Differential Immunogenicity and Clinical Relevance of Kidney

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Differential Immunogenicity and Clinical Relevance of Kidney Compartment Specific Antigens after Renal Transplantation Li Li, Tara Sigdel, Matthew Vitalone, Sang Ho Lee, and Minnie Sarwal* Department of Pediatrics, Stanford University, 300 Pasteur Drive, Stanford, California 94304, United States Received August 24, 2010

To evaluate the pathogenic role of non-HLA antibodies after organ transplantation, 81 unique serum samples from renal transplant patients were analyzed by protein array technology on integrative genomics approach (Li, L.; et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (11), 4148-53; Higgins, J. P.; et al. Mol. Biol. Cell 2004, 15 (2), 649-56), validated by ELISA, and the results correlated with clinical relevance with time post-transplantation or post-transplant graft function. There was a significant association of de novo non-HLA antibodies with time post-transplantation (n ) 1,785) and decline in graft function over the subsequent year (n ) 105). There was an enrichment of immunogenic antigens in the renal cortex (p ) 0.01) with post-transplant time, and for glomerular specific targets (p ) 0.02) with decline in graft function. Two targets with very strong correlation in each category (AGT and SPDYA) were validated by customized ELISA assays in independent patient sera and their localization confirmed by immunohistochemistry. In conclusion, defined profiles of these non-HLA antibodies to renal cortical proteins develop with increasing length of engraftment, and may reflect the increasing recognition of altered localization or exposure of renal tubular and interstitial proteins, affected by advancing chronic nonimmune graft injury. The panel of non-HLA antibodies to glomerular targets is most interesting, as these corresponding antigenic targets may be important pathways in functional graft injury and could provide novel targets for drug design. Keywords: Non-HLA antibodies • Antibody response • Declined graft function • Kidney transplantation

Introduction One of the main clinical barriers currently facing human renal transplantation is overcoming chronic allograft injury (CAI) and thus to improve the long-term outcomes for these allografts is an ultimate goal. This is despite the steady improvements in immunosuppression and acute allograft injury.1 Paramount to overcoming this obstacle is understanding the various different influences of this CAI, which has been described to be multifactorial with origins in donor characteristics, immunosuppression toxicity, and immune and nonimmune mechanisms.2–4 There has been a great deal of evidence showing that antibodies are playing a significant role in the induction and severity of allograft injury5 and are associated with allograft injury and decreased graft survival, irrespective of origin, be it preformed or de novo.6–8 Preformed antibodies (antibodies formed prior to transplantation) may originate from many sources that include pregnancy, blood transfusion, and previous allotransplantation, with de novo antibodies presenting at varying rates and time throughout the life of the allograft.6,7 The major pathogenic antibodies associated with graft dysfunction and CAI are those recognizing HLA (human * To whom correspondence should be addressed. Minnie Sarwal, M.D., FRCP, Ph.D., Department of Pediatrics, G320, 300 Pasteur Drive, Stanford, CA 94304 USA. E-mail: [email protected]. Phone: (650) 724-3320. Fax: (650) 498-6714. 10.1021/pr1008674

 2010 American Chemical Society

leukocyte antigens) of the major histocompatibility complexes. Nevertheless, even HLA-identical transplants can experience CAI and patients can experience both acute and chronic allograft injury in the absence of detectable HLA-antibodies. More recently, “minor” non-HLA antigens have been implicated in renal allograft outcome.9,10,11 Published studies have shown an association between chronic or acute allograft injury and non-HLA-antibodies directed toward Duffy and Kidd polymorphic blood group antigens,12 Agrin (heparin sulfate proteoglycan),13 Angiotensin II type 1 receptor (AT1R-AA),14 vimentin,15 protein kinase C zeta (PRKCZ) antigen,10 STMN3 (stathmin-like 3) and ARHGEF6 (Rac/Cdc42 guanine nucleotide exchange factor 6).11 The true scope of the antigenic load (either within or circulating through the allograft) that may be pathogenically relevant to clinical outcomes is relatively unknown, as theoretically, any peptide or molecule secreted, shed, or presented to the host is a potential antigen for antibody production. However, without exact target antigen identification, antibody screening for specificity of many other currently uncharacterized non-HLA-antibodies is near impossible. The advent of high-density protein microarrays has made screening for serum antibodies against thousands of human proteins more efficient, as seen in recent publications in autoimmune disease16,17 and cancer18 and organ transplantation.9 Using protein microarray technology for the first time in organ transplantation, we were able to identify 5056 proteins present on the Human ProtoArray v3 (Invitrogen).10,11 Using Journal of Proteome Research 2010, 9, 6715–6721 6715 Published on Web 10/05/2010

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Table 1. Patient Demographics of Unique Kidney Transplant Recipients Who Provided Sera Samples for Analysis by ProtoArray (Discovery Set) and ELISA (Validation Set) Baseline Patient Characteristics

ProtoArray Before Transplantation Recipient Mean age (years) Infant: e5 years old (%) Gender: males (%) Race (1, 2, 3, 4, 5) ea Donor Mean donor age Donor Type (LRD) Mean HLA match a

