Original Clinical ScienceçGeneral
Clinical, Histological, and Molecular Markers Associated With Allograft Loss in Transplant Glomerulopathy Patients Layla Kamal,1 Pilib Ó. Broin,2 Yi Bao,1 Maria Ajaimy,1 Michelle Lubetzky,1 Anjali Gupta,1 Graciela de Boccardo,1 James Pullman,3 Aaron Golden,2,4 and Enver Akalin1
Background. We aimed to investigate the clinical, histopathological, and molecular factors associated with allograft loss in transplant glomerulopathy (TGP) patients. Methods. Of the 525 patients who underwent clinically indicated kidney biopsies, 52 (10%) had diagnosis of TGP. Gene expression profiles of 28 TGP and 11 normal transplant kidney biopsy samples were analyzed by Affymetrix HuGene 1.0 ST expression arrays. Results. Over a median follow up of 23 months (1-46 months) after the diagnosis of TGP by biopsy, 17 patients (32%) lost their allografts at a median of 16 months (1-44 months). There was no difference between the 2 groups in terms of any demographic variables, serum creatinine, panel reactive antibody levels, donor-specific antibody frequency, or mean fluorescence intensity values. Patients who lost their allograft had a significantly higher median spot protein to creatinine ratio 2.81 (1.20-6.00) compared to no graft loss patients 1.16 (0.15-2.53), (P < 0.01), and a trends toward a higher mean chronic glomerulopathy (cg) score (1.65 ± 0.93 vs 1.11 ± 0.93) (P = 0.05). There was also no difference in microvascular inflammation or any other Banff injury scores between the 2 groups. Although 117 gene transcripts were upregulated in both groups, 86 and 57 were upregulated in graft loss and functioning allograft groups, respectively. There were significantly increased levels of intragraft endothelial cell–associated transcripts, gene transcripts associated with complement cascade, interleukins and their receptors and granulysin in graft loss patients compared to patients with a functioning allograft. Conclusion. Our results demonstrate differential intragraft gene expression profiles in TGP patients with allograft loss.
(Transplantation 2015;99: 1912–1918)
T
ransplant glomerulopathy (TGP) is a pathological diagnosis of the kidney allograft characterized by double contour of the glomerular basement membrane (GBM) in the absence of immune complex deposits.1 Transplant glomerulopathy is recognized by the Banff classification as the cardinal manifestation of chronic antibody-mediated rejection (AMR).2 Endothelial cells, in the presence of donor-specific antibodies (DSA) or autoantibodies are the main target of antibody-mediated injury. Transplant glomerulopathy Received 19 June 2014. Revision requested 14 July 2014. Accepted 13 November 2014. 1
Montefiore-Einstein Center for Transplantation, Montefiore Medical Center, The University Hospital for Albert Einstein College of Medicine, New York, NY.
2 Department of Genetics, Division of Computational Genetics, Montefiore-Einstein Center for Transplantation, Montefiore Medical Center, The University Hospital for Albert Einstein College of Medicine, Bronx, NY. 3
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY.
4
Department of Mathematical Sciences, Yeshiva University, New York, NY.
This study is supported by an Internal Grant from the Montefiore Medical Center and the Albert Einstein College of Medicine. The authors declare no conflicts of interest. This study was accepted at World Transplant Congress 2014 in San Francisco. Dr. Layla Kamal received the best clinical science abstract award at New York Society of Nephrology Fellows’ Night in May 2014. L.K. and E.A. participated in research design, writing of the article, performance of the research, and data analysis. Y.B., P.Ó.B., M.A., M.L., A.G., G.d.B., J.P., and A.G. participated in writing of the article, performance of the research, and data analysis.
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results from subsequent endothelial cell injury and remodeling.1 Although it appears that antibody-mediated injury plays a dominant role in TGP, it can also be caused by other types of insults, both immunologic and nonimmunologic. These include chronic cellular rejection, chronic thrombotic microangiopathy, and chronic hepatitis C infection.3 Its prevalence varies from 1 study to another, more common in sensitized transplant recipients with DSAs4 and its incidence, can reach up to 20% at 5 years.5 Transplant glomerulopathy carries a poor prognosis and is associated with decreased allograft survival. In a large series of kidney transplant recipients, graft loss was observed in 38% of TGP patients at 5 years compared to 5% in patients without TGP.5 Among patients with TGP, predictors of poor outcome included C4d deposition in the peritubular capillaries,6,7 higher serum creatinine and proteinuria at diagnosis,8 the severity of interstitial fibrosis,9 and GBM duplication (higher Banff “chronic glomerulopathy” [cg] score),10 Correspondence: Enver Akalin, MD, Montefiore-Einstein Center for Transplantation, Montefiore Medical Center, The University Hospital for Albert Einstein College of Medicine, 111 East 210th Street, Bronx, NY 10467. ([email protected]) Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/15/9909-1912 DOI: 10.1097/TP.0000000000000598
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and the presence of DSA.11 However, the importance of microvascular inflammation determined by peritubular capillaritis (ptc) and glomerulitis (g) scores, and the strength of DSA has not been explored thoroughly. Few studies explored the gene expression profiles of TGP.12-14 We have recently reported the intragraft gene expression profiles of TGP patients, with or without DSA and found that TGP patients with DSA have indistinguishable profiles to those seen in chronic antibody mediated injury, whereas TGP without C4d or DSA demonstrated upregulation of cytotoxic immune response.13 The association between gene expression profiles of biopsies and the allograft survival, however, has not been investigated. In this study, in addition to known clinical and demographic factors associated with poor outcomes in TGP, we specifically aimed to investigate if intragraft gene expression profiles by microarrays, microvascular inflammation scores, and the strength of DSA determined by mean fluorescence intensity (MFI) values are associated with allograft loss in TGP patients. PATIENTS AND METHODS Patient Population
The study population was derived from renal allograft biopsies diagnosed with TGP at Montefiore Medical Center, Bronx, NY from January 2009 to December 2012. All biopsies were clinically indicated for elevated creatinine or proteinuria. A retrospective chart review was conducted to record clinical, demographic, immunological, and histopathological parameters and graft outcomes. Graft loss was defined as requiring dialysis and/or retransplantation during follow-up. Histopathology
Biopsies were examined by light microscopy using hematoxylin and eosin, periodic acid-Schiff, Masson Trichrome, and C4d immunoperoxidase stains. Immunoperoxidase staining for C4d was performed on paraffin embedded sections using a polyclonal rabbit antihuman antibody (Cell Marque, Rocklin, CA) at a dilution of 1:100 with the Dako Envision system. Evaluation of the biopsies were based on the Banff ‘07 and ‘0915,16 acute and chronic indices including glomerulitis (g), interstitial inflammation (i), tubulitis (t), intimal arteritis (v), peritubular capillaritis (ptc), TGP (cg), mesangial matrix increase (m), interstitial fibrosis (ci), tubular atrophy (ct), vascular fibrous intimal thickening (cv), arteriolar hyaline thickening (ah), and C4d. Microvascular inflammation score was defined as the sum of g and ptc scores. The cg scores of 1, 2, and 3 correspond to double contours in 1% to 25%, 26% to 50%, and more than 50% of the peripheral capillary loops, respectively. Biopsies were diagnosed as TGP by electron microscopy if they showed electron-lucent widening of the subendothelial zone of the GBM, subendothelial accumulation of flocculent material, with or without a new subendothelial basement membrane layer.
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Statistical Analysis
Statistical analyses were performed using the R statistical programming environment. Demographic and clinical data were compared using the Wilcoxon rank-sum for continuous variables and were reported as median (interquartile range) or mean (standard deviation) as appropriate. Fisher exact test was used for categorical variables. A P value cutoff less than 0.05 was considered statistically significant. Gene Expression Profiles by Microarrays
A subgroup of patients who were enrolled in the IRBapproved “Immune Monitoring Study” and had clinically indicated biopsy samples were included in the analysis. A total of 28 biopsy samples (8 graft losses and 20 functioning allografts) were used for gene expression profiling and compared to 11 normal transplant kidney biopsies. One additional biopsy core was collected for gene expression analysis. It was immediately placed into a vial containing 1 ml of RNALater (Ambion, Catalog Number AM7023), stored at −20 °C for 24 hours and then transferred to −70 °C until RNA isolation. Renal tissue content in all cores was verified using a dissecting microscope and nonrenal tissue was removed. The gene expression profiles were studied using Affymetrix Human Gene 1.0 ST Array hybridization containing 28,869 gene probe sets as described previously.13,17 Gene Set libraries were taken from the Molecular Signatures Database, and the new libraries were generated by mapping HUGO gene identifiers to the University of Alberta, pathogenesis-based transcripts (PBT http://transplants.med.ualberta.ca/Nephlab/data/gene_lists.html) gene sets and a set of 33 genes specific for human regulatory T cells (TREG) identified by Pfoertner et al as upregulated genes compared to naive T cells.18 The following PBT were analyzed (n corresponds to the number of genes): (1) Kidney-specific transcripts (n = 570). (2) GRIT: Gamma-IFN and rejection induced transcripts (n = 50). (3) CAT1-IQR: Cytotoxic T cell–associated transcripts (n = 143). (4) TREG: Regulatory T cell associated transcripts (n = 33). (5) BAT-IQR: B cell associated transcripts (n = 50). (6) NKAT-IQR: Natural killer cell associated transcripts (n = 134). (7)AMA-IQR: Alternate macrophage-associated transcripts (n = 94). (8) DSAST: Transcripts differentially expressed between rejection-classified biopsies from DSA positive patients compared to DSA negative patients (n = 21). (9) ENDAT: Endothelial cell associated transcripts (n = 114).
