TA scores of eachcorresponding renal biopsy. A total of 34 antibodies and 41antibodies demonstrated a positive correlation with CADIscore (overall P,0.04) and IF/TA (overall P,0.04), respec-tively. The top 20 antibodies are listed in Table 3. Pathwayanalysis suggested that these antigens are involved in cellularmovement, antigen presentation, cell-to-cell signaling and in-teraction, and cell death (P,0.002). Scatter plots for the sixmost significant antibodies (IFNG [IFN-g], MIG, CSNK2A2,CCL21, glial cell-derived neurotrophic factor [GDNF], and
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ITAC) and their correlation with the CADI score are presentedin Figure 3.
Predicting Injury Progression after TransplantationAs shown by our group in a previous publication,15 chronicrenal injury and end stage renal failure result in uncovering ofkidney-specific epitopes, mostly in the renal cortex and therenal pelvis, and the immunologic recognition of these newlydiscovered non-HLA antigens with humoral responses in theform of non-HLA antibodies, specific to chronic renal injury.To evaluate if this repertoire of preformed non-HLA antibod-ies specific to chronic renal failure could predict the subse-quent development of CAI after kidney transplantation, thelevel of these non-HLA antibodies was evaluated at the baseline(day 0 sera) of each patient. Spearman correlation analysis wasperformed to identify the specificities or levels with the pro-gression of CAI on the biopsy performed at 6 months and 24months after transplantation. Interestingly, baseline levels ofthese preformed antibodies to MIG, ITAC, IFNG, GABPA, andGDNF positively correlated (P,0.05) with CADI score at6 months, and baseline levels of four of these five antibodies(MIG, ITAC, IFNG, GDNF), as well as two additional antibod-ies to IL-8 and CCL21, all positively correlated (P=0.04) withCADI score at 24 months (Table 4). As expected for this selec-tion of antibodies, antibody levels at 6 months post-transplantfor IL-8,MIG, IL21, CCL19, LRRK2, CCL21, GDNF, and IFNGalso correlated with CAI at 24 months post-transplantation,suggesting that these antibody levels could also be tracked forprogression of CAI in both the pretransplant period
(preformed) and the post-transplant period(de novo) (Figure 4, A–D) (Table 4).
Univariate and multivariate logistic re-gressions were performed to examine therelationship between the baseline signalintensities of each of the preformed fourantibodies (MIG, ITAC, GDNF, and IFNG)andCADI scores from the protocol biopsiesperformed in these samepatients at 6 and24months post-transplantation. A CADIscore of $3 was used as a cutoff to defineeither severe ($3 CADI score) or mild (,3CADI score) histologic CAI. The receiveroperating characteristic (ROC) curves foreach of these antibodies to predict the de-velopment of CAI are shown for the6-month protocol biopsy (Figure 4E) andthe 24-month protocol biopsy (Figure 4F).When a regression model was built by uni-variate analysis, detection of baseline levelsof preformed antibody to MIG at levels.200 RFU had a significant associationwith the patient developing CAI on theprotocol biopsy at both 6 months (oddsratio, 1.04; 95% confidence limit, 1.003–1.078; P=0.034), and at 24 months post-
transplantation (odds ratio, 1.023; 95% confidence limit,1.001–1.045; P=0.0375). Patients who never developed CAIhad baseline antibody levels to MIG of ,50 RFU.
Validation of Selected Potential Antibody BiomarkersELISAs were developed and optimized to demonstrate thatdiscovery of target non-HLA antibodies by protoarray could bevalidated by ELISA. ELISAs were set up, customized, andperformed for some selected (and significant) targets in whichthe full-length proteins could be commercially obtained fromstock supplies for setting up the reverse ELISAs for antibodymeasurements. These assays were done for MIG, ITAC,CSNK2A2, and PDGFRA. A significant increase in the antibodylevelswere confirmed inCAIby reverse ELISA for all four targets:MIG (P,0.02), ITAC (P,0.014), CSNK2A2 (P,0.0002), andPDGFRA (P,0.0001) (Figure 5, A–D). For further validation,seven independent patients were selected, whowere not used fordiscovery by protoarrays, in whom serial sera andmatched pro-tocol biopsies were available and who had a CAI grade of at least.3 on both the 6-month and the 24-month protocol biopsies.Three of these targets were confirmed by our customized reverseELISA to also show significant increases in this longitudinalanalysis. Antibody levels for patients with CAI at 24 monthswere significantly higher in 24-month sera versus 6-monthsera for CSNK2A2 (P=0.05), ITAC (P=0.05), and MIG(P=0.005) (Figure 5, E and F). In addition, there was a strongcorrelation of two of these antibody levels with CADI scores onthe 24-month protocol biopsies (MIG: r=0.62, P=0.05; ITAC:r=0.62, P=0.05).