ELISA

P-value

18

15

11.0 ( 5.5 17%

12.9 ( 6.9 27%

0.38 0.48

78% 47%, 18%, 6%, 17%,12%

53% 27%, 47%, 13%, 7%, 6%

0.14 0.34

32.0 ( 13.6 67% 2.7 ( 1.6

29.6 ( 10.5 47% 2.6 ( 1.8

0.57 0.25 0.92

1, Caucasian; 2, Hispanic; 3, Asian; 4, African American; 5, other.

this data, we were able to identify the PRKCZ in association with acute rejection, which was confirmed using ELISA.10 In a separate study, we combined these post-transplant “antibodyome” profiles with publically accessible cDNA microarray data from microdissected compartments of the normal kidney,11 and were able to localize individual proteins and groups of proteins to specific anatomical regions of the kidney. A previously published study identified that the renal pelvis, the outer renal cortex, and the glomerular antigens were the most immunogenic regions of the transplanted kidney. As it is likely that many of the identified repertoire of non-HLA-antibodies after organ engraftment may not be pathogenically relevant to transplant injury or survival, the findings from these integrative protein array based studies have been applied in this paper to explore the clinical impact of some of the newly found nonHLA-antibodies after organ transplantation in renal transplant patients, in the absence of graft dysfunction and interval acute rejection.

Methods and Materials Patient and Samples. This study was done on 81 unique serum samples obtained from 33 unique kidney transplant recipients. The study patients and serum samples were subdivided into two groups. In the first group, pretransplant and a single post-transplant serum samples were examined on the protoarray from each of 18 unique transplant recipients (discovery set, N ) 36) (GSE:1045211). The post-transplant serum sample was collected between 3 and 72 months after engraftment and demographics on these patients have been previously provided.11 Each patient had clinical follow-up for graft function for at least 1 year after the date of sample collection for the protoarray analysis. In the second group, three longitudinal sera samples were collected at 3, 12, and 24 months post-transplantation from an additional 15 unique transplant recipients and processed by ELISA for two selected non-HLA-antibodies (validation set, n ) 45). These 15 patients did not have any detectable anti-HLA Ab by Luminex assay. Protocol biopsies are done for all recipients at our center at 0, 3, 6, 12, and 24 months post-transplantation. Thus, all of the sera samples in the validation cohort had stable graft function, and had paired protocol biopsies that showed absence of any subclinical acute rejection. The demographics of these patients are provided in Table 1. At each sample collection time, graft function was measured based on the calculated creatinine 6716

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19,20

clearance. Decline in graft function was defined as the difference between the calculated creatinine clearance measured at the post-transplant sample time and the clearance measured 1 year after this sample time. Hypertension was defined as use of one or more antihypertensive agents, and this information was collected on the independent set of samples by interrogation of the clinical database. De novo posttransplant antibody responses were measured across a human Protoarray platform as we previously described.11,21 The study was approved by the Institutional Review Board of Stanford University. Protoarray Measurements and Integrated Genomics To Map ProtoArray Targets to Kidney Compartment Distribution. Invitrogen ProtoArray Human Protein Microarray v3.0 platform (Invitrogen, Carlsbad, CA) was run on all 36 pre- and posttransplant serum samples following the protocol and details for experiment processing and data retrieving that were described in the previous published studies from our group.11 Immune response profiling program was used in Prospector software; duplicated spots for each protein were calculated by Pearson correlation coefficient and raverage was 0.97 (range 0.71-0.99) for all patients across 5056 proteins.11 Seven compartmental specific gene lists based on 3835 common genes/proteins identified from previous published study11,22 were used for hypergeometric analyses to find whether compartment specific antigens had clinical relevance with time post-transplantation or post-transplant graft function. Pearson correlation coefficients were calculated between signal intensity for all patients and the corresponding post-transplantation sample time and graft function. Independent Validation of Selected Non-HLA-Antibodies by ELISA. For independent validation of the clinical association between selected non-HLA-antibodies and clinical outcomes, 45 additional serum samples were drawn at 3, 12, and 24 months post-transplantation from each of 15 unique pediatric renal transplant recipients. Each patient had stable graft function and no interval clinical acute rejection, and also had paired protocol biopsies at the time of serum sample collection that qualified the patient as not having any subclinical acute rejection. Two target non-HLA-antibodies were selected for subsequent validation, based on their strong association with time posttransplantation [angiotensinogen (serpin peptidase inhibitor, clade A, member 8), AGT] without association with decline in graft function or decline in graft function based on the delta calculated creatinine clearance between the time of sampling and a year subsequently without association with time posttransplantation [speedy homologue A (Xenopus laevis), SPDYA]. The enzyme linked immunosorbent assay (ELISA) was developed to detect serum IgG binding to AGT (BC011519) and SPDYA (NM_182756). Insect derived purified AGT fused to glutathione S-transferase were acquired from Invitrogen (Carlsbad, CA) and GST-tagged full length SPDYA was purchased from Abnova Corporation (Taipei, Taiwan). A titration was performed to determine the optimal amount of proteins to be coated on to the immunosorbant 96-well plate (NUNC Brand Cat. No. 446612). Briefly, the 96 well microwell ELISA plates were coated with corresponding protein in 50 µL of coating buffer (15 mM Na2CO3, 30 mM NaHCO3, 0.02% NaN3, pH 9.6) and incubated overnight at 4 °C. Standard curves were generated using Anti-GST tag (mouse monoclonal IgG) (Millipore, Temecula, CA) and AP-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, West Grove, PA). After washing

