Association of angiotensin II type 1 receptor antibodies with graft histology, function and survival in paediatric renal transplant recipients

Association of angiotensin II type 1 receptor antibodies with graft histology, function and... ABSTRACT Background We analysed in a carefully phenotyped cohort of paediatric patients the association of serum angiotensin II type 1 receptor antibodies (AT1R-Ab) with specific histological lesions and with graft function and survival in conjunction with overall and complement-binding donor-specific human leucocyte antigen donor-specific antibodies (HLA-DSA). Methods Sera of 62 patients at the time of renal graft biopsy for clinical indication >1 year post-transplant were assessed for AT1R-Ab by enzyme-linked immunosorbent assay (ELISA) and for DSA and C1q-fixing DSA by single-antigen bead technology. Results Serum AT1R-Ab concentration was significantly higher in antibody-mediated rejection (ABMR) than in T-cell-mediated rejection or control. By receiver operating characteristic (ROC) curve analysis, the optimal AT1R-Ab cut-off value discriminating between patients with features of ABMR and those without was 9.5 U/mL. A total of 6 of 28 patients (21.4%) with ABMR were only positive for AT1R-Ab. Patients with AT1R-Ab and HLA-DSA double positivity had a significantly higher vascular micro-inflammation score than DSA-negative patients. The 5-year graft survival was only 59% in the AT1R-Ab-positive group compared with 87% in the AT1R-Ab-negative group. Patients with AT1R-Ab and HLA-DSA double positivity tended to have a more rapid decline of estimated glomerular filtration rate (eGFR) than patients who were only positive for AT1R-Ab or HLA-DSA. In a multivariate Cox regression model of non-invasive factors, C1q-positive HLA-DSA, eGFR and AT1R-Ab positivity were significantly associated with accelerated graft function decline. Conclusions Serum AT1R-Ab positivity in the context of an indication biopsy >1 year post-transplant is associated with the histopathology of ABMR and is an independent non-invasive risk factor for adverse graft outcome. angiotensin II type 1 receptor antibodies, C1q-binding donor-specific antibodies, donor-specific HLA antibodies, graft failure, indication biopsy, paediatric renal transplantation INTRODUCTION Antibody-mediated rejection (ABMR) is the major cause of graft loss in both adult [1–3] and paediatric renal transplant recipients [3, 4]. The majority of these rejections are caused by preformed and/or de novo donor-specific antibodies against human leucocyte antigen donor-specific antibodies (HLA-DSA). However, there is a significant subset of patients with histological features of ABMR in the graft biopsy in whom HLA-DSA cannot be detected in the circulation despite the use of highly sensitive assays such as the single-antigen bead (SAB) technology. In recent years, therefore, there have been increasing efforts directed towards the detection and biological