Figure 1. Study schematic. A novel approach is used to identify novel non-HLAantibodies through an integrative approach of analyzing sera samples with matchedbiopsies on protein microarray for the discovery step. The validation step is per-formed after filtering the data for highly correlated antibodies for renal graft injury.The highly correlated antibodies are then validated by indirect ELISAs on an in-dependent set of patients for cross-sectional and longitudinal analyses. nCAI, non-CAI; STA, stable.
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DISCUSSION
CAI is related to a humoral response driven by alloantibodiesagainst HLA antigens, with recent data also supporting a rolefor immunogenic non-HLA renal antigens. An understandingof the specificities and correlative levels of these antibodies canprovide an understanding of molecular injury triggers in CAIand ameans to noninvasively monitor the development of thisinjury. Despite the contribution of newly available immuno-suppressive drugs in the short-term survival of the trans-planted kidney,3 CAI progresses relentlessly post-transplant,accelerating the risk for graft loss. There is a lack of reliableand robust noninvasive biomarkers to track the health status ofthe transplanted organ, without the requirement of serial pro-tocol biopsies. Elaborating injury mechanisms and developingpredictive, noninvasive monitoring methods for CAI is an im-portant unmet clinical need.2,23 The process of biologic tissueinjury from events such as exposure to calcineurin inhibitoragents, from graft ischemia, or from recipient hypertension,likely results in the alteration of the levels, exposure, or
targeting for various proteins/antigens in the renal allograft.These altered antigens mount specific antibody responses.The ability to recognize these relevant antigens, to measurethese specific antibody levels in the circulation, and to correlatethese levels with histologic CAI in the allograft itself is a power-ful approach to identify noninvasive, clinically relevant sero-logical biomarkers for detecting and predicting CAI. Our initialstudies strongly suggested a pathogenic role for non-HLA anti-bodies after transplantation.11,16,18 From gene expression anal-yses, we reported an increased level of Ig gene transcripts as afunction graft injury.24We thus hypothesized that accumulatedorgan injury in the form of CAI could be associated with aspecific set of circulating non-HLA antibodies that can be de-tected, quantified, and used to follow chronic graft injury.
We undertook a carefully designed study of highly selectedpatients with established histologic CAI, with available serialsera and matched protocol biopsies, and with no confoundinginfluences of delayed graft function, acute rejection, or in-fection on the evolution of chronic injury. Unbiased discoveryof a panel of correlative non-HLA antibodies with CAI was
Figure 2. A cross-sectional analysis of sera samples taken from patients with matched biopsies showing either the presence (n=20) orabsence of CAI (n=20). (A) A volcano plot demonstrates a significant shift in antibody responses CAI. A total of 111 antibodies (Ab) aresignificantly increased (in red dots) in sera collected from renal transplant patients with CAI (n=20) compared with patients without CAI(n=20) (P#0.05), and 40 antibodies are increased in CAI (P#0.01). (B) The dynamics of change of antibody levels of most significantantibodies over the period of 24 months are shown. Ab, antibody.
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evaluated from pretransplant and post-transplant sera in thesepatients. Selection of antibody targets was biased for kidneyexpressed antigens, and customized reverse ELISAs were gen-erated to validate the findings in independent sera samplesfrom an independent group of patients with CAI. These resultsdemonstrated, verified, and validated a small panel of newlyidentified non-HLA antibodies that correlate not only withhistologic CAI in thematched sera sample, but that also havecorrelative levels post-transplantation with progressive
CAI and, most importantly, can predict the future de-velopment of CAI by pretransplant sampling for the sameantibody panel.