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Figure 1. Scatter plots for signal intensity from ProtoArray and related clinical relevance. Two highly significant non-HLA antibody targets identified by the ProtoArray, associated with sample time post-transplant for target AGT with r ) 0.77 (p ) 0.0088) (A) and ∆crcl for target SPDYA with r ) 0.71 (p ) 0.003) (B). Selections were based on strong correlation between signal intensity and clinical relevance.

the plates with TBST buffer for 5 times, the nonspecific protein binding was blocked using 100 µL of 5% dry milk in TBST buffer for 1 h at room temperature. After the blocking step, 50 µL of serum samples (10-fold diluted with 5% milk in TBST buffer) was incubated in the wells for 1 h at room temperature. The plates were washed 5 times with TBST buffer and incubated in 50 µL of AP-conjugated AffiniPure goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA). The color was developed using AP-pNPP Liquid Substrate System for ELISA (Sigma-Aldrich, St. Louis, MO). Absorption was measured at 405 nm with a SPECTRAMax 190 microplate reader (Molecular Devices, Sunnyvale, CA) with less than 4% intra-assay coefficient of variation (CV) and 0.5 for sample time and for graft function association was set for final selection of the most biologically significant candidates for further ELISA validation. All P-values were two-sided, and those less than 0.05 were considered as significant level in all statistical tests. Pathway analysis was done by Ingenuity Pathway Analysis (IPA) (Redwood City, CA).

Data Availability. Data deposit: http://www.ncbi.nlm.nih. gov/projects/geo (GSE 10452).

Results Protoarray Detects Novel Non-HLA Antibodies to Immunogenic Epitopes That Associate with Time Post-Transplantation. Antibody measurements were selected for their association with time post-transplantation based on the presumption that increases in antibody strength, over time, may reflect increased immunogenicity or increasing antigen exposure after the initial transplant event. There was a strong association between antibody signal intensity and time post-transplantation (p < 0.05 and r > 0.5 for 1785 targets) (Supplemental Table 1a). By IPA analysis, targets associated with increasing time posttransplantation are enriched in cellular immune response, such as T cell receptor signaling (p ) 2.5 × 10-3), natural killer cell signaling (p ) 0.02), humoral B cell receptor signaling (p ) 1.67 × 10-6), NRF2-mediated oxidative stress response (p ) 2.9 × 10-8), and rennin-angiotensin signaling (p ) 5.4 × 10-5). On the basis of the biological importance of the rennin-angiotensin pathway in renovascular hypertension, a previous collaborative genetic study demonstrated significant differences in plasma concentrations of AGT (angiotensinogen) among hypertensive subjects with different AGT genotypes, demonstrating an association of AGT molecular variants with the disease.23 Therefore, AGT (r ) 0.77, p ) 0.0008) was selected for further validation by ELISA in our study. The correlation plot for AGT protoarray signal intensity and post-transplant time is shown in Figure 1A. Protoarray Detects Novel Non-HLA Antibodies Significantly Associated with Subsequent Decline in Graft Function. There was a significant association for a much smaller set of nonHLA-antibodies with subsequent decline in graft function (p < 0.05; |r| > 0.5 for 105 targets) (Supplemental Table 1b). Canonical pathway analysis from IPA shows that these non-HLA antibodies associated with a subsequent decline in graft function are enriched for the complement system (r > 0.45; p ) 0.038) against C1QA and CD46. These proteins have also been shown to be correlated at the transcriptional level with Journal of Proteome Research • Vol. 9, No. 12, 2010 6717

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Table 2. Hypergeometric Analysis of de Novo Antibody Responses to Kidney Compartment Specific Targets in Association with (|r| > 0.5) Time Post-Transplantation or Decline in Graft Function over the Subsequent Year (∆crcl)a glomeruli

number and p-value of de novo Antibody responses to kidney compartment specific targets

no.

sample_time (|r| > 0.75) (N ) 196) sample_time (|r| > 0.50) (N ) 1785) ∆crcl (|r| > 0.5) (N ) 105)

6 71 9

a

inner cortex

outer cortex

inner medulla

outer medulla

p

no.

p

no.

p

no.

p

no.

p

no.

p

no.

p

0.12 0.053 0.02

7 76 4

0.08 0.002 0.16

11 141 10

0.03 0.01 0.13

0 3 0

0.62 0.2 0.78

3 18 1

0.13 0.04 0.37

10 83 4

0.12 0.04 0.19

19 204 13

0.05 0.02 0.12

pelvis

Given the large number of targets associated with time post-transplantation, a higher threshold of |r| > 0.75 is also shown.