characterization of antibodies against other endothelial targets beside HLA [5]. Newly developed solid-phase assays enable the detection of functional non-HLA antibodies targeting G protein-coupled receptors such as the angiotensin II type 1 receptor. In one study of 63 HLA-DSA-negative adult patients, the presence of angiotensin II type 1 receptor antibodies (AT1R-Ab) was strongly associated with ABMR [6]. In a large prospective study in 599 kidney transplant recipients, the pre-transplant positivity for AT1R-Ab was an independent risk factor for allograft loss [7]. More recent smaller studies have confirmed the increased rejection risk in kidney transplant recipients with preformed AT1R-Ab [8, 9] and its link to a C4d-negative ABMR phenotype [10]. However, findings from adult populations cannot necessarily be extrapolated to paediatric patients. There are unique differences in the immune system in children, namely the naivety of their immune system and differences in alloimmunological reactivity, as reflected by small numbers of antigen-experienced T cells, mature dendritic cells or macrophages and HLA-alloreactive B cells [11]. Up to now, data on AT1R-Ab in paediatric renal transplant recipients have been scarce, from small descriptive case series [12–14]. The aims of this study were to investigate the association of serum AT1R-Ab with specific histological lesions and with graft function and survival in paediatric patients undergoing graft biopsies >1 year post-transplant for clinical indication. Targets of AT1R-Ab are constitutively expressed on the vascular endothelium and expression may be induced or increased during inflammatory events. Analysis of the phenotypic characteristics of biopsies in the presence of AT1R-Ab could provide further evidence linking them to allograft dysfunction. Because AT1R-Ab may exert pathophysiologic effects alone or in synergy with HLA antibodies, we analysed serum AT1R-Ab in conjunction with overall and complement-binding donor-specific HLA-DSA. MATERIALS AND METHODS Study design and patient population This is a retrospective single-centre cohort analysis based on prospectively collected serum samples of all paediatric patients ≤18 years of age at the date of transplantation who were non-presensitized [HLA antibodies negative on complement-dependent cytotoxicity (CDC) and enzyme-linked immunosorbent assay (ELISA) testing], received a kidney transplant at our institution between January 1999 and January 2010 and underwent a kidney graft biopsy for clinical indication (functional impairment or severe de novo proteinuria) >1 year post-transplant (index biopsy). Only patients with index biopsies after January 2004 were included, when routine C4d staining was introduced in our institution. T- or B-cell crossmatch-positive patients and recipients of ABO-incompatible or combined organ transplants were excluded from the analysis. In all, eight patients who met the inclusion criteria were secondarily