Irrespective of the initial trigger, it is believed that the path-ophysiology of CAI involves endothelial injury leading to anincreased expression of cytokines and several other immune-related genes, resulting in proliferative processes, remodeling,and scarring of the graft. The increased IgG antibody level ofcytokines IFNG,MIG, and ITAC strengthens the belief that the
Table 1. Early and late responding non-HLA antibodies in CAI
Serial No. Gene Symbol Protein Name P Value
Early response (,6 mo post-transplant)1 CSNK2A2 Casein kinase 2, a2 0.012 CSNK2A1 Casein kinase 2, a1 0.023 KLK6 Kallikrein-related peptidase 6 0.014 PPID Peptidylprolyl isomerase D 0.005 CCDC55 Coiled-coil domain containing 55 0.036 NEK3 NIMA (never in mitosis gene a)–related kinase 3 0.027 CSNK1G3 Casein kinase 1, g3 0.0028 CCL21/6CKINE Chemokine (C-C motif) ligand 21 0.009 BIN1 Bridging integrator 1 0.05
10 MAPRE2 Microtubule-associated protein, RP/EB family, member 2 0.0011 SGK2 Serum/glucocorticoid regulated kinase 2, transcript variant 1 0.0313 GYG2 Glycogenin 2 0.0114 MEF2D Myocyte enhancer factor 2D 0.000615 EGFR L861Q EGF receptor (erythroblastic leukemia viral [v-erb-b]
oncogene homolog, avian), transcript variant 10.002
16 EGFR EGF receptor (erythroblastic leukemiaviral [v-erb-b] oncogene homolog, avian);see catalog number for detailed informationon wild-type or point mutant status
0.00
17 Jo-1/HARS Histidyl-rRNA synthetase (Jo-1) 0.01Late response (.6 mo post-transplant)18 MIG Chemokine (C-X-C motif) ligand 919 GDNF Glial cell-derived neurotrophic factor 0.000120 CSNK1G1 Casein kinase 1, g1 0.0521 BHMT2 Betaine-homocysteine methyltransferase 2 0.0022 PKN1 Serine/threonine-protein kinase N1 0.0023 ATXN3 Ataxin-3 0.0124 MARK4 MAP/microtubule affinity-regulating kinase 4 0.02
Table 2. Compartment-specific enrichment of CAI-specific antibodies cross-mapping of kidney and kidneycompartment–specific genes in between cDNA microarraya
Kidney/Compartment Kidney-Specific Targets CAI Specific (20 np versus 20 P) (P,0.05) P Value
Kidney 261 3 (ACY1, BIN1, CSNK2A1) 0.10Glomerulus 245 7 (FLT1, FLT4, MIG, PDGFRB, PRKCE, KLK6, BHMT2) 0.11Inner cortex 267 3 (ACY1, BHMT2, CLK4) 0.03Outer cortex 433 8 (ACY1, PRKCE, PRKCZ, KLK6, VDR,
SNF1LK2, BHMT2, CLK4)0.13
Outer medulla 42 0 0.41Inner medulla 11 0 0.79Papillary tip 222 4 (PDGFRA, PRKCB1, RARB, AFAP1l2)Pelvis 635 6 (IFNG, JAK3, CCL19, CCL21, WEE1, NDE1) 0.001
aThe cross-mapping was performed using previously published data and methods (18,21,22).
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transplanted kidney is under continuous immunologic attack.Similar results were also shown by gene expression analysis onCAI protocol biopsies from the samepatient group.24 IFNG is atype II IFN that is encoded by the IFNG gene25 and is exten-sively produced by a wide range of immune-related cells suchas CD4+ T helper cell type 1 lymphocytes, CD8+ cytotoxiclymphocytes, natural killer cells, B cells, natural killer T cells,and antigen presenting cells.26–30 IFN-g gene polymorphism+874 T/A (rs2430561) has been suggested to be associatedwith AR31,32 and activated T lymphocytes in alloimmune in-jury are a major source of IFNG expression, regulated by IL-12and IL-18.29,33 IFNG is critical in trafficking of specificimmune cells to sites of inflammation by upregulating ex-pression of adhesion molecules and chemokines.34 In thiscontext, our observation of a significant increase in the anti-body level againstMIG and ITAC is important. BothMIG andITAC are chemokine proteins that also serve as chemoattrac-tants for leukocytes, monocytes, neutrophils, and other
mononuclear cells to the site of tissue damage.35 Upregula-tion of these chemokine proteins has been demonstrated tobe correlated with T lymphocyte recruitment during acuteand chronic rejection events. In addition, pretransplant se-rum levels of MIG and CXCL10 have been reported to beassociated with graft failure,36,37 and recently our group hasreported that there are high levels of circulating MIG proteinin the serum of kidney and heart transplant patients at thetime of acute rejection.38
We also observed an increase in the IgG antibodies of anumber of novel antigens that have not been previously reportedto be relevant in organ transplantation. Some of these continueto support the activation of the immune axis as a critical triggerfor CAI, such as antibodies to the Jo-1 antigen, also known ashistidyl-tRNA synthetase, and are responsible for the synthesis ofhistidyl-transfer RNAand themigration of activatedmonocytes,immature dendritic cells, and activated lymphocytes.39 An in-teresting antibody in CAI is the GDNF, which is involved in
Figure 3. CAI-specific antibodies correlate with the severity of CAI at the time of sampling. (Upper panel) IgG antibody level of sixantigens: (A) IFNG, (B) MIG (CXCL9), (C) CSNK2A2, (D) CCL21, (E) GDNF, and (F) ITAC (CXCL11) are shown to be correlated with chronicinjury in terms of CADI score. (Lower panel) Evidence of the presence of four of the corresponding antigens of CAI-specific antibodies:(A) IFNG, (B) ITAC (CXCL11), (C) GDNF, and (D) PDGFRA.