The data discussed in this publication has been deposited in the GEO data archive and are available under accession number GSE58601. RESULTS
HLA Antibody Screening
Demographics and Clinical Characteristics
Anti–HLA antibodies were studied by Luminex HLA Single Antigen Bead assays (LABScreen, One Lambda Inc., Canoga Park, CA) that use a panel of color-coded beads coated with purified single HLA antigens. The beads include HLA-A, −B, −DR, and -DQ antigens. The cutoff value for a positive DSA was an MFI of 1000 or higher.
Of the 525 patients who underwent clinically indicated transplant kidney biopsies between January 2009 and December 2012, 52 patients (10%) had diagnosis of TGP. Median follow-up time after biopsy was 23 months (range, 1-46 months). Seventeen patients (32%) lost their allografts (defined by return to dialysis or retransplantation) at a
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TABLE 1.
Demographic and clinical characteristics of transplant glomerulopathy patients with and without graft loss Variables
Mean age ± SD Sex, male Race, Black Native kidney disease Diabetes Mellitus Hypertension Glomerulonephritis Hepatitis C virus antibody positive Previous transplant Transplant type, deceased donor Prior acute rejection Median time to biopsy, y Median serum creatinine at diagnosis, mg/dL Median spot urine protein/creatinine, g/g Immunosuppressive regimen Tacrolimus/mycophenolate/prednisone Tacrolimus/prednisone Treatment of transplant glomerulopathy Discontinuation of sirolimus, n Mycophenolate reintroduced or dose increased, n IVIG 1.5 g/kg, n Plasmapheresis, n Rituximab, n
P
Graft loss n = 17
No graft loss n = 35
52 ± 15 53% 47%
50 ± 13 49% 29%
18% 35% 24% 12% 6% 65% 18% 7.2 (4.0-10.9) 2.3 (1.60-3.00) 2.81 (1.20-6.00)
11% 26% 37% 3% 14% 60% 11% 7.5 (2.1-10.9) 1.90 (1.60-2.80) 1.16 (0.15-2.53)
0.67 0.52 0.37 0.25 0.65 1.0 0.67 0.8 0.65 < 0.01
76% 12%
77% 11%
1.0 1.0 1.0
1 2 4 1 1
4 5 6 2 1
median time of 16 months (range, 1-44 months). One patient died with a functioning graft. There was no difference in terms of age, sex, race, etiology of native kidney disease, type of transplant, history of prior transplant or rejection episodes, immunosuppression regimen, hepatitis C virus status, and serum creatinine levels between the graft loss group and the functioning graft group (Table 1). Patients who lost their allograft had a statistically significant higher median spot protein to creatinine ratio of 2.81 (range, 1.20-6.00 g/g), compared to the no graft loss patients whose ratio was 1.16 (range, 0.15-2.53 g/g) (P < 0.01). Immunological Characteristics
Donor-specific antibodies were detected by Luminex single beads assay in 61% of the TGP patients (32/52). Among patients with DSA, 9 patients had DSA to class I HLA, 16 patients to HLA class II, and 7 patients to both classes I and II. There was no statistically significant difference between the 2 groups
0.63 1.0 0.22
in terms of mean class I and II panel reactive antibody levels (Table 2). When comparing graft loss to those with a functioning graft, there was no statistically significant difference in DSA frequency (71% vs 57%) or the mean MFI values of DSAs to HLA-A (2725.75 ± 1693.44 vs 4974.29 ± 3965.94), HLA-B (2665.67 ± 2573.60 vs 5078.80 ± 3456.49), HLA-DQ (10593.8 ± 6333.35 vs 6660.38 ± 4122.22), and HLA-DR (7273.13 ± 5750.39 vs 11060.33 ± 6428.84). Histopathologic Features and Banff Allograft Injury Scores
Glomerular basement membrane duplication was focal involving less than 10% of the capillary loops in 10 patients, corresponding to a Banff cg score of 0, and TGP was diagnosed by electron microscopy. Glomerular basement membrane duplication was mild in 25 patients (cg score of 1), moderate in 9 patients (cg score 2), and severe in 8 patients
TABLE 2.
Immunological characteristics of transplant glomerulopathy patients with and without graft loss Immunological variables
Mean class I PRA Mean class II PRA DSA frequency Mean peak class 1 DSA MFI HLA-A HLA-B Mean peak class II DSA MFI HLA-DQ HLA-DR
Graft loss N = 17
No graft loss N = 35
P
21.18 ± 25.29 47.71 ± 35.77 71%
27.43 ± 30.11 44.97 ± 35.12 57%
0.56 0.66 0.38
2725.75 ± 1693.44 2665.67 ± 2573.60
4974.29 ± 3965.94 5078.80 ± 3456.49
0.41 0.25
10593.8 ± 6333.35 7273.13 ± 5750.39
6660.38 ± 4122.22 11060.33 ± 6428.84
0.22 0.17
PRA, panel reactive antibody; DSA, donor-specific antibody. Mean values were calculated by ± standard deviation.
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TABLE 3.
Histopathological characteristics of TGP patients with and without graft loss Banff injury scores
Graft loss
No graft loss
Mean ± standard deviation
N = 17
N = 35
P
Glomerulitis (g) Peritubular capillaritis (ptc) G + ptc Tubulitis (t) Interstitial inflammation (i) Intimal arteritis (v) Allograft glomerulopathy (cg) Tubular atrophy (ct) Interstitial fibrosis (ci) Vascular fibrosis(cv) Arteriolar hyalinization (ah) Peritubular capillary C4D staining (ptc)
0.82 ± 0.95 1.00 ± 0.79 1.82 ± 1.42 0.18 ± 0.39 1.29 ± 0.77 0.00 1.65 ± 0.93 1.59 ± 0.94 1.59 ± 0.87 0.71 ± 0.92 1.12 ± 1.32 0.53 ± 1.07
0.60 ± 0.74 1.11 ± 1.11 1.71 ± 1.47 0.20 ± 0.47 1.26 ± 1.01 0.00 1.11 ± 0.93 1.66 ± 0.87 1.60 ± 0.81 0.94 ± 0.76 0.97 ± 0.92 0.60 ± 1.09
0.48 0.90 0.74 1.0 0.75 NA 0.05 0.72 0.92 0.20 0.87 0.74
(cg of 3). Interstitial fibrosis/tubular atrophy was seen in 94% of the patients; it was mild in 44% (23/52), moderate in 28% (15/52), and severe in 21% (11/52) of the cases. Peritubular capillary C4d staining was negative in 69% of
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the cases (36/52), diffuse positive in 19% (10/52), focal in 5.5% (3/52), and minimal in 5.5% (3/52) of the cases. There was no statistically significant difference in terms of microvascular inflammation scores (peritubular capillaritis and glomerulitis), tubulitis, interstitial inflammation, interstitial fibrosis, tubular atrophy, vascular fibrosis, or C4d scores between the 2 groups of patients as shown in Table 3. There was a trend toward a higher mean cg score in graft loss patients (1.65 ± 0.93) compared to patients with a functioning allograft (1.11 ± 0.93) (P = 0.05). Treatment of TGP
After the diagnosis of TGP, all the patients were aimed to be on triple immunosuppressive regimen including tacrolimus, mycophenolate mofetil, and prednisone. A total of 5 patients were on sirolimus prior to the diagnosis of TGP, one of which lost their graft. Once the diagnosis of TGP was made, sirolimus was stopped and switched to tacrolimus in all patients. Mycophenolate was reintroduced, or doses were increased in 7 patients (2 in the graft loss group and 5 in the functioning group). All of the patients with proteinuria were started on angiotensin converting enzyme inhibitor or angiotensin receptor blocker unless contraindicated. Administration of intravenous immunoglobulins was the most common
TABLE 4.