excluded due to insufficient amounts of serum available or loss of follow-up. Therefore 62 of 70 eligible patients were analysed. Patient and transplant characteristics at the time of index biopsy are depicted in Table 1 and parameters at the time of engrafting are shown in Table 2. Serum HLA-DSA and AT1R-Ab were measured at the time of index biopsy. Serum was collected in the framework of the ongoing prospective Collaborative Transplant Study (CTS) Serum Project. Data on C1q-fixing DSA in this cohort have been published previously [16]. Ethics committee approval was obtained and investigations were performed in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines. Written informed consent was obtained by all parents or guardians and patients when appropriate for their age. This study was designed, analysed and reported according to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines [17]. Table 1 Patient and transplant characteristics at the time of index biopsy (Banff 2015) Characteristics  Entire cohort  No rejection  T-cell-mediated rejectiona  Antibody-mediated rejectionb  P-value    (n = 62)  (n = 19)  (n = 15)  (n = 28)    Age (years), mean ± SD  13.7 ± 5.5  13.3 ± 5.18  12.5 ± 5.36  14.5 ± 5.72  0.489  eGFR (mL/min/1.73 m2), mean ± SDc  41.0 ± 15.3  44.6 ± 16.8  42.8 ± 12.0  37.5 ± 15.5  0.260  Proteinuria (>100 g/mol creatinine), n (%)  8 (12.9)  2 (10.5)  0  6 (21.4)  0.127  Proteinuria (>20 g/mol creatinine), n (%)  29 (46.8)  5 (26.3)  5 (33.3)  19 (67.9)  0.010  Number of pre-biopsies per patient, median (IQR)  2.0 (0–4)  2.0 (1.0–3.0)  2.0 (1.0–2.0)  2.0 (0.25–3.0)  0.943  Patients with BPAR (≥Banff IA) in pre-biopsies, n (%)  17 (27.4)  4.0 (21.1)  3 (20.0)  10 (35.7)  0.413  Patients with treated AR in pre-biopsies, n (%)  32 (51.6)  13 (68.4)  6 (40.0)  14 (50.0)  0.231  Major biopsy features, n (%)   C4d positivity  21 (33.9)  2 (10.5)  0  19 (67.9)  <0.001   TG  19 (30.6)  1 (5.3)  0  18 (64.3)  <0.001  Immunosuppression, n (%)   Tacrolimus  41 (66.1)  16 (84.2)  11 (73.3)  14 (50.0)  0.041   Cyclosporine  18 (29.0)  3 (15.8)  4 (26.7)  11 (39.3)  0.214   CNI-free/SRL  3 (4.8)  0  0  3 (10.7)  0.147   Prednisolone  39 (62.9)  12 (63.2)  13 (86.7)  14 (50.0)  0.060   MMF  52 (83.9)  16 (84.2)  11 (73.3)  25 (89.3)  0.399  A-B-DR-DQ mismatches/8  2.68 ± 1.21  2.58 ± 1.71  2.47 ± 0.83  2.86 ± 0.97  0.558   A-B  1.33 ± 0.70  1.47 ± 0.91  1.33 ± 0.49  1.25 ± 0.65  0.568   DR  0.76 ± 0.53  0.58 ± 0.69  0.73 ± 0.46  0.89 ± 0.42  0.138   DQ  0.58 ± 0.62  0.53 ± 0.70  0.40 ± 0.63  0.71 ± 0.53  0.255  Patients with HLA-DSA, n (%)  29 (46.8)  4 (21.1)  5 (33.3)  20 (71.4)  0.002   Class I  12 (19.4)  1 (5.3)  0  11 (29.3)  0.001   Class II  27 (43.5)  3 (15.8)  5 (33.3)  19 (67.9)  0.001  Time Tx to biopsy (months), median (IQR)  53.5 (33.8–75.0)  51.0 (27.0–63.0)  35.0 (24.0–60.0)  62.0 (39.3–109)  0.067  Follow-up post-biopsy (months), median (IQR)  79.5 (65.0–99.0)  73.0 (62.0–88.0)  90.0 (69.0–105)  80.5 (63.5–115)  0.390  Characteristics  Entire cohort  No rejection  T-cell-mediated rejectiona  Antibody-mediated rejectionb  P-value    (n = 62)  (n = 19)  (n = 15)  (n = 28)    Age (years), mean ± SD  13.7 ± 5.5  13.3 ± 5.18  12.5 ± 5.36  14.5 ± 5.72  0.489  eGFR (mL/min/1.73 m2), mean ± SDc  41.0 ± 15.3  44.6 ± 16.8  42.8 ± 12.0  37.5 ± 15.5  0.260  Proteinuria (>100 g/mol creatinine), n (%)  8 (12.9)  2 (10.5)  0  6 (21.4)  0.127  Proteinuria (>20 g/mol creatinine), n (%)  29 (46.8)  5 (26.3)  5 (33.3)  19 (67.9)  0.010  Number of pre-biopsies per patient, median (IQR)  2.0 (0–4)  2.0 (1.0–3.0)  2.0 (1.0–2.0)  2.0 (0.25–3.0)  0.943  Patients with BPAR (≥Banff IA) in pre-biopsies, n (%)  17 (27.4)  4.0 (21.1)  3 (20.0)  10 (35.7)  0.413  Patients with treated AR in pre-biopsies, n (%)  32 (51.6)  13 (68.4)  6 (40.0)  14 (50.0)  0.231  Major biopsy features, n (%)   C4d positivity  21 (33.9)  2 (10.5)  0  19 (67.9)  <0.001   TG  19 (30.6)  1 (5.3)  0  18 (64.3)  <0.001  Immunosuppression, n (%)   Tacrolimus  41 (66.1)  16 (84.2)  11 (73.3)  14 (50.0)  0.041   Cyclosporine  18 (29.0)  3 (15.8)  4 (26.7)  11 (39.3)  0.214   CNI-free/SRL  3 (4.8)  0  0  3 (10.7)  0.147   Prednisolone  39 (62.9)  12 (63.2)  13 (86.7)  14 (50.0)  0.060   MMF  52 (83.9)  16 (84.2)  11 (73.3)  25 (89.3)  0.399  A-B-DR-DQ mismatches/8  2.68 ± 1.21  2.58 ± 1.71  2.47 ± 0.83  2.86 ± 0.97  0.558   A-B  1.33 ± 0.70  1.47 ± 0.91  1.33 ± 0.49  1.25 ± 0.65  0.568   DR  0.76 ± 0.53  0.58 ± 0.69  0.73 ± 0.46  0.89 ± 0.42  0.138   DQ  0.58 ± 0.62  0.53 ± 0.70  0.40 ± 0.63  0.71 ± 0.53  0.255  Patients with HLA-DSA, n (%)  29 (46.8)  4 (21.1)  5 (33.3)  20 (71.4)  0.002   Class I  12 (19.4)  1 (5.3)  0  11 (29.3)  0.001   Class II  27 (43.5)  3 (15.8)  5 (33.3)  19 (67.9)  0.001  Time Tx to biopsy (months), median (IQR)  53.5 (33.8–75.0)  51.0 (27.0–63.0)  35.0 (24.0–60.0)  62.0 (39.3–109)  0.067  Follow-up post-biopsy (months), median (IQR)  79.5 (65.0–99.0)  73.0 (62.0–88.0)  90.0 (69.0–105)  80.5 (63.5–115)  0.390  AR, acute rejection; BPAR, biopsy-proven acute rejection; CNI, calcineurin inhibitors; HLA-DSA, human leucocyte antigen donor-specific antibodies; MMF, mycophenolate mofetil; SRL, sirolimus; TG, transplant glomerulopathy; Tx, renal transplantation. a Including borderline changes. b Including suspicious for ABMR. c eGFR according to Schwartz et al. [15]. Table 1 Patient and transplant characteristics at the time of index biopsy (Banff 2015) Characteristics  Entire cohort  No rejection  T-cell-mediated rejectiona  Antibody-mediated rejectionb  P-value    (n = 62)  (n = 19)  (n = 15)  (n = 28)    Age (years), mean ± SD  13.7 ± 5.5  13.3 ± 5.18  12.5 ± 5.36  14.5 ± 5.72  0.489  eGFR (mL/min/1.73 m2), mean ± SDc  41.0 ± 15.3  44.6 ± 16.8  42.8 ± 12.0  37.5 ± 15.5  0.260  Proteinuria (>100 g/mol creatinine), n (%)  8 (12.9)  2 (10.5)  0  6 (21.4)  0.127  Proteinuria (>20 g/mol creatinine), n (%)  29 (46.8)  5 (26.3)  5 (33.3)  19 (67.9)  0.010  Number