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kidney development. Signaling by the secreted protein GDNFthrough the RETreceptor tyrosine kinase and the GDNF familyreceptor a1, a GDNF co-receptor, are involved in kidney devel-opment,40 specifically in metanephros41 and ureteric bud devel-opment.42 GDNF antibodies are increased in CAI and correlatewithhistologicCAI (Figure3), andbaseline/pretransplant antibodylevels are associated with a greater risk of CAI post-transplantation(Figure 4).
In conclusion, we identified and validated a panel of cir-culating non-HLA antibodies in renal transplant patients thatcorrelate with established CAI, can be serially measured post-transplant to identify which patients will develop acceleratedCAI over time, and, most importantly, to stratify risk for de-velopment of CAI by measuring this antibody panel even be-fore organ engraftment. The repertoire of the panel suggestsimmunologic “preconditioning” of the recipient as an impor-tant risk factor for CAI progression after engraftment becausethis panel of antibodies is enriched against cell-to-cell signal-ing and interaction as well as cell-mediated immune response,antigen presentation, and cell death (P,0.02). The alteredexpression of these factors may predispose the recipient tocontinued alloimmune injury, which then drives the turnoverof chronic interstitial injury, fibrosis, and TA, with loss of graftfunction over time. Importantly, very similar results are notedby microarray profiling of the very same biopsy samples, fromthe same patient cohort, in which very coordinated expressionof immune-specific genes in the allograft in the early protocolbiopsy can predict the progression of CAI in the later protocolbiopsy sample.24 It is intriguing that the reactive cytokinessuch as MIG, ITAC, and IFNG observed in this study are
also associated with AR in our previous reports.38,43 This ob-servation suggests a common activation of cytokines not onlyas immediate trigger of inflammation and injury in AR butalso as activated cytokines, in the case of CAI (Figure 6). Thefuture direction of this work requires a large-scale validationof these data in a prospective trial to assess the specificity andsensitivity of this antibody panel before transplant to assess therisk of CAI, and perhaps to subsequently titrate the immuno-suppression induction and maintenance strategy for eachtransplant patient on the basis of the risk of post-transplantCAI.
CONCISE METHODS
Patients and SamplesWe analyzed serological response on serum samples that were
collected from renal transplant patients from Lucile Packard Child-
ren’s Hospital, Stanford University, Stanford, California. After exclu-
sion of patients with incidences of acute rejection (antibody mediated
or cellular), infection, delayed graft function, donor pathology, and
C4d-positive peritubular capillary staining on biopsies, 172 sera sam-
ples from98patientswere selected and split into training and verification
groups with a one-third (n=60 sera samples for discovery) to two-thirds
split (n=112 sera samples for validation). The transplant patients in-
cluded in the study were unsensitized patients, with a peak plasma renin
activity,20% and mean recipient and donor HLA match of 3.561.29.
The demographic information of the transplant patients in the learning
and the verification groups is summarized in Table 5. For the discovery
group, a total of 60 sera samples were used. We selected a total of 30
Table 3. CAI-specific antibodies correlate with CADI score and IF-TA scores
Serial No. Gene Symbol Protein Name CADI Score (r, P) IF-TA Score (r, P)
1 IFNG IFN-g 0.68, ,0.0001 0.61, ,0.00012 MIG C-X-C motif chemokine 9 0.61, ,0.0001 0.55, 0.00023 ITAC C-X-C motif chemokine 11 0.51, 0.0009 0.42, 0.0074 CSNK2A2 Casein kinase 2, a prime polypeptide 0.51, 0.0008 0.52, 0.00065 GDNF Glial-derived neurotrophic factor 0.63, ,0.0001 0.58, ,0.00016 BHMT2 Betaine-homocysteine methyltransferase 2 0.47, 0.002 0.54, 0.00037 6CKINE C-C motif chemokine 21 0.56, 0.0002 0.54, 0.00038 CSNK2A1 Casein kinase 2, a1 polypeptide,
transcript variant 20.50, 0.001 0.54, 0.0004
9 J0-1(HARS) Histidyl-rRNA synthetase 0.63, ,0.0001 0.63, ,0.000110 CSNK1G1 Casein kinase 1, g1 0.49, 0.001 0.51, 0.000811 IL21 IL-21 0.57, 0.0001 0.51, 0.000812 CSNK1G3 Casein kinase 1, g3 0.35, 0.026 0.43, 0.00613 IL-8 IL-8 0.43, 0.006 0.48, 0.00214 PRKCE Protein kinase C, « 0.41, 0.009 0.48, 0.00215 FLJ21908 RNA polymerase II-associated protein 3 0.48, 0.002 0.47, 0.00216 WIBG Within bgcn homolog (Drosophila) 0.39, 0.01 0.46, 0.00317 ATXN3 Ataxin-3 0.46, 0.003 0.45, 0.00318 RNAPOL RNA polymerase 0.39, 0.01 0.45, 0.00419 MAPRE2 Microtubule-associated protein,
RP/EB family, member 20.34, 0.03 0.45, 0.004
20 CCL19 chemokine (C-C motif) ligand 19 0.40, 0.009 0.43, 0.006
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sera collected at time 0, 6, and 24 months post-transplantation from
10 patients with confirmed CAI by 24 months post-transplantation.