Selected intragraft gene transcripts upregulated in only patients with graft loss Transcript cluster ID
7898793 7898799 7906435 7906486 7906757 7912145 7922773 7948455 7951217 7956878 7960464 7960874 7997712 8006608 8025601 8031293 8039212 8043236 8044049 8045688 8054722 8075886 8081386 8114010 8118149 8118324 8118345 8118556 8118594 8122265 8140556 8166065
Gene name
Adjusted P value
Complement component 1, q subcomponent, A chain (C1QA) Complement component 1, q subcomponent, C chain (C1QC), transcript variant 1 Duffy blood group, chemokine receptor (DARC), transcript variant 2 SLAM family member 8 (SLAMF8) Fc fragment of IgG, low affinity IIa, receptor (CD32) (FCGR2A), transcript variant1 Tumor necrosis factor receptor superfamily, member 9 (TNFRSF9) Neutrophil cytosolic factor 2 (NCF2), transcript variant Membrane-spanning 4-domains, subfamily A, member 6A (MS4A6A) Matrix metallopeptidase 7 (matrilysin, uterine) (MMP7) Interleukin-1 receptor-associated kinase 3 (IRAK3), transcript variant 1 Von Willebrand factor (VWF), mRNA Complement component 3a receptor 1 (C3AR1) Interferon regulatory factor 8 (IRF8) Chemokine (C-C motif ) ligand 4-like 1 (CCL4L1) Intercellular adhesion molecule 1 (ICAM1) Killer cell immunoglobulin-like receptor, 2 domains, long cytoplasmic tail, 3 (KIR2DL3) Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 2 (LILRB2) Granulysin (GNLY), transcript variant 519 Interleukin 18 receptor accessory protein (IL18RAP) Tumor necrosis factor, α-induced protein 6 (TNFAIP6) Interleukin 1, β (IL1B) Interleukin 2 receptor, β (IL2RB) Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta (NFKBIZ) Interferon regulatory factor 1 (IRF1) Leukocyte specific transcript 1 (LST1), transcript variant 2 Complement component 2 (C2) Complement factor B (CFB) Major histocompatibility complex, class II, DQ α 1 (HLA-DQA1) Major histocompatibility complex, class II, DP β 1 (HLA-DPB1) Tumor necrosis factor, α-induced protein 3 (TNFAIP3) Hepatocyte growth factor (hepapoietin A; scatter factor) (HGF) Toll-like receptor 8 (TLR8), mRNA
0.018 0.032 0.0007 0.015 0.006 0.012 0.004 0.006 0.03 0.002 0.005 0.021 0.004 0.006 0.004 0.009 0.001 0.003 0.0007 0.046 0.004 0.019 0.0007 0.012 0.0007 0.025 0.007 0.042 0.003 0.003 0.003 0.003
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therapeutic intervention for patients who had DSA and was given at 1.5 to 2.0 g/kg (divided into 3-4 doses) to 4 patients in the graft loss group and 6 patients in the functioning graft group. In addition, 3 DSA + patients received plasma exchange therapy (1 in graft loss group) and 2 patients received Rituximab (1 in each group). There was no statistically significant difference between the 2 groups in terms of medical intervention. Gene Expression Profiles
The gene expression profiles of 28 TGP biopsies (8 graft losses and 20 functioning allografts) compared to 11 normal transplant kidney biopsies were studied by microarrays. Although 117 gene transcripts were upregulated and 10 were downregulated in both groups, 86 and 57 gene transcripts were upregulated, 51 and 5 were downregulated in graft loss and functioning allograft groups, respectively. The full list of upregulated intragraft gene transcripts is shown in Table S1, SDC, http://links.lww.com/TP/B113 (Table S1a: upregulated gene transcripts in only graft loss group; Table S1b: in only functioning allograft group; and S1c, in both groups). The majority of the gene transcripts upregulated in both groups were related to immune activity, including CD52, CD53, CD1c, CD1e, CD48, CD69, CD180, Fc fragment of IgG (CD64, CD32, CD16a), adhesion molecules, chemokines
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and chemokine receptors (Selectin L and E, CCR2, CX3CR1, CXCL1, CXCL10, CXCL11), and toll-like receptor 7. Table 4 shows selected upregulated gene transcripts related to immune activity in only graft loss group. These gene transcripts included complement components 1 and 2, complement component 3a receptor 1a, complement factor B, Duffy blood group, chemokine receptor, tumor necrosis factor receptor superfamily, member 9, Interleukin-1 receptor-associated kinase 3, interleukin 1, β, interleukin 2 receptor, β, interleukin 18 receptor accessory protein, interferon regulatory factors 1 and 8, granulysin, and Toll-like receptor 8. When intragraft gene expression profiles of graft loss and functioning allograft group were studied by using PBT, there was no significant difference in gene transcripts associated with cytotoxic T cells, regulatory T cells, B cells, natural killer (NK) cells, macrophages, and DSA-specific transcripts seen in AMR between the 2 groups (Figure 1). There was a statistically significant increase in ENDAT in graft loss patients (P = 0.02). There was a trend toward increased interferon gamma and rejection-induced transcripts (GRIT) in the graft loss TGP patients compared to patients with a functioning allograft (P = 0.05). Figure 2 shows the plot of all ENDAT expression levels for each of the samples. The median transcript levels for each of the sample groups were shown as a correspondingly colored vertical line on the plot. Although 6 of
FIGURE 1. Pathogenesis based transcript gene expression in transplant kidney biopsies of graft loss patients compared to no graft loss patients. Shown on the Y-axis are the mean log2 expression values for the set of genes in each pathogenesis based transcripts. Associated P values are taken from the limma romer analysis. KT, Kidney-specific transcripts, GRIT, γ-interferon and rejection induced transcripts, CAT, cytotoxic T cell–associated transcripts; TREG, regulatory T cell–associated transcripts, BAT = B cell associated transcripts, NKAT, natural killer cell–associated transcripts.
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FIGURE 2. The plot of all endothelial cell-associated transcript expression levels for each of the samples. Graft loss (G2) samples are shown in dark gray and functioning allograft group (G3) samples are shown in light gray. The median for each of the sample groups is shown as a correspondingly colored vertical line on the plot.
the 8 (75%) graft loss patients had higher than median ENDAT levels, only 20% of the functioning allograft group had high ENDAT levels. DISCUSSION Our study demonstrates differential intragraft gene expression profiles in TGP patients with graft loss when compared to patients with functioning allograft. The ENDAT and gene transcripts associated with complement cascade, interleukins and granulysin were significantly upregulated in biopsies of the graft loss group. This result supports the importance of molecular diagnosis in exploring the mechanisms associated with graft loss. Sis et al identified 119 ENDATs from literature and demonstrated these transcripts were increased in AMR biopsy samples and predicted graft loss.19 The authors suggested that increased ENDATs could identify missed AMR cases with negative C4d staining. This data was reviewed and presented at the Banff 2013 meeting and it is now accepted that increased ENDAT could replace C4d staining to define chronic active AMR.20 We recently reported the mechanisms involved in development of DSA and/or C4d negative TGP by allograft gene expression profiles using microarrays comparing to DSA +/C4d + chronic AMR biopsies.13 We demonstrated increased expression of gene transcripts related to cytotoxic T cells, NK cells, macrophages, interferon-gamma, and AMR in both, DSA+/C4d + chronic AMR and DSA+/C4d- TGP biopsy specimens, suggesting that DSA + TGP patients could be classified as chronic AMR despite negative C4d staining. However, ENDAT expression was upregulated only in DSA +/C4d + chronic AMR biopsies in our previous study.13 Our current study also demonstrated a trend toward graft loss with increased expression of GRIT and complement cascade. Although DSA and antibody mediated injury play a major role in the pathogenesis of TGP, we have previously shown that TGP can develop without antibodies21 and could develop through cellular immune response.13,22 Increased
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cytotoxic T cell associated transcripts were the only gene transcripts found to be increased in DSA-TGP biopsies in our previous study.13 Infiltration of the allograft by T cells and NK cells suggested by increased T cell and NK cell transcripts as shown in some studies,14 leads to local interferon-gamma production. This response is essential for induction of class I and II antigens on endothelium23 and in turn increases antibody binding to the endothelium, which is followed by complement fixation to induce allograft inflammation and injury. Interferon-γ–associated gene transcripts were identified in both AMR and cellular rejection.24 Increased intragraft GRIT and complement cascade related gene transcript expression in graft loss patients in our cohort suggest more active alloimmune injury. Hidalgo et al25 studied the gene expression profiles of biopsy specimens with AMR and identified 23 selective gene transcripts in AMR but not cellular rejection, those were recognized as DSAST (DSA-associated transcripts). The DSAST were significantly upregulated in biopsy specimen of patients with TGP associated with DSA and chronic AMR biopsy specimens.13 We identified a tendency toward graft loss in TGP patients with high DSAST expression in their biopsy specimens, although this did not reach statistical significance (P = 0.06). TGP develops through endothelial damage, and increased ENDAT expression in graft loss group might suggest more ongoing endothelial damage. Microvascular inflammation