decline in graft function.24,25 Additional pathways enriched with immunogenic targets that are dysregulated with declining transplant function relate to growth hormone signaling (p ) 0.04), ERK5 signaling (p ) 0.005), and Calcium-induced T lymphocyte apoptosis (p ) 0.004). One of the most significant non-HLA-antibodies that associated with subsequent decline in graft function was speedy homologue A, SPDYA (r ) 0.71, p ) 0.003) (Figure 1B), which regulates CDK2 (a known biomarker for prognosis of Melanoma), plays roles of proliferation and survival, and promotes cell cycle progression through the G1/S phase transition;26 thus, this was selected for further validation by ELISA in an independent sample set. Integrative Analysis Maps Differential Immunogenic Potential for Renal Antigens. We performed our previously published integrative analysis of antibody and genomic data11 to map the relative immunogenic potential of different kidney compartments, as microdissected by Higgins et al.22 Targets which were correlated with sample time or a decline in graft function were further analyzed to see if they could be mapped predominantly to one or more kidney compartment antigens, as mapped to the outer cortex, inner cortex, glomerulus, outer medulla, inner medulla, renal pelvis and renal papillae.11 This study identified that kidney outer cortex antigens are most immunogenic for post-transplant time (r > 0.75, 196 targets), followed by inner cortex and renal pelvis antigens when r > 0.5 (Table 2). In addition, kidney glomerular antigens are immunogenic for subsequent decline in graft function, irrespective of posttransplant time when |r| > 0.5. For the 1785 antibodies associated with time of sampling post-transplantation (p < 0.05, r > 0.5), there was a significant enrichment of compartment specific targets for the outer cortex (p ) 0.01), inner cortex (p ) 0.002), outer medulla (p ) 0.04), pelvis (p ) 0.02), and pap tips (p ) 0.04) (Table 2). By gene ontology analysis, these antibodies are mounted against proteins involved in apoptosis (n ) 86, p ) 0.0001), cell death (n ) 93, p ) 0.001) and cell cycle (n ) 95, p ) 0.006). For the 105 antibodies associated with a decline in graft function after sampling, these were significantly enriched in glomerular compartment (p ) 0.02), not in other renal compartments (Table 2). Many of these antibodies are being mounted to antigenic targets associated by Gene Ontology analysis with cellular growth and proliferation (n ) 34, p ) 0.001), cellular function and maintenance (n ) 15, p ) 0.001), and cell morphology (n ) 15, p ) 0.001). Nine targets of these were specific to glomerulus region with the enrichment p ) 0.02 and |r| > 0.5 (Supplemental Table 1b), and one target MICA antigen has been confirmed for glomerulus localization by immunochemistry in our previous study and has been also shown to correlate with subsequent decline in graft function.21 Another target, vascular endothelial growth factor or FLT1, has been identified by integrative genomics to localize to the glomerulus and in this analysis has been found to be significantly associ6718

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ated with subsequent decline in graft function (r ) 0.54, p ) 0.04). Interestingly, a rat model of microvasculature injury has previously identified FLT1 to be also involved in glomerular endothelial injury,27 thus, supporting the preliminary findings in human sera samples from transplant patients. Interestingly, when the most significantly correlated nonHLA antibodies were compared across all data sets based upon correlating with either sample time post-transplant or a decline in subsequent graft function (Figure 2A), the majority of both data sets had independently associated immunogenic and pathogenic antibodies, and only 12 overlapping targets between the 2 data sets correlated with both time post-transplant and transplant functional decline (Figure 2B). About two-thirds of these targets are highly expressed in endothelial cells, monocytes and T cells (Figure 2B). Three of the antibodies have corresponding antigenic epitopes which provide key targets for modulation by three currently used immunosuppressive agents, namely, RPTOR (a regulatory protein of the MTOR complex 1, which is an essential scaffold for MTOR-catalyzed phosphorylation, mediates TOR action in vivo, and is highly expressed in the kidney) which is regulated by Rapamycin; TRMU (tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase, a mitochondrial protein, highly expressed in the kidney) which is regulated by Azathioprine; and ITPRIP (inositol 1,4,5-triphosphate receptor interacting protein which regulates DAPK1 or death associated protein kinase, which also regulates the inositol monophosphate dehydroganse pathway) which is regulated by Mycophenolate Mofetil. Immunohistochemistry analysis on normal kidney tissue (http://www.proteinatlas.org/) (Figure 2C)28–31 reveals high expression for these 3 proteins in the renal glomerulus. ELISA Validation of AGT and SPDYA in Independent, Longitudinal Sera Samples. For validation of the clinical relevance of the 2 most significant antibody targets, independent sera samples (n ) 45) were obtained at 3, 12, and 24 months posttransplant from 15 pediatric kidney transplant recipients with stable graft function. Antibody signal intensity (optical density at 450 nm) for AGT was validated to increase with posttransplant time (0.29 ( 0.06 24 months vs 0.18 ( 0.04 at 12 months; p ) 0.006, and 0.10 ( 0.02 at 3 months; p ) 0.01) (Figure 3A). Interestingly, patients with hypertension (hypertension defined as use of antihypertensive drugs at each of 3, 12, or 24 months) had significant increase in the serum AGT levels by ELISA over the first 2 years post-transplantation (mean ∆AGT levels between 12 and 24 months post-transplantation were 0.18 in hypertensive patients versus 0.03 in normotensive patients; p ) 0.024). The AGT protein was predicted to localize to the renal cortex and renal pelvis from the integrative bioinformatic analysis;11 this was confirmed by immunohistochemistry (IHC), as positive cytoplasmic staining for the AGT protein was noted in kidney tubules (http://www.proteinatlas. org/) (Figure 3B).28–31 ELISA analysis for anti-SPDYA antibodies