The quality of the graft at the time of transplantation was excellent in
this selected group of patients, with amean Remuzzi score of 0.560.76
at the time of transplantation and a median score of 0. In the overall
group, the mean CADI score was used as a semi-quantitative measure
of the chronic injury grade.44,45 For analytical purposes, an arbitrary
cutoff CADI score,2 was used to score nonsig-
nificant histologic injury progression; a CADI
score $5.0 was used to score significant histo-
logic injury progression. These CAI samples
were compared with 30 sera samples from 10 de-
mographically matched transplant recipients with
stable graft function, with histologically clean pro-
tocol biopsies and minimal to no injury in each of
the 6- and 24-month protocol biopsies. For vali-
dation of discovered antibodies by cross-sectional
analysis, we identified sera samples collected from
demographically matched 31 renal transplant pa-
tients with biopsy CAI and 30 without CAI. The
details are summarized in Table 6. This study was
approved by the Institutional Review Board of
Stanford University and the other participating
centers of the clinical trial.
All 172 study biopsies were blindly analyzed
by a Stanford University pathologist and were
graded by the Banff classification5,46,47 for acute
rejection, and intragraft C4d stains were per-
formed48,49 to assess for acute humoral rejec-
tion.50,51 The histologic lesions of CAI were
extensively identified and a semi-quantitative
score for CAI applied to each biopsy, based on
standardized definitions from the Banff (2),
CADI, (3), and chronic calcineurin inhibitor
toxicity (19) scores. For the semi-quantitative
scoring criteria, chronic lesions are preceded by
“c”; interstitial fibrosis is denoted as “ci” and
scored as ci0–ci3; TA is denoted as “ct” and
scored as ct0–ct3 based on the area of cortical
tubular atrophy; glomerulopathy is denoted as
“cg: and scored as cg0–cg3 based on the extent
of double contours in glomerular capillary loops;
and arteriolar hyalinosis is denoted as “ah”
and scored as ah0–ah3 based on the extent of
PAS-positive hyalinosis. On the basis of the com-
bination of these features, a diagnosis of CAIwas
graded on the basis of severity as grade I (6%–
25% of cortex), grade II (26%–50% of cortex),
and grade III (.50% of cortex). If the biopsy of
the patient showed a CAI score,6% at the time
of serum collection, the sample was considered
as stable. Samples with biopsy scores for CAI
between 6% and 25%were deliberately excluded
to allow for clean separation of CAI samples in
this discovery process.
Serum Sample Collection and StorageBlood samples (4.5 ml) were collected in a 5-ml cryotube and incu-
bated at roomtemperature for30minutes until the clotwas formed.The
sample was then centrifuged at 20003g for 5 minutes using a swinging
bucket rotor. The serum was transferred to another cryotube and was
stored at 280°C until use.
Figure 4. CAI-specific antibodies at the time of transplant correlate with the injuryprogression post-transplant. IgG antibody level of two MIG antibodies at the time oftransplantation is positively correlated with CADI score post-transplantation at (A) 6 and(B) 24 months. IgG antibody level of two ITAC antibodies at the time of transplantationis positively correlated with CADI score post-transplantation at (A) 6 and (B) 24 months.ROC curves for the MIG, GDNF, ITAC, and IFNG antibodies predict development ofCAI. The predictive ability of baseline antibody level to predict injury at (E) 6 months (F)and (B) 24 months post-transplantation is shown in terms of ROC curves and area underthe curve for different antibodies as well as their combined contribution in the pre-diction. Ab, antibody.