determined by peritubular capillaritis and glomerulitis has been shown to be a good histological marker for endothelial damage and was found to predict graft failure in AMR cases.26 However, we did not find any difference in microvascular inflammation scores in our cohort of TGP patients. Proteinuria has been recognized as a risk factor for poor outcomes in many kidney diseases, including TGP,5,6,8 as well as higher cg scores,5,8 demonstrating the association between the degree of allograft damage and graft survival. Increased proteinuria levels and Banff cg score was the most important clinical and histopathological predictors for poor outcome in our study. We failed to demonstrate an association between degree of other Banff chronic injury scores and graft loss, as was previously shown by other investigators.9 Similarly, C4d deposition in the peritubular capillaries was linked with reduced graft survival in some studies6,7 but we did not find this correlation in our cohort of patients. Poorer kidney function at diagnosis (estimated by serum creatinine or glomerular filtration rate) was linked to poor graft outcomes in many studies.5,6,9 Although our graft loss patients had higher serum creatinine levels at the time of biopsy, this was not statistically significant. There are probably 2 main reasons that we could not able demonstrate the association between the graft failure and serum creatinine levels and histopathologic injury scores. One is the follow-up time, which is shorter in our study, approximately 2 years, and the other is the small number of patients involved. In terms of immunological variables, DSAs, particularly those directed to HLA class II antigens, play a central role in the pathogenesis of TGP and are detected in 70% to 88% of patients with TGP in some studies.5,27-29 In our study, 61% of TGP patients had DSAs, but there was no association between DSA frequency or MFI values of class I and II DSAs between the graft loss and no graft loss groups. In our previous report, there was no difference in graft survival between DSA + and DSA- TGP patients.13 This contrasts with the findings of
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some of the studies that linked presence of DSA11 and higher levels of class II HLA antibodies to reduced graft survival in TGP patients.6 There is no known effective treatment for TGP. Intensification of immunosuppression is adopted by most centers once the diagnosis of TGP is made. Intavenous immunoglobulin (IVIG), plasmapheresis, and rituximab have been tried for the treatment of DSA + TGP patients with variable outcomes.30,31 Acute AMR is now considered a treatable condition with the use of antibody removal strategies, IVIG, rituximab, and bortezomib. On the other hand, the antibodymediated injury seen in TGP tends to have a protracted time course making acute antibody removal strategies less effective. Haas and Mirocha32 reported the successful prevention of overt TGP in some patients who received AMR therapy (IVIG, rituximab, plasmapheresis) after the detection of microcirculation inflammation, a few glomerular double contours, and endothelial cell ultrastuctural changes identifiable by electron microscopy in early biopsy specimens. Therefore, there is a value in detecting early ultrastructural changes by electron microscopy before double contouring of the GBM is evident by light microscopy. The most recent Banff meeting20 favored incorporation of electron microscopy findings into the definition of cg, given that endothelial and GBM lesions detectable within the first 3 months after transplantation by electron microscopy are highly correlated with later development of overt TG. CONCLUSIONS Although our study is limited by the small number of samples available for gene analysis, we were able to demonstrate an association of allograft loss in TGP patients with increased intragraft expression of ENDAT and gene transcripts associated with complement cascade, interleukins and granulysin. Larger, multicenter studies are needed to further validate our findings. REFERENCES 1. Husain S, Sis B. Advances in the understanding of transplant glomerulopathy. Am J Kidney Dis 2013;62(2):352. 2. Racusen LC, Colvin RB, Solez K, et al. Antibody-mediated rejection criteria—an addition to the Banff 97 classification of renal allograft rejection. Am J Transplant 2003;3(6):708. 3. Baid-Agrawal S, Farris AB 3rd, Pascual M, et al. Overlapping pathways to transplant glomerulopathy: chronic humoral rejection, hepatitis C infection, and thrombotic microangiopathy. Kidney Int 2011;80(8):879. 4. Gloor JM, Cosio FG, Rea DJ, et al. Histologic findings one year after positive crossmatch or ABO blood group incompatible living donor kidney transplantation. Am J Transplant 2006;6(8):1841. 5. Gloor JM, Sethi S, Stegall MD, et al. Transplant glomerulopathy: subclinical incidence and association with alloantibody. Am J Transplant 2007;7(9):2124. 6. Issa N, Cosio FG, Gloor JM, et al. Transplant glomerulopathy: risk and prognosis related to anti-human leukocyte antigen class II antibody levels. Transplantation 2008;86(5):681. 7. Kieran N, Wang X, Perkins J, et al. Combination of peritubular c4d and transplant glomerulopathy predicts late renal allograft failure. J Am Soc Nephrol 2009;20(10):2260. 8. Banfi G, Villa M, Cresseri D, Ponticelli C. The clinical impact of chronic transplant glomerulopathy in cyclosporine era. Transplantation 2005;80 (10):1392. 9. John R, Konvalinka A, Tobar A, Kim SJ, Reich HN, Herzenberg AM. Determinants of long-term graft outcome in transplant glomerulopathy. Transplantation 2010;90(7):757.
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10. Cosio FG, Gloor JM, Sethi S, Stegall MD. Transplant glomerulopathy. Am J Transplan 2008;8(3):492. 11. Eng HS, Bennett G, Chang SH, et al. Donor human leukocyte antigen specific antibodies predict development and define prognosis in transplant glomerulopathy. Hum Immunol 2011;72(5):386. 12. Elster EA, Hawksworth JS, Cheng O, et al. Probabilistic (Bayesian) modeling of gene expression in transplant glomerulopathy. J Mol Diagn 2010;12(5):653. 13. Hayde N, Bao Y, Pullman J, et al. The clinical and genomic significance of donor-specific antibody-positive/c4d-negative and donor-specific antibodynegative/c4d-negative transplant glomerulopathy. Clin J Am Soc Nephrol 2013;8(12):2141. 14. Dean PG, Park WD, Cornell LD, Gloor JM, Stegall MD. Intragraft gene expression in positive crossmatch kidney allografts: ongoing inflammation mediates chronic antibody-mediated injury. Am J Transplant 2012;12(6): 1551. 15. Sis B, Mengel M, Haas M, et al. Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups. Am J Transplant 2010;10(3):464. 16. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant 2008;8(4):753. 17. Hayde N, Broin PO, Bao Y, et al. Increased intragraft rejection-associated gene transcripts in patients with donor-specific antibodies and normal biopsies. In: Kidney Int 2014. 18. Pfoertner S, Jeron A, Probst-Kepper M, et al. Signatures of human regulatory T cells: an encounter with old friends and new players. Genome Biol 2006;7(7):R54. 19. Sis B, Jhangri GS, Bunnag S, Allanach K, Kaplan B, Halloran PF. Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am J Transplant 2009;9(10):2312. 20. Haas M, Sis B, Racusen LC, et al. Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am J Transplant 2014;14(2):272. 21. Akalin E, Dinavahi R, Dikman S, et al. Transplant glomerulopathy may occur in the absence of donor-specific antibody and C4d staining. Clin J Am Soc Nephrol 2007;2(6):1261. 22. Akalin E, Dikman S, Murphy B, Bromberg JS, Hancock WW. Glomerular infiltration by CXCR3+ ICOS + activated T cells in chronic allograft nephropathy with transplant glomerulopathy. Am J Transplant 2003;3(9): 1116. 23. Goes N, Urmson J, Hobart M, Halloran PF. The unique role of interferongamma in the regulation of MHC expression on arterial endothelium. Transplantation 1996;62(12):1889. 24. Mueller TF, Einecke G, Reeve J, et al. Microarray analysis of rejection in human kidney transplants using pathogenesis-based transcript sets. Am J Transplant 2007;7(12):2712. 25. Hidalgo LG, Sis B, Sellares J, et al. NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection. Am J Transplant 2010;10(8):1812. 26. Sis B, Jhangri GS, Riopel J, et al. A new diagnostic algorithm for antibodymediated microcirculation inflammation in kidney transplants. Am J Transplant 2012;12(5):1168. 27. Mauiyyedi S, Pelle PD, Saidman S, et al. Chronic humoral rejection: identification of antibody-mediated chronic renal allograft rejection by C4d deposits in peritubular capillaries. J Am Soc Nephrol 2001;12(3):574. 28. Sis B, Campbell PM, Mueller T, et al. Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J Transplant 2007;7(7):1743. 29. Willicombe M, Brookes P, Sergeant R, et al. De novo DQ donor-specific antibodies are associated with a significant risk of antibody-mediated rejection and transplant glomerulopathy. Transplantation 2012;94(2):172. 30. Billing H, Rieger S, Susal C, et al. IVIG and rituximab for treatment of chronic antibody-mediated rejection: a prospective study in paediatric renal transplantation with a 2-year follow-up. Transpl Int 2012;25(11):1165. 31. Kahwaji J, Najjar R, Kancherla D, et al. Histopathologic features of transplant glomerulopathy associated with response to therapy with intravenous immune globulin and rituximab. Clin Transplant 2014;28(5):546. 32. Haas M, Mirocha J. Early ultrastructural changes in renal allografts: correlation with antibody-mediated rejection and transplant glomerulopathy. Am J Transplant 2011;11(10):2123.