Non-HLA Antibodies and Related Clinical Relevance

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Figure 2. Twelve overlapped targets for either associated with time of sampling or decline in graft function. Significant non-HLA antibodies that associate with time of sampling post-transplantation (n ) 1,785) or subsequent decline in graft function a year after sample analysis (n ) 105) (A). Twelve non-HLA antibodies are detected in association with both longer sampling time post-transplantation and greater decline in graft function a year after sampling (B). Proteins highly expressed in endothelial cells are in bold font. *Targets are modulated by known immunosuppression drugs used in organ transplantation. IHC stainings for the 3 proteins (C), which are targets of drug design, show high levels of expression in the renal glomerulus. Antibodies to these proteins show rising titers with increasing time post-transplant.

Figure 3. Validation from ELISA and IHC for target AGT. ELISA validation for antibody levels against AGT in 45 independent sera samples show the significant increase in anti-AGT levels with post-transplant sample time (A). The AGT protein localizes by integrative informatics and IHC to the renal cortex in normal kidney from Human Protein Atlas (B).

in 3 month samples (n ) 15) also confirmed the association of these antibody titers with subsequent decline in graft function at 24 months post-transplantation in this patient cohort (r ) 0.75, p ) 0.01). A similar association was confirmed for ELISA analysis for anti-SPDYA antibody titers at 12 months and

decline in graft function at 24 months post-transplantation (r ) 0.67, p ) 0.006). Decline graft function in both cases was calculated as the difference based on calculated creatinine clearance between 3 and 24 months and 12 and 24 months post-transplant, respectively. Journal of Proteome Research • Vol. 9, No. 12, 2010 6719

research articles Discussion Recent studies have highlighted a growing understanding of the pathogenic role of non-HLA antibodies after organ transplantation.9,10,11 A rate-limiting step in the discovery of new non-HLA antibodies has been the difficulty in identification of new antigenic epitopes that are immunogenic. Protein array technology now allows for the simultaneous interrogation of thousands of antibody specificities in patient sera.10,11 These studies have provided a means to identify novel non-HLA antibodies, such as the anti-PRKCZ antibody, as a pathogenic biomarker for aggressive acute graft rejection.10 We have previously shown that, even in well functioning grafts without delayed graft function or interval acute rejection, many new non-HLA antibodies develop after kidney transplantation and the majority of this non-HLA antibody repertoire is patient specific.11 This study shows for the first time that most of the de novo non-HLA antibodies do not have a pathogenic role in graft injury, suggesting that a majority of the observed nonHLA antibodies have a “simple bystander” effect after transplant surgery. It is unknown currently if different mechanisms may be involved in the formation of pathogenic and nonpathogenic non-HLA antibodies. Nevertheless, even in stable transplant patients followed over time post-transplantation, a specific panel of non-HLA antibodies can be found to significantly correlate with posttransplant time. The antigenic epitopes for these antibodies localize predominantly in the renal cortex, highlighting that the cellular and structural proteins in the renal cortex may be mounting antibody responses during the process of chronic tubular injury and accrual of drug toxicity injury, a well recognized pathology in renal transplants over time.32 Though a small subset of these antibodies does appear to be pathologically relevant, as they correlate strongly with subsequent decline in graft function, a majority of non-HLA antibodies that associate with clinical relevance to graft function are independent of post-transplant time. This is interesting and suggests a different mechanism of generation of increased titers of nonHLA antibody with graft injury, at the time of graft injury, rather than simply time post-transplantation. These targets are likely to be of greater pathogenic relevance. Interestingly, integrative genomics11 suggests that many of the protein targets for these antibodies are enriched in the renal glomerulus, the pivotal region of the kidney responsible for renal filtration and renal function. It is of note that previously described non-HLA antibodies which have correlated with graft function, such as Agrin11,13 and Vimentin,33 are also primarily glomerular in localization. We thus hypothesize that immunogenic injury in different kidney compartments may evolve differentially over time and bear differential clinical relevance. Further studies are required to fully define how non-HLA antibodies can modulate the immune response and affect graft dysfunction. Interestingly, some of the antigenic targets that are modulated by increasing time post-transplant and impact graft function adversely have been already recognized as important drug targets, and are modulated by 3 major immunosuppressive drugs in current transplant practice. Non-HLA antibodies may play a role in the induction of costimulatory molecules and facilitate T lymphocyte activation by the endothelium.34 In vitro studies have suggested that the effector function of non-HLA antibodies may relate to complement-mediated or complement independent injury of endothelial cells. For example, in xeno-transplantation models, antiGal can trigger the inflammatory process of endothelial cells, 6720