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Immune Response Biomarker Profiling by ProteinMicroarraysWe used the ProtoArray Human Protein Microarray (v4.0 and v4.1;
Invitrogen,Carlsbad,CA) for this study. The slides contained a total of
approximately 8200 antigens printed on each slide. The proteins
included in the arrays belong to different cellular locationswith awide
variety of functions. For the detection of immune response, we used
manufacturer’s protocol with slight changes. Briefly, the microarray
slides were blocked with 5.0 ml of blocking buffer (100 mM sodium
phosphate pH 7.4, 200 mMNaCl, 0.08% Triton X-100, 25% glycerol,
20 mM reduced glutathione, 1.0 mM DTT, 1% Hammarsten-grade
casein) with gentle agitation in four-well trays for 1 hour at 4°C. After
the blocking step, the blocking buffer was removed by aspiration and
5.0ml diluted serum (1:150) in PBSTbuffer (13PBS, 1%Hammarsten-
grade casein, 0.1% Tween 20) was added onto the plate to incubate for
90 minutes with gentle agitation at 4°C. In the next step, the serum
sample was removed and the plates were washed five times with 5.0
ml of fresh PBST buffer with 5-minute incubations per wash with
gentle agitation. A 5.0-ml portion of secondary antibody (Alexa
Fluor 647 conjugate anti-human antibody) diluted in PBST buffer
was added and the plates were incubated for 90 minutes with gentle
agitation at 4°C. The secondary antibody solution was removed by
aspiration. The plates were then washed five times with 5.0 ml of
fresh PBST buffer, with 5-minute incubations per wash with gentle
agitation. At the end, the slides were dried by centrifugation and
scanned using an Axon GenePix 4000B scanner (Molecular Devices,
Sunnyvale, CA). The data were acquired by using GenePix Pro 6.0
microarray analysis software (Molecular Devices). The slides were
scanned at 635 nm with a photomultiplier gain of 600, a laser power
of 100%, and a focus point of 0 mm. The .gal files were obtained
from a ProtoArray central portal on the Invitrogen website (www.
invitrogen.com/ProtoArray) by submitting the barcode of each pro-
tein microarray.
Statistical AnalysesSignal intensities per spot for each individual array were obtained
using GenePix Pro 6.0 software. We used Prospector Analyzer 5.2
(Invitrogen Inc) to subtract the background and to normalize and to
analyze the GenePix results files for each array. We used a standard
cutoff Z score of 3.0. Individual antigen reactivity was ranked based
on Z score above or below the mean signal for each array. Arrays from
patients of a distinct clinical phenotype were analyzed as a group.
Group analyses (.5 per group as suggested by the manufacturer)
were made by comparing two sets of individual antibody level for
every antigen present on the array usingM-statistics of the Prospector
Analyzer with the robust linear normalization method.20 Differences
in significance were displayed as P values, with #0.05 considered
significant. We performed two-factor ANOVA on the protein array
data using the CADI score and sample time as factors. The ANOVA P
values were corrected for multiple hypotheses using the Benjamini–
Hochberg correction. We found 72 antibodies that were significantly
affected by CADI score but not by time at false discovery rate ,0.2
Table 4. List of antibodies the level (from protein arrays) of which at the time of transplant and 6 months post-transplantcorrelates with CAI progression in the future
Gene Symbol Antigen Name Spearman Correlation (r)P
Value
Antibody level measured at the time of transplantation correlates with 6-mo chronic injuryGABPA GA binding protein transcription
factor, a subunit0.46 4.31E202
MIG Chemokine (C-X-C motif) ligand 9 0.74 2.00E204ITAC Chemokine (C-X-C motif) ligand 11 0.49 2.90E202IFNG IFN-g 0.60 5.50E203GDNF Glial cell-derived neurotrophic
factor0.55 1.12E202
Antibody level measured at the time of transplantation correlates with 24-mo chronic injuryIL-8 IL-8 0.46 3.98E202CCL21 Chemokine (C-C motif) ligand 21 0.49 2.94E202MIG Chemokine (C-X-C motif) ligand 0.71 5.00E204ITAC Chemokine (C-X-C motif) ligand 11 0.54 1.36E202IFNG IFN-g 0.73 3.00E204GDNF Glial cell-derived neurotrophic factor 0.62 3.20E203
Antibody level measured at 6 mo post-transplantation correlates with 24-mo chronic injuryIL-8 IL-8 0.47 3.87E202MIG Chemokine (C-X-C motif) ligand 9 0.46 4.25E202IL21 IL-21 0.51 2.16E202CCL19 Chemokine (C-C motif) ligand 19 0.48 3.30E202LRRK2 Leucine-rich repeat kinase 0.46 4.25E202CCL21 Chemokine (C-C motif) ligand 21 0.47 3.77E202GDNF Glial cell-derived neurotrophic factor 0.60 5.30E203IFNG IFN-g 0.59 6.30E203
758 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 750–763, 2012
CLINICAL RESEARCH www.jasn.org
for CADI score, of which 53 were increased in the CAI group. Hyper-
geometric enrichment, univariate and multivariate analyses, and logis-
tic regression modeling were performed using customized algorithms.