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Clinical, Histological, and Molecular Markers Associated With Allograft Loss in Transplant Glomerulopathy Patients Layla Kamal,1 Pilib Ó. Broin,2 Yi Bao,1 Maria Ajaimy,1 Michelle Lubetzky,1 Anjali Gupta,1 Graciela de Boccardo,1 James Pullman,3 Aaron Golden,2,4 and Enver Akalin1
Background. We aimed to investigate the clinical, histopathological, and molecular factors associated with allograft loss in transplant glomerulopathy (TGP) patients. Methods. Of the 525 patients who underwent clinically indicated kidney biopsies, 52 (10%) had diagnosis of TGP. Gene expression profiles of 28 TGP and 11 normal transplant kidney biopsy samples were analyzed by Affymetrix HuGene 1.0 ST expression arrays. Results. Over a median follow up of 23 months (1-46 months) after the diagnosis of TGP by biopsy, 17 patients (32%) lost their allografts at a median of 16 months (1-44 months). There was no difference between the 2 groups in terms of any demographic variables, serum creatinine, panel reactive antibody levels, donor-specific antibody frequency, or mean fluorescence intensity values. Patients who lost their allograft had a significantly higher median spot protein to creatinine ratio 2.81 (1.20-6.00) compared to no graft loss patients 1.16 (0.15-2.53), (P < 0.01), and a trends toward a higher mean chronic glomerulopathy (cg) score (1.65 ± 0.93 vs 1.11 ± 0.93) (P = 0.05). There was also no difference in microvascular inflammation or any other Banff injury scores between the 2 groups. Although 117 gene transcripts were upregulated in both groups, 86 and 57 were upregulated in graft loss and functioning allograft groups, respectively. There were significantly increased levels of intragraft endothelial cell–associated transcripts, gene transcripts associated with complement cascade, interleukins and their receptors and granulysin in graft loss patients compared to patients with a functioning allograft. Conclusion. Our results demonstrate differential intragraft gene expression profiles in TGP patients with allograft loss.
(Transplantation 2015;99: 1912–1918)
T
ransplant glomerulopathy (TGP) is a pathological diagnosis of the kidney allograft characterized by double contour of the glomerular basement membrane (GBM) in the absence of immune complex deposits.1 Transplant glomerulopathy is recognized by the Banff classification as the cardinal manifestation of chronic antibody-mediated rejection (AMR).2 Endothelial cells, in the presence of donor-specific antibodies (DSA) or autoantibodies are the main target of antibody-mediated injury. Transplant glomerulopathy Received 19 June 2014. Revision requested 14 July 2014. Accepted 13 November 2014. 1
Montefiore-Einstein Center for Transplantation, Montefiore Medical Center, The University Hospital for Albert Einstein College of Medicine, New York, NY.
2 Department of Genetics, Division of Computational Genetics, Montefiore-Einstein Center for Transplantation, Montefiore Medical Center, The University Hospital for Albert Einstein College of Medicine, Bronx, NY. 3
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY.
4
Department of Mathematical Sciences, Yeshiva University, New York, NY.
This study is supported by an Internal Grant from the Montefiore Medical Center and the Albert Einstein College of Medicine. The authors declare no conflicts of interest. This study was accepted at World Transplant Congress 2014 in San Francisco. Dr. Layla Kamal received the best clinical science abstract award at New York Society of Nephrology Fellows’ Night in May 2014. L.K. and E.A. participated in research design, writing of the article, performance of the research, and data analysis. Y.B., P.Ó.B., M.A., M.L., A.G., G.d.B., J.P., and A.G. participated in writing of the article, performance of the research, and data analysis.
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results from subsequent endothelial cell injury and remodeling.1 Although it appears that antibody-mediated injury plays a dominant role in TGP, it can also be caused by other types of insults, both immunologic and nonimmunologic. These include chronic cellular rejection, chronic thrombotic microangiopathy, and chronic hepatitis C infection.3 Its prevalence varies from 1 study to another, more common in sensitized transplant recipients with DSAs4 and its incidence, can reach up to 20% at 5 years.5 Transplant glomerulopathy carries a poor prognosis and is associated with decreased allograft survival. In a large series of kidney transplant recipients, graft loss was observed in 38% of TGP patients at 5 years compared to 5% in patients without TGP.5 Among patients with TGP, predictors of poor outcome included C4d deposition in the peritubular capillaries,6,7 higher serum creatinine and proteinuria at diagnosis,8 the severity of interstitial fibrosis,9 and GBM duplication (higher Banff “chronic glomerulopathy” [cg] score),10 Correspondence: Enver Akalin, MD, Montefiore-Einstein Center for Transplantation, Montefiore Medical Center, The University Hospital for Albert Einstein College of Medicine, 111 East 210th Street, Bronx, NY 10467. ([email protected]) Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/15/9909-1912 DOI: 10.1097/TP.0000000000000598
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and the presence of DSA.11 However, the importance of microvascular inflammation determined by peritubular capillaritis (ptc) and glomerulitis (g) scores, and the strength of DSA has not been explored thoroughly. Few studies explored the gene expression profiles of TGP.12-14 We have recently reported the intragraft gene expression profiles of TGP patients, with or without DSA and found that TGP patients with DSA have indistinguishable profiles to those seen in chronic antibody mediated injury, whereas TGP without C4d or DSA demonstrated upregulation of cytotoxic immune response.13 The association between gene expression profiles of biopsies and the allograft survival, however, has not been investigated. In this study, in addition to known clinical and demographic factors associated with poor outcomes in TGP, we specifically aimed to investigate if intragraft gene expression profiles by microarrays, microvascular inflammation scores, and the strength of DSA determined by mean fluorescence intensity (MFI) values are associated with allograft loss in TGP patients. PATIENTS AND METHODS Patient Population
The study population was derived from renal allograft biopsies diagnosed with TGP at Montefiore Medical Center, Bronx, NY from January 2009 to December 2012. All biopsies were clinically indicated for elevated creatinine or proteinuria. A retrospective chart review was conducted to record clinical, demographic, immunological, and histopathological parameters and graft outcomes. Graft loss was defined as requiring dialysis and/or retransplantation during follow-up. Histopathology
Biopsies were examined by light microscopy using hematoxylin and eosin, periodic acid-Schiff, Masson Trichrome, and C4d immunoperoxidase stains. Immunoperoxidase staining for C4d was performed on paraffin embedded sections using a polyclonal rabbit antihuman antibody (Cell Marque, Rocklin, CA) at a dilution of 1:100 with the Dako Envision system. Evaluation of the biopsies were based on the Banff ‘07 and ‘0915,16 acute and chronic indices including glomerulitis (g), interstitial inflammation (i), tubulitis (t), intimal arteritis (v), peritubular capillaritis (ptc), TGP (cg), mesangial matrix increase (m), interstitial fibrosis (ci), tubular atrophy (ct), vascular fibrous intimal thickening (cv), arteriolar hyaline thickening (ah), and C4d. Microvascular inflammation score was defined as the sum of g and ptc scores. The cg scores of 1, 2, and 3 correspond to double contours in 1% to 25%, 26% to 50%, and more than 50% of the peripheral capillary loops, respectively. Biopsies were diagnosed as TGP by electron microscopy if they showed electron-lucent widening of the subendothelial zone of the GBM, subendothelial accumulation of flocculent material, with or without a new subendothelial basement membrane layer.
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Statistical Analysis
Statistical analyses were performed using the R statistical programming environment. Demographic and clinical data were compared using the Wilcoxon rank-sum for continuous variables and were reported as median (interquartile range) or mean (standard deviation) as appropriate. Fisher exact test was used for categorical variables. A P value cutoff less than 0.05 was considered statistically significant. Gene Expression Profiles by Microarrays
A subgroup of patients who were enrolled in the IRBapproved “Immune Monitoring Study” and had clinically indicated biopsy samples were included in the analysis. A total of 28 biopsy samples (8 graft losses and 20 functioning allografts) were used for gene expression profiling and compared to 11 normal transplant kidney biopsies. One additional biopsy core was collected for gene expression analysis. It was immediately placed into a vial containing 1 ml of RNALater (Ambion, Catalog Number AM7023), stored at −20 °C for 24 hours and then transferred to −70 °C until RNA isolation. Renal tissue content in all cores was verified using a dissecting microscope and nonrenal tissue was removed. The gene expression profiles were studied using Affymetrix Human Gene 1.0 ST Array hybridization containing 28,869 gene probe sets as described previously.13,17 Gene Set libraries were taken from the Molecular Signatures Database, and the new libraries were generated by mapping HUGO gene identifiers to the University of Alberta, pathogenesis-based transcripts (PBT http://transplants.med.ualberta.ca/Nephlab/data/gene_lists.html) gene sets and a set of 33 genes specific for human regulatory T cells (TREG) identified by Pfoertner et al as upregulated genes compared to naive T cells.18 The following PBT were analyzed (n corresponds to the number of genes): (1) Kidney-specific transcripts (n = 570). (2) GRIT: Gamma-IFN and rejection induced transcripts (n = 50). (3) CAT1-IQR: Cytotoxic T cell–associated transcripts (n = 143). (4) TREG: Regulatory T cell associated transcripts (n = 33). (5) BAT-IQR: B cell associated transcripts (n = 50). (6) NKAT-IQR: Natural killer cell associated transcripts (n = 134). (7)AMA-IQR: Alternate macrophage-associated transcripts (n = 94). (8) DSAST: Transcripts differentially expressed between rejection-classified biopsies from DSA positive patients compared to DSA negative patients (n = 21). (9) ENDAT: Endothelial cell associated transcripts (n = 114).