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irrespective of complement activation. MICA and anti-Gal antibodies are also known to be involved in the procoagulant activation of endothelial cells.36,37 Non-HLA antibodies have also been shown in other studies to decrease in vitro endothelial cell viability by promoting apoptosis.38 These data suggest that the interactions of non-HLA antibodies with the graft endothelium might be the essential axis in the pathogenic mechanisms contributing to graft injury. A validated antibody that correlates with decline in graft function in our study, against human SPDYA, may result from the role of SPDYA in glomerular damage39 and apoptosis.40 A future study direction is to test the effect of a neutralizing antibody to SPDYA in the progression of apoptosis injury in the graft endothelium. In conclusion, this integrative genomics approach of proteinarray analysis of sera from stable kidney transplant patients, and its application to clinical parameters of graft function and post-transplant time, has advanced our understanding of the evolution and relevance non-HLA antibodies in organ transplant recipients. Generation of antibodies to a myriad of nonHLA targets is a generalized observance in all organ transplant recipients. Defined profiles of these non-HLA antibodies to renal cortical proteins develop with increasing length of engraftment, and may reflect the increasing recognition of altered localization or exposure of renal tubular and interstitial proteins, affected by advancing chronic nonimmune graft injury. The panel of non-HLA antibodies to glomerular targets is most interesting, as these corresponding antigenic targets may be important pathways in functional graft injury and could provide novel targets for drug design. Though a host of other non-HLA antibodies appear to have a bystander role after engraftment, their pathogenic relevance to other clinical parameters in larger cohorts of patients with longer follow-up is warranted. Abbreviations: CAI, chronic allograft injury; HLA, human leukocyte antigens; IHC, immunohistochemistry.

Acknowledgment. We are grateful to Drs. Atul Butte and Maarten Naesens for discussions and valuable comments. We also are grateful to our pediatric transplant nephrology team for helping with clinical sample collection. This work was supported by Beta Sigma Phi, Bio-X and the Packard Foundation (to M.S., L.L., and T.S). Supporting Information Available: Specific compartment targets which show significant correlation with either sample time post-transplantation or decline in graft function were provided in the supplemental tables. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Pascual, M.; Theruvath, T.; Kawai, T.; Tolkoff-Rubin, N.; Cosimi, A. B. Strategies to improve long-term outcomes after renal transplantation. N. Engl. J. Med. 2002, 346 (8), 580–90. (2) Chapman, J. R.; O’Connell, P. J.; Nankivell, B. J. Chronic renal allograft dysfunction. J. Am. Soc. Nephrol. 2005, 16 (10), 3015–26. (3) Nankivell, B. J.; Borrows, R. J.; Fung, C. L.; O’Connell, P. J.; Allen, R. D.; Chapman, J. R. The natural history of chronic allograft nephropathy. N. Engl. J. Med. 2003, 349 (24), 2326–33. (4) Nankivell, B. J.; Fenton-Lee, C. A.; Kuypers, D. R.; Cheung, E.; Allen, R. D.; O’Connell, P. J.; Chapman, J. R. Effect of histological damage on long-term kidney transplant outcome. Transplantation 2001, 71 (4), 515–23. (5) Terasaki, P.; Mizutani, K. Antibody mediated rejection: update 2006. Clin. J. Am. Soc. Nephrol. 2006, 1 (3), 400–3. (6) Akalin, E.; Pascual, M. Sensitization after kidney transplantation. Clin. J. Am. Soc. Nephrol. 2006, 1 (3), 433–40. (7) Hourmant, M.; Cesbron-Gautier, A.; Terasaki, P. I.; Mizutani, K.; Moreau, A.; Meurette, A.; Dantal, J.; Giral, M.; Blancho, G.;