of pre-biopsies per patient, median (IQR)  2.0 (0–4)  2.0 (1.0–3.0)  2.0 (1.0–2.0)  2.0 (0.25–3.0)  0.943  Patients with BPAR (≥Banff IA) in pre-biopsies, n (%)  17 (27.4)  4.0 (21.1)  3 (20.0)  10 (35.7)  0.413  Patients with treated AR in pre-biopsies, n (%)  32 (51.6)  13 (68.4)  6 (40.0)  14 (50.0)  0.231  Major biopsy features, n (%)   C4d positivity  21 (33.9)  2 (10.5)  0  19 (67.9)  <0.001   TG  19 (30.6)  1 (5.3)  0  18 (64.3)  <0.001  Immunosuppression, n (%)   Tacrolimus  41 (66.1)  16 (84.2)  11 (73.3)  14 (50.0)  0.041   Cyclosporine  18 (29.0)  3 (15.8)  4 (26.7)  11 (39.3)  0.214   CNI-free/SRL  3 (4.8)  0  0  3 (10.7)  0.147   Prednisolone  39 (62.9)  12 (63.2)  13 (86.7)  14 (50.0)  0.060   MMF  52 (83.9)  16 (84.2)  11 (73.3)  25 (89.3)  0.399  A-B-DR-DQ mismatches/8  2.68 ± 1.21  2.58 ± 1.71  2.47 ± 0.83  2.86 ± 0.97  0.558   A-B  1.33 ± 0.70  1.47 ± 0.91  1.33 ± 0.49  1.25 ± 0.65  0.568   DR  0.76 ± 0.53  0.58 ± 0.69  0.73 ± 0.46  0.89 ± 0.42  0.138   DQ  0.58 ± 0.62  0.53 ± 0.70  0.40 ± 0.63  0.71 ± 0.53  0.255  Patients with HLA-DSA, n (%)  29 (46.8)  4 (21.1)  5 (33.3)  20 (71.4)  0.002   Class I  12 (19.4)  1 (5.3)  0  11 (29.3)  0.001   Class II  27 (43.5)  3 (15.8)  5 (33.3)  19 (67.9)  0.001  Time Tx to biopsy (months), median (IQR)  53.5 (33.8–75.0)  51.0 (27.0–63.0)  35.0 (24.0–60.0)  62.0 (39.3–109)  0.067  Follow-up post-biopsy (months), median (IQR)  79.5 (65.0–99.0)  73.0 (62.0–88.0)  90.0 (69.0–105)  80.5 (63.5–115)  0.390  Characteristics  Entire cohort  No rejection  T-cell-mediated rejectiona  Antibody-mediated rejectionb  P-value    (n = 62)  (n = 19)  (n = 15)  (n = 28)    Age (years), mean ± SD  13.7 ± 5.5  13.3 ± 5.18  12.5 ± 5.36  14.5 ± 5.72  0.489  eGFR (mL/min/1.73 m2), mean ± SDc  41.0 ± 15.3  44.6 ± 16.8  42.8 ± 12.0  37.5 ± 15.5  0.260  Proteinuria (>100 g/mol creatinine), n (%)  8 (12.9)  2 (10.5)  0  6 (21.4)  0.127  Proteinuria (>20 g/mol creatinine), n (%)  29 (46.8)  5 (26.3)  5 (33.3)  19 (67.9)  0.010  Number of pre-biopsies per patient, median (IQR)  2.0 (0–4)  2.0 (1.0–3.0)  2.0 (1.0–2.0)  2.0 (0.25–3.0)  0.943  Patients with BPAR (≥Banff IA) in pre-biopsies, n (%)  17 (27.4)  4.0 (21.1)  3 (20.0)  10 (35.7)  0.413  Patients with treated AR in pre-biopsies, n (%)  32 (51.6)  13 (68.4)  6 (40.0)  14 (50.0)  0.231  Major biopsy features, n (%)   C4d positivity  21 (33.9)  2 (10.5)  0  19 (67.9)  <0.001   TG  19 (30.6)  1 (5.3)  0  18 (64.3)  <0.001  Immunosuppression, n (%)   Tacrolimus  41 (66.1)  16 (84.2)  11 (73.3)  14 (50.0)  0.041   Cyclosporine  18 (29.0)  3 (15.8)  4 (26.7)  11 (39.3)  0.214   CNI-free/SRL  3 (4.8)  0  0  3 (10.7)  0.147   Prednisolone  39 (62.9)  12 (63.2)  13 (86.7)  14 (50.0)  0.060   MMF  52 (83.9)  16 (84.2)  11 (73.3)  25 (89.3)  0.399  A-B-DR-DQ mismatches/8  2.68 ± 1.21  2.58 ± 1.71  2.47 ± 0.83  2.86 ± 0.97  0.558   A-B  1.33 ± 0.70  1.47 ± 0.91  1.33 ± 0.49  1.25 ± 0.65  0.568   DR  0.76 ± 0.53  0.58 ± 0.69  0.73 ± 0.46  0.89 ± 0.42  