The data discussed in this publication has been deposited in the GEO data archive and are available under accession number GSE58601. RESULTS
HLA Antibody Screening
Demographics and Clinical Characteristics
Anti–HLA antibodies were studied by Luminex HLA Single Antigen Bead assays (LABScreen, One Lambda Inc., Canoga Park, CA) that use a panel of color-coded beads coated with purified single HLA antigens. The beads include HLA-A, −B, −DR, and -DQ antigens. The cutoff value for a positive DSA was an MFI of 1000 or higher.
Of the 525 patients who underwent clinically indicated transplant kidney biopsies between January 2009 and December 2012, 52 patients (10%) had diagnosis of TGP. Median follow-up time after biopsy was 23 months (range, 1-46 months). Seventeen patients (32%) lost their allografts (defined by return to dialysis or retransplantation) at a
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TABLE 1.
Demographic and clinical characteristics of transplant glomerulopathy patients with and without graft loss Variables
Mean age ± SD Sex, male Race, Black Native kidney disease Diabetes Mellitus Hypertension Glomerulonephritis Hepatitis C virus antibody positive Previous transplant Transplant type, deceased donor Prior acute rejection Median time to biopsy, y Median serum creatinine at diagnosis, mg/dL Median spot urine protein/creatinine, g/g Immunosuppressive regimen Tacrolimus/mycophenolate/prednisone Tacrolimus/prednisone Treatment of transplant glomerulopathy Discontinuation of sirolimus, n Mycophenolate reintroduced or dose increased, n IVIG 1.5 g/kg, n Plasmapheresis, n Rituximab, n
P
Graft loss n = 17
No graft loss n = 35
52 ± 15 53% 47%
50 ± 13 49% 29%
18% 35% 24% 12% 6% 65% 18% 7.2 (4.0-10.9) 2.3 (1.60-3.00) 2.81 (1.20-6.00)
11% 26% 37% 3% 14% 60% 11% 7.5 (2.1-10.9) 1.90 (1.60-2.80) 1.16 (0.15-2.53)
0.67 0.52 0.37 0.25 0.65 1.0 0.67 0.8 0.65 < 0.01
76% 12%
77% 11%
1.0 1.0 1.0
1 2 4 1 1
4 5 6 2 1
median time of 16 months (range, 1-44 months). One patient died with a functioning graft. There was no difference in terms of age, sex, race, etiology of native kidney disease, type of transplant, history of prior transplant or rejection episodes, immunosuppression regimen, hepatitis C virus status, and serum creatinine levels between the graft loss group and the functioning graft group (Table 1). Patients who lost their allograft had a statistically significant higher median spot protein to creatinine ratio of 2.81 (range, 1.20-6.00 g/g), compared to the no graft loss patients whose ratio was 1.16 (range, 0.15-2.53 g/g) (P < 0.01). Immunological Characteristics
Donor-specific antibodies were detected by Luminex single beads assay in 61% of the TGP patients (32/52). Among patients with DSA, 9 patients had DSA to class I HLA, 16 patients to HLA class II, and 7 patients to both classes I and II. There was no statistically significant difference between the 2 groups
0.63 1.0 0.22
in terms of mean class I and II panel reactive antibody levels (Table 2). When comparing graft loss to those with a functioning graft, there was no statistically significant difference in DSA frequency (71% vs 57%) or the mean MFI values of DSAs to HLA-A (2725.75 ± 1693.44 vs 4974.29 ± 3965.94), HLA-B (2665.67 ± 2573.60 vs 5078.80 ± 3456.49), HLA-DQ (10593.8 ± 6333.35 vs 6660.38 ± 4122.22), and HLA-DR (7273.13 ± 5750.39 vs 11060.33 ± 6428.84). Histopathologic Features and Banff Allograft Injury Scores
Glomerular basement membrane duplication was focal involving less than 10% of the capillary loops in 10 patients, corresponding to a Banff cg score of 0, and TGP was diagnosed by electron microscopy. Glomerular basement membrane duplication was mild in 25 patients (cg score of 1), moderate in 9 patients (cg score 2), and severe in 8 patients
TABLE 2.
Immunological characteristics of transplant glomerulopathy patients with and without graft loss Immunological variables
Mean class I PRA Mean class II PRA DSA frequency Mean peak class 1 DSA MFI HLA-A HLA-B Mean peak class II DSA MFI HLA-DQ HLA-DR
Graft loss N = 17
No graft loss N = 35
P
21.18 ± 25.29 47.71 ± 35.77 71%
27.43 ± 30.11 44.97 ± 35.12 57%
0.56 0.66 0.38
2725.75 ± 1693.44 2665.67 ± 2573.60
4974.29 ± 3965.94 5078.80 ± 3456.49
0.41 0.25
10593.8 ± 6333.35 7273.13 ± 5750.39
6660.38 ± 4122.22 11060.33 ± 6428.84
0.22 0.17
PRA, panel reactive antibody; DSA, donor-specific antibody. Mean values were calculated by ± standard deviation.
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TABLE 3.
Histopathological characteristics of TGP patients with and without graft loss Banff injury scores
Graft loss
No graft loss
Mean ± standard deviation
N = 17
N = 35
P
Glomerulitis (g) Peritubular capillaritis (ptc) G + ptc Tubulitis (t) Interstitial inflammation (i) Intimal arteritis (v) Allograft glomerulopathy (cg) Tubular atrophy (ct) Interstitial fibrosis (ci) Vascular fibrosis(cv) Arteriolar hyalinization (ah) Peritubular capillary C4D staining (ptc)
0.82 ± 0.95 1.00 ± 0.79 1.82 ± 1.42 0.18 ± 0.39 1.29 ± 0.77 0.00 1.65 ± 0.93 1.59 ± 0.94 1.59 ± 0.87 0.71 ± 0.92 1.12 ± 1.32 0.53 ± 1.07
0.60 ± 0.74 1.11 ± 1.11 1.71 ± 1.47 0.20 ± 0.47 1.26 ± 1.01 0.00 1.11 ± 0.93 1.66 ± 0.87 1.60 ± 0.81 0.94 ± 0.76 0.97 ± 0.92 0.60 ± 1.09
0.48 0.90 0.74 1.0 0.75 NA 0.05 0.72 0.92 0.20 0.87 0.74
(cg of 3). Interstitial fibrosis/tubular atrophy was seen in 94% of the patients; it was mild in 44% (23/52), moderate in 28% (15/52), and severe in 21% (11/52) of the cases. Peritubular capillary C4d staining was negative in 69% of
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the cases (36/52), diffuse positive in 19% (10/52), focal in 5.5% (3/52), and minimal in 5.5% (3/52) of the cases. There was no statistically significant difference in terms of microvascular inflammation scores (peritubular capillaritis and glomerulitis), tubulitis, interstitial inflammation, interstitial fibrosis, tubular atrophy, vascular fibrosis, or C4d scores between the 2 groups of patients as shown in Table 3. There was a trend toward a higher mean cg score in graft loss patients (1.65 ± 0.93) compared to patients with a functioning allograft (1.11 ± 0.93) (P = 0.05). Treatment of TGP
After the diagnosis of TGP, all the patients were aimed to be on triple immunosuppressive regimen including tacrolimus, mycophenolate mofetil, and prednisone. A total of 5 patients were on sirolimus prior to the diagnosis of TGP, one of which lost their graft. Once the diagnosis of TGP was made, sirolimus was stopped and switched to tacrolimus in all patients. Mycophenolate was reintroduced, or doses were increased in 7 patients (2 in the graft loss group and 5 in the functioning group). All of the patients with proteinuria were started on angiotensin converting enzyme inhibitor or angiotensin receptor blocker unless contraindicated. Administration of intravenous immunoglobulins was the most common
TABLE 4.