Ingenuity software (version 7.5; Ingenuity Systems Inc, RedwoodCity,
CA) was used to map target antigens to canonical pathways within the
Ingenuity Knowledge Base. Tissue expression of antigens against sig-
nificant antibodies correlated with CAI was examined using the hu-
man protein atlas.52–54
Figure 5. CAI-specific antibodies are validated with immunoassays using indirect ELISA. Levels of four of the CAI-specific antibodies areassayed in CAI sera and their increased presence in biopsy-proven CAI (n=31) is analyzed by comparing their level in renal transplantpatients with biopsy-proven stable graft function (n=30). Levels of (A) CSNK2A2 (P,0.002), (B) MIG (P,0.02), (C) ITAC (P,0.014), and(D) PDGFRA (P,0.0001) are increased. The increased level of antibody level of (E) CSNK2A2 and (F) MIG, along with increased CAI, isobserved from ELISA on independent samples (n=17). Increases in antibody level measured by ELISA, as well as CADI score for boththe antibodies are statistically significant (P,0.05). STA, stable.
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Cross-Mapping of Gene IDs of the Compartment-Specific Gene on cDNA Microarray PlatformCross-mapping of kidney and kidney compartment-specific genes in
between cDNAmicroarray was performed using the data published21,22
and the ProteinMicroarray V.1 was conducted using the method pub-
lished earlier by our group18 using AILUN software (http://ailun.
stanford.edu/) to re-annotate probes to the most recent NCBI Entrez
gene identifiers.
Development and Optimization of ELISA Assay forValidation of CAI-Based Non-HLA AntibodyFor validation purposes, we developed antibody ELISA to detect serum
Ig binding to MIG (also called CXCL9), ITAC (also called CXCL11),
CSNK2A2,andPDGFRAfollowingthemethodpublishedpreviously.11,15
In brief, purified proteins, CSNK2A2 (cat# PV3624), PDGFRA (cat#
PV3811), ITAC (cat# PHC1694), and MIG (cat# OHC1604) were ac-
quired from Invitrogen (Carlsbad, CA). A titration with various coated
amounts starting at 30.00, 15.00, 7.5, 3.75, 1.87, 0.94, 0.47, and 0 ng,
respectively, was performed to determine the optimal amount to be
coated onto the immunosorbent 96-well plate. The 96-well microwell
ELISAplateswere coatedwith corresponding protein in 50ml of coating
buffer (15 mM Na2CO3, 30 mM NaHCO3, 0.02% NaN3, pH 9.6). The
subsequent washing and antibody incubation followed themethod pre-
viously published.11,15 The color was developed by using AP-pNPP
Liquid Substrate System for ELISA (Sigma-Aldrich, St. Louis, MO).
Absorptionwas measured at 405 nmwith a SpectraMax 190microplate
reader (Molecular Devices). To control for nonspecific binding, wells
with no proteins coated were served as negative controls.
Validation of CAI-Specific Antibody by ELISAAfter the optimization step, we validated the discovery made by the
protein arrays on 112 sera collected from 78 renal transplant patients.
There were 61 cross-sectional sera samples collected at 1 year post-
transplantation from 31 unique patients with biopsy-confirmed CAI
and 30 unique patients with stable biopsies (non-CAI). In addition,
there were 51 serial (longitudinal) samples from 17 patients with CAI
confirmed on their 6-month and 24-month biopsies, with sera
samples at 0, 6, and 24 months post-transplantation.
Figure 6. A developing picture of involvement of non-HLA antibodies against cytokines and other novel antigens in CAI. Abs, anti-bodies.