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Cantarovich, D.; Karam, G.; Follea, G.; Soulillou, J. P.; Bignon, J. D. Frequency and clinical implications of development of donorspecific and non-donor-specific HLA antibodies after kidney transplantation. J. Am. Soc. Nephrol. 2005, 16 (9), 2804–12. Varnavidou-Nicolaidou, A.; Iniotaki-Theodoraki, A. G.; Doxiadis, I. I.; Georgiou, D.; Patargias, T.; Stavropoulos-Giokas, C.; Kyriakides, G. Expansion of humoral donor-specific alloreactivity after renal transplantation correlates with impaired graft outcome. Hum. Immunol. 2005, 66 (9), 985–8. Opelz, G. Non-HLA transplantation immunity revealed by lymphocytotoxic antibodies. Lancet 2005, 365 (9470), 1570–6. Sutherland, S. M.; Li, L.; Sigdel, T. K.; Wadia, P. P.; Miklos, D. B.; Butte, A. J.; Sarwal, M. M. Protein microarrays identify antibodies to protein kinase Czeta that are associated with a greater risk of allograft loss in pediatric renal transplant recipients. Kidney Int. 2009, 76 (12), 1277–83. Li, L.; Wadia, P.; Chen, R.; Kambham, N.; Naesens, M.; Sigdel, T. K.; Miklos, D. B.; Sarwal, M. M.; Butte, A. J. Identifying compartmentspecific non-HLA targets after renal transplantation by integrating transcriptome and “antibodyome” measures. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (11), 4148–53. Lerut, E.; Van Damme, B.; Noizat-Pirenne, F.; Emonds, M. P.; Rouger, P.; Vanrenterghem, Y.; Pirenne, J.; Ansart-Pirenne, H. Duffy and Kidd blood group antigens: minor histocompatibility antigens involved in renal allograft rejection. Transfusion 2007, 47 (1), 28– 40. Joosten, S. A.; Sijpkens, Y. W.; van Ham, V.; Trouw, L. A.; van der Vlag, J.; van den Heuvel, B.; van Kooten, C.; Paul, L. C. Antibody response against the glomerular basement membrane protein agrin in patients with transplant glomerulopathy. Am. J. Transplant. 2005, 5 (2), 383–93. Dragun, D.; Muller, D. N.; Brasen, J. H.; Fritsche, L.; NieminenKelha, M.; Dechend, R.; Kintscher, U.; Rudolph, B.; Hoebeke, J.; Eckert, D.; Mazak, I.; Plehm, R.; Schonemann, C.; Unger, T.; Budde, K.; Neumayer, H. H.; Luft, F. C.; Wallukat, G. Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection. N. Engl. J. Med. 2005, 352 (6), 558–69. Carter, V.; Shenton, B. K.; Jaques, B.; Turner, D.; Talbot, D.; Gupta, A.; Chapman, C. E.; Matthews, C. J.; Cavanagh, G. Vimentin antibodies: a non-HLA antibody as a potential risk factor in renal transplantation. Transplant. Proc. 2005, 37 (2), 654–7. Leitner, W. W.; Hwang, L. N.; deVeer, M. J.; Zhou, A.; Silverman, R. H.; Williams, B. R.; Dubensky, T. W.; Ying, H.; Restifo, N. P. Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways. Nat. Med. 2003, 9 (1), 33–9. Rowshani, A. T.; Bemelman, F. J.; Lardy, N. M.; Ten Berge, I. J. Humoral immunity in renal transplantation: clinical significance and therapeutic approach. Clin. Transplant. 2008, 22 (6), 689–99. Hudson, M. E.; Pozdnyakova, I.; Haines, K.; Mor, G.; Snyder, M. Identification of differentially expressed proteins in ovarian cancer using high-density protein microarrays. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (44), 17494–9. Schwartz, G. J.; Feld, L. G.; Langford, D. J. A simple estimate of glomerular filtration rate in full-term infants during the first year of life. J. Pediatr. 1984, 104 (6), 849–54. Schwartz, G. J.; Gauthier, B. A simple estimate of glomerular filtration rate in adolescent boys. J. Pediatr. 1985, 106 (3), 522–6. Li, L.; Chen, A.; Chaudhuri, A.; Kambham, N.; Sigdel, T.; Chen, R.; Sarwal, M. M. Compartmental localization and clinical relevance of MICA antibodies after renal transplantation. Transplantation , 89 (3), 312–9. Higgins, J. P.; Wang, L.; Kambham, N.; Montgomery, K.; Mason, V.; Vogelmann, S. U.; Lemley, K. V.; Brown, P. O.; Brooks, J. D.; van de Rijn, M. Gene expression in the normal adult human kidney assessed by complementary DNA microarray. Mol. Biol. Cell 2004, 15 (2), 649–56. Jeunemaitre, X.; Soubrier, F.; Kotelevtsev, Y. V.; Lifton, R. P.; Williams, C. S.; Charru, A.; Hunt, S. C.; Hopkins, P. N.; Williams, R. R.; Lalouel, J. M.; et al. Molecular basis of human hypertension: role of angiotensinogen. Cell 1992, 71 (1), 169–80. Naesens, M.; Li, L.; Ying, L.; Sansanwal, P.; Sigdel, T. K.; Hsieh, S. C.; Kambham, N.; Lerut, E.; Salvatierra, O.; Butte, A. J.; Sarwal, M. M. Expression of complement components differs between kidney allografts from living and deceased donors. J. Am. Soc. Nephrol. 2009, 20 (8), 1839–51. Naesens, M.; Butte, A. J.; Sarwal, M. M. C3 polymorphisms and outcomes of renal allografts N. Engl. J. Med. 2009,360 (23), 2478; author reply 2478-9.