Selected intragraft gene transcripts upregulated in only patients with graft loss Transcript cluster ID
7898793 7898799 7906435 7906486 7906757 7912145 7922773 7948455 7951217 7956878 7960464 7960874 7997712 8006608 8025601 8031293 8039212 8043236 8044049 8045688 8054722 8075886 8081386 8114010 8118149 8118324 8118345 8118556 8118594 8122265 8140556 8166065
Gene name
Adjusted P value
Complement component 1, q subcomponent, A chain (C1QA) Complement component 1, q subcomponent, C chain (C1QC), transcript variant 1 Duffy blood group, chemokine receptor (DARC), transcript variant 2 SLAM family member 8 (SLAMF8) Fc fragment of IgG, low affinity IIa, receptor (CD32) (FCGR2A), transcript variant1 Tumor necrosis factor receptor superfamily, member 9 (TNFRSF9) Neutrophil cytosolic factor 2 (NCF2), transcript variant Membrane-spanning 4-domains, subfamily A, member 6A (MS4A6A) Matrix metallopeptidase 7 (matrilysin, uterine) (MMP7) Interleukin-1 receptor-associated kinase 3 (IRAK3), transcript variant 1 Von Willebrand factor (VWF), mRNA Complement component 3a receptor 1 (C3AR1) Interferon regulatory factor 8 (IRF8) Chemokine (C-C motif ) ligand 4-like 1 (CCL4L1) Intercellular adhesion molecule 1 (ICAM1) Killer cell immunoglobulin-like receptor, 2 domains, long cytoplasmic tail, 3 (KIR2DL3) Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 2 (LILRB2) Granulysin (GNLY), transcript variant 519 Interleukin 18 receptor accessory protein (IL18RAP) Tumor necrosis factor, α-induced protein 6 (TNFAIP6) Interleukin 1, β (IL1B) Interleukin 2 receptor, β (IL2RB) Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta (NFKBIZ) Interferon regulatory factor 1 (IRF1) Leukocyte specific transcript 1 (LST1), transcript variant 2 Complement component 2 (C2) Complement factor B (CFB) Major histocompatibility complex, class II, DQ α 1 (HLA-DQA1) Major histocompatibility complex, class II, DP β 1 (HLA-DPB1) Tumor necrosis factor, α-induced protein 3 (TNFAIP3) Hepatocyte growth factor (hepapoietin A; scatter factor) (HGF) Toll-like receptor 8 (TLR8), mRNA
0.018 0.032 0.0007 0.015 0.006 0.012 0.004 0.006 0.03 0.002 0.005 0.021 0.004 0.006 0.004 0.009 0.001 0.003 0.0007 0.046 0.004 0.019 0.0007 0.012 0.0007 0.025 0.007 0.042 0.003 0.003 0.003 0.003
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therapeutic intervention for patients who had DSA and was given at 1.5 to 2.0 g/kg (divided into 3-4 doses) to 4 patients in the graft loss group and 6 patients in the functioning graft group. In addition, 3 DSA + patients received plasma exchange therapy (1 in graft loss group) and 2 patients received Rituximab (1 in each group). There was no statistically significant difference between the 2 groups in terms of medical intervention. Gene Expression Profiles
The gene expression profiles of 28 TGP biopsies (8 graft losses and 20 functioning allografts) compared to 11 normal transplant kidney biopsies were studied by microarrays. Although 117 gene transcripts were upregulated and 10 were downregulated in both groups, 86 and 57 gene transcripts were upregulated, 51 and 5 were downregulated in graft loss and functioning allograft groups, respectively. The full list of upregulated intragraft gene transcripts is shown in Table S1, SDC, http://links.lww.com/TP/B113 (Table S1a: upregulated gene transcripts in only graft loss group; Table S1b: in only functioning allograft group; and S1c, in both groups). The majority of the gene transcripts upregulated in both groups were related to immune activity, including CD52, CD53, CD1c, CD1e, CD48, CD69, CD180, Fc fragment of IgG (CD64, CD32, CD16a), adhesion molecules, chemokines
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and chemokine receptors (Selectin L and E, CCR2, CX3CR1, CXCL1, CXCL10, CXCL11), and toll-like receptor 7. Table 4 shows selected upregulated gene transcripts related to immune activity in only graft loss group. These gene transcripts included complement components 1 and 2, complement component 3a receptor 1a, complement factor B, Duffy blood group, chemokine receptor, tumor necrosis factor receptor superfamily, member 9, Interleukin-1 receptor-associated kinase 3, interleukin 1, β, interleukin 2 receptor, β, interleukin 18 receptor accessory protein, interferon regulatory factors 1 and 8, granulysin, and Toll-like receptor 8. When intragraft gene expression profiles of graft loss and functioning allograft group were studied by using PBT, there was no significant difference in gene transcripts associated with cytotoxic T cells, regulatory T cells, B cells, natural killer (NK) cells, macrophages, and DSA-specific transcripts seen in AMR between the 2 groups (Figure 1). There was a statistically significant increase in ENDAT in graft loss patients (P = 0.02). There was a trend toward increased interferon gamma and rejection-induced transcripts (GRIT) in the graft loss TGP patients compared to patients with a functioning allograft (P = 0.05). Figure 2 shows the plot of all ENDAT expression levels for each of the samples. The median transcript levels for each of the sample groups were shown as a correspondingly colored vertical line on the plot. Although 6 of
FIGURE 1. Pathogenesis based transcript gene expression in transplant kidney biopsies of graft loss patients compared to no graft loss patients. Shown on the Y-axis are the mean log2 expression values for the set of genes in each pathogenesis based transcripts. Associated P values are taken from the limma romer analysis. KT, Kidney-specific transcripts, GRIT, γ-interferon and rejection induced transcripts, CAT, cytotoxic T cell–associated transcripts; TREG, regulatory T cell–associated transcripts, BAT = B cell associated transcripts, NKAT, natural killer cell–associated transcripts.
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Kamal et al
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FIGURE 2. The plot of all endothelial cell-associated transcript expression levels for each of the samples. Graft loss (G2) samples are shown in dark gray and functioning allograft group (G3) samples are shown in light gray. The median for each of the sample groups is shown as a correspondingly colored vertical line on the plot.
the 8 (75%) graft loss patients had higher than median ENDAT levels, only 20% of the functioning allograft group had high ENDAT levels. DISCUSSION Our study demonstrates differential intragraft gene expression profiles in TGP patients with graft loss when compared to patients with functioning allograft. The ENDAT and gene transcripts associated with complement cascade, interleukins and granulysin were significantly upregulated in biopsies of the graft loss group. This result supports the importance of molecular diagnosis in exploring the mechanisms associated with graft loss. Sis et al identified 119 ENDATs from literature and demonstrated these transcripts were increased in AMR biopsy samples and predicted graft loss.19 The authors suggested that increased ENDATs could identify missed AMR cases with negative C4d staining. This data was reviewed and presented at the Banff 2013 meeting and it is now accepted that increased ENDAT could replace C4d staining to define chronic active AMR.20 We recently reported the mechanisms involved in development of DSA and/or C4d negative TGP by allograft gene expression profiles using microarrays comparing to DSA +/C4d + chronic AMR biopsies.13 We demonstrated increased expression of gene transcripts related to cytotoxic T cells, NK cells, macrophages, interferon-gamma, and AMR in both, DSA+/C4d + chronic AMR and DSA+/C4d- TGP biopsy specimens, suggesting that DSA + TGP patients could be classified as chronic AMR despite negative C4d staining. However, ENDAT expression was upregulated only in DSA +/C4d + chronic AMR biopsies in our previous study.13 Our current study also demonstrated a trend toward graft loss with increased expression of GRIT and complement cascade. Although DSA and antibody mediated injury play a major role in the pathogenesis of TGP, we have previously shown that TGP can develop without antibodies21 and could develop through cellular immune response.13,22 Increased
1917
cytotoxic T cell associated transcripts were the only gene transcripts found to be increased in DSA-TGP biopsies in our previous study.13 Infiltration of the allograft by T cells and NK cells suggested by increased T cell and NK cell transcripts as shown in some studies,14 leads to local interferon-gamma production. This response is essential for induction of class I and II antigens on endothelium23 and in turn increases antibody binding to the endothelium, which is followed by complement fixation to induce allograft inflammation and injury. Interferon-γ–associated gene transcripts were identified in both AMR and cellular rejection.24 Increased intragraft GRIT and complement cascade related gene transcript expression in graft loss patients in our cohort suggest more active alloimmune injury. Hidalgo et al25 studied the gene expression profiles of biopsy specimens with AMR and identified 23 selective gene transcripts in AMR but not cellular rejection, those were recognized as DSAST (DSA-associated transcripts). The DSAST were significantly upregulated in biopsy specimen of patients with TGP associated with DSA and chronic AMR biopsy specimens.13 We identified a tendency toward graft loss in TGP patients with high DSAST expression in their biopsy specimens, although this did not reach statistical significance (P = 0.06). TGP develops through endothelial damage, and increased ENDAT expression in graft loss group might suggest more ongoing endothelial damage. Microvascular inflammation determined by peritubular capillaritis and glomerulitis has been shown to be a good histological marker for endothelial