Table 5. Demographic data
Characteristic
Discovery Set (60 Sera) Validation Set (112 Sera)
Protein Array Analysis Cross-Sectional ELISALongitudinal ELISA
Non-CAI CAI P Value Non-CAI CAI P Value
Number of patients 10 10 30 31 17Steroid free/steroidbased
4/6 6/4 0.65 12/18 12/19 1.00 6/11
Donor sex (M/F) 8/2 4/6 0.17 18/12 22/9 0.13 9/8Donor age (yr) 24610
(21; 14–47)35.0610(36; 17–54)
0.03 30610(31; 14–47)
30610(31; 14–54)
0.97 29610
Recipient sex (M/F) 5/5 5/5 1.00 13/17 10/21 0.43 10/7Recipient age (yr) 1464
(13; 8–19)1066
(9; 2–19)0.17 1464
(15; 3–19)1265(12; 2–20)
0.08 1066
Living/deceased 4/6 6/4 0.66 14/16 17/14 0.61 7/10
760 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 750–763, 2012
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We coated 15.6 ng of the purified proteins (MIG, ITAC, CSNK2A2,
andPDGFRA)ontoan immunosorbent96-wellplate(NUNCbrandcat#
446612). We followed a previously published protocol developed by our
laboratory.11,15 Briefly, the 96-well microwell ELISA plates were coated
with corresponding protein in 50ml of coating buffer (15 mMNa2CO3,
30mMNaHCO3, 0.02%NaN3, pH 9.6) and incubated overnight at 4°C.
Standard curves were generated using anti-GST tag (mousemonoclonal
IgG) (Millipore, Temecula, CA) andAP-conjugatedAffiniPure goat anti-
mouse IgG (Jackson ImmunoResearch, West Grove, PA). After washing
the plates with TBST buffer five times, the nonspecific protein binding
was blocked by 100ml of 5%drymilk in TBST buffer for 1 hour at room
temperature. After the blocking step, 50-ml serum samples (40-fold di-
luted with 2% milk in TBST buffer) were incubated in the wells for 1
hour at room temperature. The plates were washed five timeswith TBST
buffer and incubated in 50 ml of AP-conjugated AffiniPure mouse anti-
human IgG (Jackson ImmunoResearch). The color was developed by
using AP-pNPP Liquid Substrate System for ELISA (Sigma-Aldrich).
Absorption was measured at 405 nm with a SpectraMax 190
microplate reader (Molecular Devices). Statistical calculation (t test
and Spearman correlation) was performed using GraphPad Prism
software. P,0.05 was considered significant.
ACKNOWLEDGMENTS
We thankDr.MatthewVitalone for critically reading thismanuscript.
We appreciate the support from the Sarwal Laboratorymembers during
the study period and the patients and their families who participated in
this research study. We also thank the Stanford Functional Genomics
Facility at Stanford University for providing scanning facilities for the
protein arrays.
DISCLOSURESNone.
Table 6. Histologic data for the discovery set for protein array analysis
Non-CAI CAI P Value
Histology at implantation (n=10)IF/TA grade .0 0/10 1/10 1.00arteriolar hyalinosis (present/absent) 1/10 1/10 1.00intimal vascular thickening(present/absent)
1/10 0/10 1.00
glomerulosclerosis (present/absent) 1/10 2/10 1.00Remuzzi score 0.260.42 (0; 0–1) 0.860.92 (0.5; 0–2) 0.08
Histology at 6 months (n=10)tubulitis score .0 0/10 0/10 1.00interstitial inflammation score .0 0/10 1/10 1.00vasculitis score .0 0/10 0/10 1.00IF/TA grade .1 0/10 2/10 0.47arteriolar hyalinosis (present/absent) 0/10 0/10 1.00intimal vascular thickening(present/absent)
1/10 4/10 0.30
glomerulosclerosis (present/absent) 0/10 2/10 0.47CADI score 0.660.70 (10.5;0–2) 3.1761.65 (3.5; 1–6) ,0.0001
Histology at 24 months (n=10)tubulitis score .0 0/10 0/10 1.00interstitial inflammation score .0 0/10 0/10 1.00vasculitis score .0 0/10 0/10 1.00IF/TA grade .1 1/10 9/10 0.001arteriolar hyalinosis (present/absent) 0/10 1/10 1.00intimal vascular thickening(present/absent)
3/10 5/10 0.65
glomerulosclerosis (present/absent) 0/10 6/10 0.02CADI score 1.661.6 (1.5; 0–5) 7.161.5 (7;4–9) ,0.0001CADI score slope in first 2 years 0.760.9 (0.6; 20.3 to 2.5) 2.960.6 (3.1; 1.9–3.8) ,0.0001
Schwartz GFRat 6 months 99.2629.5 (93.6; 52–154) 117.0630.5 (102.9; 69.12–164.8) 0.42at 24 months 93.9630.2 (98.6; 62–121) 110.3619.5 (100.1; 97.15–144) 0.38
Absolute GFRat 6 months 73.7621.9 (70.6; 41–109) 66.0613.7 (67.2; 39–81) 0.36at 24 months 78.3649.1 (102; 22–111) 71.7629.9 (87.8; 34–101) 0.82
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This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2011060596/-/DCSupplemental.
J Am Soc Nephrol 23: 750–763, 2012 Antibodies for CAI 763
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