(26) Porter, L. A.; Kong-Beltran, M.; Donoghue, D. J. Spy1 interacts with p27Kip1 to allow G1/S progression. Mol. Biol. Cell 2003, 14 (9), 3664–74. (27) Maynard, S. E.; Min, J. Y.; Merchan, J.; Lim, K. H.; Li, J.; Mondal, S.; Libermann, T. A.; Morgan, J. P.; Sellke, F. W.; Stillman, I. E.; Epstein, F. H.; Sukhatme, V. P.; Karumanchi, S. A. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J. Clin. Invest. 2003, 111 (5), 649–58. (28) Ponten, F.; Jirstrom, K.; Uhlen, M. The Human Protein Atlassa tool for pathology. J. Pathol. 2008, 216 (4), 387–93. (29) Nilsson, P.; Paavilainen, L.; Larsson, K.; Odling, J.; Sundberg, M.; Andersson, A. C.; Kampf, C.; Persson, A.; Al-Khalili Szigyarto, C.; Ottosson, J.; Bjorling, E.; Hober, S.; Wernerus, H.; Wester, K.; Ponten, F.; Uhlen, M. Towards a human proteome atlas: highthroughput generation of mono-specific antibodies for tissue profiling. Proteomics 2005, 5 (17), 4327–37. (30) Berglund, L.; Bjorling, E.; Oksvold, P.; Fagerberg, L.; Asplund, A.; Szigyarto, C. A.; Persson, A.; Ottosson, J.; Wernerus, H.; Nilsson, P.; Lundberg, E.; Sivertsson, A.; Navani, S.; Wester, K.; Kampf, C.; Hober, S.; Ponten, F.; Uhlen, M. A genecentric Human Protein Atlas for expression profiles based on antibodies. Mol. Cell. Proteomics 2008, 7 (10), 2019–27. (31) Uhlen, M.; Bjorling, E.; Agaton, C.; Szigyarto, C. A.; Amini, B.; Andersen, E.; Andersson, A. C.; Angelidou, P.; Asplund, A.; Asplund, C.; Berglund, L.; Bergstrom, K.; Brumer, H.; Cerjan, D.; Ekstrom, M.; Elobeid, A.; Eriksson, C.; Fagerberg, L.; Falk, R.; Fall, J.; Forsberg, M.; Bjorklund, M. G.; Gumbel, K.; Halimi, A.; Hallin, I.; Hamsten, C.; Hansson, M.; Hedhammar, M.; Hercules, G.; Kampf, C.; Larsson, K.; Lindskog, M.; Lodewyckx, W.; Lund, J.; Lundeberg, J.; Magnusson, K.; Malm, E.; Nilsson, P.; Odling, J.; Oksvold, P.; Olsson, I.; Oster, E.; Ottosson, J.; Paavilainen, L.; Persson, A.; Rimini, R.; Rockberg, J.; Runeson, M.; Sivertsson, A.; Skollermo, A.; Steen, J.; Stenvall, M.; Sterky, F.; Stromberg, S.; Sundberg, M.; Tegel, H.; Tourle, S.; Wahlund, E.; Walden, A.; Wan, J.; Wernerus, H.; Westberg, J.; Wester, K.; Wrethagen, U.; Xu, L. L.; Hober, S.; Ponten, F. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol. Cell. Proteomics 2005, 4 (12), 1920–32. (32) Naesens, M.; Kambham, N.; Concepcion, W.; Salvatierra, O., Jr.; Sarwal, M. The evolution of nonimmune histological injury and its clinical relevance in adult-sized kidney grafts in pediatric recipients. Am. J. Transplant. 2007, 7 (11), 2504–14. (33) Yaoita, E.; Franke, W. W.; Yamamoto, T.; Kawasaki, K.; Kihara, I. Identification of renal podocytes in multiple species: higher vertebrates are vimentin positive/lower vertebrates are desmin positive. Histochem. Cell Biol. 1999, 111 (2), 107–15. (34) Sumitran-Holgersson, S.; Ge, X.; Karrar, A.; Xu, B.; Nava, S.; Broome, U.; Nowak, G.; Ericzon, B. G. A novel mechanism of liver allograft rejection facilitated by antibodies to liver sinusoidal endothelial cells. Hepatology 2004, 40 (5), 1211–21. (35) Ehrnfelt, C.; Serrander, L.; Holgersson, J. Porcine endothelium activated by anti-alpha-GAL antibody binding mediates increased human neutrophil adhesion under flow. Transplantation 2003, 76 (7), 1112–9. (36) Sumitran-Holgersson, S.; Wilczek, H. E.; Holgersson, J.; Soderstrom, K. Identification of the nonclassical HLA molecules, mica, as targets for humoral immunity associated with irreversible rejection of kidney allografts. Transplantation 2002, 74 (2), 268–77. (37) Gollackner, B.; Goh, S. K.; Qawi, I.; Buhler, L.; Knosalla, C.; Daniel, S.; Kaczmarek, E.; Awwad, M.; Cooper, D. K.; Robson, S. C. Acute vascular rejection of xenografts: roles of natural and elicited xenoreactive antibodies in activation of vascular endothelial cells and induction of procoagulant activity. Transplantation 2004, 77 (11), 1735–41. (38) Le Bas-Bernardet, S.; Hourmant, M.; Coupel, S.; Bignon, J. D.; Soulillou, J. P.; Charreau, B. Non-HLA-type endothelial cell reactive alloantibodies in pre-transplant sera of kidney recipients trigger apoptosis. Am. J. Transplant. 2003, 3 (2), 167–77. (39) Barnes, E. A.; Porter, L. A.; Lenormand, J. L.; Dellinger, R. W.; Donoghue, D. J. Human Spy1 promotes survival of mammalian cells following DNA damage. Cancer Res. 2003, 63 (13), 3701–7. (40) Gastwirt, R. F.; Slavin, D. A.; McAndrew, C. W.; Donoghue, D. J. Spy1 expression prevents normal cellular responses to DNA damage: inhibition of apoptosis and checkpoint activation. J. Biol. Chem. 2006, 281 (46), 35425–35.

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