damage and was found to predict graft failure in AMR cases.26 However, we did not find any difference in microvascular inflammation scores in our cohort of TGP patients. Proteinuria has been recognized as a risk factor for poor outcomes in many kidney diseases, including TGP,5,6,8 as well as higher cg scores,5,8 demonstrating the association between the degree of allograft damage and graft survival. Increased proteinuria levels and Banff cg score was the most important clinical and histopathological predictors for poor outcome in our study. We failed to demonstrate an association between degree of other Banff chronic injury scores and graft loss, as was previously shown by other investigators.9 Similarly, C4d deposition in the peritubular capillaries was linked with reduced graft survival in some studies6,7 but we did not find this correlation in our cohort of patients. Poorer kidney function at diagnosis (estimated by serum creatinine or glomerular filtration rate) was linked to poor graft outcomes in many studies.5,6,9 Although our graft loss patients had higher serum creatinine levels at the time of biopsy, this was not statistically significant. There are probably 2 main reasons that we could not able demonstrate the association between the graft failure and serum creatinine levels and histopathologic injury scores. One is the follow-up time, which is shorter in our study, approximately 2 years, and the other is the small number of patients involved. In terms of immunological variables, DSAs, particularly those directed to HLA class II antigens, play a central role in the pathogenesis of TGP and are detected in 70% to 88% of patients with TGP in some studies.5,27-29 In our study, 61% of TGP patients had DSAs, but there was no association between DSA frequency or MFI values of class I and II DSAs between the graft loss and no graft loss groups. In our previous report, there was no difference in graft survival between DSA + and DSA- TGP patients.13 This contrasts with the findings of
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Transplantation
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September 2015
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Volume 99
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Number 9
some of the studies that linked presence of DSA11 and higher levels of class II HLA antibodies to reduced graft survival in TGP patients.6 There is no known effective treatment for TGP. Intensification of immunosuppression is adopted by most centers once the diagnosis of TGP is made. Intavenous immunoglobulin (IVIG), plasmapheresis, and rituximab have been tried for the treatment of DSA + TGP patients with variable outcomes.30,31 Acute AMR is now considered a treatable condition with the use of antibody removal strategies, IVIG, rituximab, and bortezomib. On the other hand, the antibodymediated injury seen in TGP tends to have a protracted time course making acute antibody removal strategies less effective. Haas and Mirocha32 reported the successful prevention of overt TGP in some patients who received AMR therapy (IVIG, rituximab, plasmapheresis) after the detection of microcirculation inflammation, a few glomerular double contours, and endothelial cell ultrastuctural changes identifiable by electron microscopy in early biopsy specimens. Therefore, there is a value in detecting early ultrastructural changes by electron microscopy before double contouring of the GBM is evident by light microscopy. The most recent Banff meeting20 favored incorporation of electron microscopy findings into the definition of cg, given that endothelial and GBM lesions detectable within the first 3 months after transplantation by electron microscopy are highly correlated with later development of overt TG. CONCLUSIONS Although our study is limited by the small number of samples available for gene analysis, we were able to demonstrate an association of allograft loss in TGP patients with increased intragraft expression of ENDAT and gene transcripts associated with complement cascade, interleukins and granulysin. Larger, multicenter studies are needed to further validate our findings. REFERENCES 1. Husain S, Sis B. Advances in the understanding of transplant glomerulopathy. Am J Kidney Dis 2013;62(2):352. 2. Racusen LC, Colvin RB, Solez K, et al. Antibody-mediated rejection criteria—an addition to the Banff 97 classification of renal allograft rejection. Am J Transplant 2003;3(6):708. 3. Baid-Agrawal S, Farris AB 3rd, Pascual M, et al. Overlapping pathways to transplant glomerulopathy: chronic humoral rejection, hepatitis C infection, and thrombotic microangiopathy. Kidney Int 2011;80(8):879. 4. Gloor JM, Cosio FG, Rea DJ, et al. Histologic findings one year after positive crossmatch or ABO blood group incompatible living donor kidney transplantation. Am J Transplant 2006;6(8):1841. 5. Gloor JM, Sethi S, Stegall MD, et al. Transplant glomerulopathy: subclinical incidence and association with alloantibody. Am J Transplant 2007;7(9):2124. 6. Issa N, Cosio FG, Gloor JM, et al. Transplant glomerulopathy: risk and prognosis related to anti-human leukocyte antigen class II antibody levels. Transplantation 2008;86(5):681. 7. Kieran N, Wang X, Perkins J, et al. Combination of peritubular c4d and transplant glomerulopathy predicts late renal allograft failure. J Am Soc Nephrol 2009;20(10):2260. 8. Banfi G, Villa M, Cresseri D, Ponticelli C. The clinical impact of chronic transplant glomerulopathy in cyclosporine era. Transplantation 2005;80 (10):1392. 9. John R, Konvalinka A, Tobar A, Kim SJ, Reich HN, Herzenberg AM. Determinants of long-term graft outcome in transplant glomerulopathy. Transplantation 2010;90(7):757.
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10. Cosio FG, Gloor JM, Sethi S, Stegall MD. Transplant glomerulopathy. Am J Transplan 2008;8(3):492. 11. Eng HS, Bennett G, Chang SH, et al. Donor human leukocyte antigen specific antibodies predict development and define prognosis in transplant glomerulopathy. Hum Immunol 2011;72(5):386. 12. Elster EA, Hawksworth JS, Cheng O, et al. Probabilistic (Bayesian) modeling of gene expression in transplant glomerulopathy. J Mol Diagn 2010;12(5):653. 13. Hayde N, Bao Y, Pullman J, et al. The clinical and genomic significance of donor-specific antibody-positive/c4d-negative and donor-specific antibodynegative/c4d-negative transplant glomerulopathy. Clin J Am Soc Nephrol 2013;8(12):2141. 14. Dean PG, Park WD, Cornell LD, Gloor JM, Stegall MD. Intragraft gene expression in positive crossmatch kidney allografts: ongoing inflammation mediates chronic antibody-mediated injury. Am J Transplant 2012;12(6): 1551. 15. Sis B, Mengel M, Haas M, et al. Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups. Am J Transplant 2010;10(3):464. 16. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant 2008;8(4):753. 17. Hayde N, Broin PO, Bao Y, et al. Increased intragraft rejection-associated gene transcripts in patients with donor-specific antibodies and normal biopsies. In: Kidney Int 2014. 18. Pfoertner S, Jeron A, Probst-Kepper M, et al. Signatures of human regulatory T cells: an encounter with old friends and new players. Genome Biol 2006;7(7):R54. 19. Sis B, Jhangri GS, Bunnag S, Allanach K, Kaplan B, Halloran PF. Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am J Transplant 2009;9(10):2312. 20. Haas M, Sis B, Racusen LC, et al. Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am J Transplant 2014;14(2):272. 21. Akalin E, Dinavahi R, Dikman S, et al. Transplant glomerulopathy may occur in the absence of donor-specific antibody and C4d staining. Clin J Am Soc Nephrol 2007;2(6):1261. 22. Akalin E, Dikman S, Murphy B, Bromberg JS, Hancock WW. Glomerular infiltration by CXCR3+ ICOS + activated T cells in chronic allograft nephropathy with transplant glomerulopathy. Am J Transplant 2003;3(9): 1116. 23. Goes N, Urmson J, Hobart M, Halloran PF. The unique role of interferongamma in the regulation of MHC expression on arterial endothelium. Transplantation 1996;62(12):1889. 24. Mueller TF, Einecke G, Reeve J, et al. Microarray analysis of rejection in human kidney transplants using pathogenesis-based transcript sets. Am J Transplant 2007;7(12):2712. 25. Hidalgo LG, Sis B, Sellares J, et al. NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection. Am J Transplant 2010;10(8):1812. 26. Sis B, Jhangri GS, Riopel J, et al. A new diagnostic algorithm for antibodymediated microcirculation inflammation in kidney transplants. Am J Transplant 2012;12(5):1168. 27. Mauiyyedi S, Pelle PD, Saidman S, et al. Chronic humoral rejection: identification of antibody-mediated chronic renal allograft rejection by C4d deposits in peritubular capillaries. J Am Soc Nephrol 2001;12(3):574. 28. Sis B, Campbell PM, Mueller T, et al. Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J Transplant 2007;7(7):1743. 29. Willicombe M, Brookes P, Sergeant R, et al. De novo DQ donor-specific antibodies are associated with a significant risk of antibody-mediated rejection and transplant glomerulopathy. Transplantation 2012;94(2):172. 30. Billing H, Rieger S, Susal C, et al. IVIG and rituximab for treatment of chronic antibody-mediated rejection: a prospective study in paediatric renal transplantation with a 2-year follow-up. Transpl Int 2012;25(11):1165. 31. Kahwaji J, Najjar R, Kancherla D, et al. Histopathologic features of transplant glomerulopathy associated with response to therapy with intravenous immune globulin and rituximab. Clin Transplant 2014;28(5):546. 32. Haas M, Mirocha J. Early ultrastructural changes in renal allografts: correlation with antibody-mediated rejection and transplant glomerulopathy. Am J Transplant 2011;11(10):2123.
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