0.138   DQ  0.58 ± 0.62  0.53 ± 0.70  0.40 ± 0.63  0.71 ± 0.53  0.255  Patients with HLA-DSA, n (%)  29 (46.8)  4 (21.1)  5 (33.3)  20 (71.4)  0.002   Class I  12 (19.4)  1 (5.3)  0  11 (29.3)  0.001   Class II  27 (43.5)  3 (15.8)  5 (33.3)  19 (67.9)  0.001  Time Tx to biopsy (months), median (IQR)  53.5 (33.8–75.0)  51.0 (27.0–63.0)  35.0 (24.0–60.0)  62.0 (39.3–109)  0.067  Follow-up post-biopsy (months), median (IQR)  79.5 (65.0–99.0)  73.0 (62.0–88.0)  90.0 (69.0–105)  80.5 (63.5–115)  0.390  AR, acute rejection; BPAR, biopsy-proven acute rejection; CNI, calcineurin inhibitors; HLA-DSA, human leucocyte antigen donor-specific antibodies; MMF, mycophenolate mofetil; SRL, sirolimus; TG, transplant glomerulopathy; Tx, renal transplantation. a Including borderline changes. b Including suspicious for ABMR. c eGFR according to Schwartz et al. [15]. Table 2 Patient and transplant characteristics at time of transplantation according to AT1R-Ab status Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  Age (years), mean ± SD  8.6 ± 5.0  9.03 ± 4.80  8.30 ± 5.22  0.570  Male gender, n (%)  46 (74.2)  21 (70)  25 (78.1)  0.465  Donor age (years), mean ± SD  36.2 ± 12.9  36.2 ± 14.1  36.2 ± 11.9  0.992  Deceased donor, n (%)  40 (64.5)  21 (70)  19 (59.4)  0.533  Cold ischaemia time (h), mean ± SD  13.9 ± 6.4  13.3 ± 6.76  14.7 ± 5.83  0.576  Delayed graft function, n (%)  6 (9.7)  4 (13.3)  2 (6.3)  0.346  A-B-DR-DQ mismatch/8  2.68 ± 1.21  2.70 ± 1.15  2.66 ± 1.29  0.888   A-B  1.33 ± 0.70  1.50 ± 0.68  1.19 ± 0.70  0.079   DR  0.76 ± 0.53  0.67 ± 0.55  0.84 ± 0.52  0.194   DQ  0.58 ± 0.62  0.53 ± 0.63  0.63 ± 0.61  0.562  Cause of ESRD, n (%)   CAKUT  33 (53.2)  18 (60)  15 (46.9)  0.301   Nephronophthisis  5 (8.1)  2 (6.7)  3 (9.4)  0.696   Glomerular diseases  11 (17.7)  5 (16.7)  6 (18.8)  0.830   Others  13 (21.0)  5 (16.7)  8 (25)  0.421  Immunosuppression, n (%)   IL-2RA  12 (19.4)  4 (13.3)  8 (25)  0.245   Cyclosporine  40 (64.5)  19 (63.3)  21 (65.6)  0.851   Tacrolimus  22 (35.4)  11 (36.7)  11 (34.3)  0.851   MMF  62 (100)  30 (100)  32 (100)  1.000   Prednisolone  62 (100)  30 (100)  32 (100)  1.000  Observation time post-Tx (months), median (IQR)  141 (119–173)  137 (117–162)  147 (121–175)  0.307  Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  Age (years), mean ± SD  8.6 ± 5.0  9.03 ± 4.80  8.30 ± 5.22  0.570  Male gender, n (%)  46 (74.2)  21 (70)  25 (78.1)  0.465  Donor age (years), mean ± SD  36.2 ± 12.9  36.2 ± 14.1  36.2 ± 11.9  0.992  Deceased donor, n (%)  40 (64.5)  21 (70)  19 (59.4)  0.533  Cold ischaemia time (h), mean ± SD  13.9 ± 6.4  13.3 ± 6.76  14.7 ± 5.83  0.576  Delayed graft function, n (%)  6 (9.7)  4 (13.3)  2 (6.3)  0.346  A-B-DR-DQ mismatch/8  2.68 ± 1.21  2.70 ± 1.15  2.66 ± 1.29  0.888   A-B  1.33 ± 0.70  1.50 ± 0.68  1.19 ± 0.70  0.079   DR  0.76 ± 0.53  0.67 ± 0.55  0.84 ± 0.52  0.194   DQ  0.58 ± 0.62  0.53 ± 0.63  0.63 ± 0.61  0.562  Cause of ESRD, n (%)   CAKUT  33 (53.2)  18 (60)  15 (46.9)  0.301   Nephronophthisis  5 (8.1)  2 (6.7)  3 (9.4)  0.696   Glomerular diseases  11 (17.7)  5 (16.7)  6 (18.8)  0.830   Others  13 (21.0)  5 (16.7)  8 (25)  0.421  Immunosuppression, n (%)   IL-2RA  12 (19.4)  4 (13.3)  8 (25)  0.245   Cyclosporine  40 (64.5)  19 (63.3)  21 (65.6)  0.851   Tacrolimus  22 (35.4)  11 (36.7)  11 (34.3)  0.851   MMF  62 (100)  30 (100)  32 (100)  1.000   Prednisolone  62 (100)  30 (100)  32 (100)  1.000  Observation time post-Tx (months), median (IQR)  141 (119–173)  137 (117–162)  147 (121–175)  0.307  CAKUT, congenital anomalies of the kidney and urinary tract; ESRD, end-stage renal disease; IL-2RA, interleukin-2 receptor antagonist; MMF, mycophenolate mofetil; Tx, renal transplantation. Table 2 Patient and transplant characteristics at time of transplantation according to AT1R-Ab status Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  Age (years), mean ± SD  8.6 ± 5.0  9.03 ± 4.80  8.30 ± 5.22  0.570  Male gender, n (%)  46 (74.2)  21 (70)  25 (78.1)  0.465  Donor age (years), mean ± SD  36.2 ± 12.9  36.2 ± 14.1  36.2 ± 11.9  0.992  Deceased donor, n (%)  40 (64.5)  21 (70)  19 (59.4)  0.533  Cold ischaemia time (h), mean ± SD  13.9 ± 6.4  13.3 ± 6.76  14.7 ± 5.83  0.576  Delayed graft function, n (%)  6 (9.7)  4 (13.3)  2 (6.3)  0.346  A-B-DR-DQ mismatch/8  2.68 ± 1.21  2.70 ± 1.15  2.66 ± 1.29  0.888   A-B  1.33 ± 0.70  1.50 ± 0.68  1.19 ± 0.70  0.079   DR  0.76 ± 0.53  0.67 ± 0.55  0.84 ± 0.52  0.194   DQ  0.58 ± 0.62  0.53 ± 0.63  0.63 ± 0.61  0.562  Cause of ESRD, n (%)   CAKUT  33 (53.2)  18 (60)  15 (46.9)  0.301   Nephronophthisis  5 (8.1)  2 (6.7)  3 (9.4)  0.696   Glomerular diseases  11 (17.7)  5 (16.7)  6 (18.8)  0.830   Others  13 (21.0)  5 (16.7)  8 (25)  0.421  Immunosuppression, n (%)   IL-2RA  12 (19.4)  4 (13.3)  8 (25)  0.245   Cyclosporine  40 (64.5)  19 (63.3)  21 (65.6)  0.851   Tacrolimus  22 (35.4)  11 (36.7)  11 (34.3)  0.851   MMF  62 (100)  30 (100)  32 (100)  1.000   Prednisolone  62 (100)  30 (100)  32 (100)  1.000  Observation time post-Tx (months), median (IQR)  141 (119–173)  137 (117–162)  147 (121–175)  0.307  Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  Age (years), mean ± SD  8.6 ± 5.0  9.03 ± 4.80  8.30 ± 5.22  0.570  Male gender, n (%)  46 (74.2)  21 (70)  25 (78.1)  0.465  Donor age (years), mean ± SD  36.2 ± 12.9  36.2 ± 14.1  36.2 ± 11.9  0.992  Deceased donor, n (%)  40 (64.5)  21 (70)  19 (59.4)  0.533  Cold ischaemia time (h), mean ± SD  13.9 ± 6.4  13.3 ± 6.76  14.7 ± 5.83  0.576  Delayed graft function, n (%)  6 (9.7)  4 (13.3)  2 (6.3)  0.346  A-B-DR-DQ mismatch/8  2.68 ± 1.21  2.70 ± 1.15  2.66 ± 1.29  0.888   A-B  1.33 ± 0.70  1.50 ± 0.68  1.19 ± 0.70  0.079   DR  0.76 ± 0.53  0.67 ± 0.55  0.84 ± 0.52  0.194   DQ  0.58 ± 0.62  0.53 ± 0.63  0.63 ± 0.61  0.562  Cause of ESRD, n (%)   CAKUT  33 (53.2)  18 (60)  15 (46.9)  0.301   Nephronophthisis  5 (8.1)  2 (6.7)  3 (9.4)  0.696   Glomerular diseases  11 (17.7)  5 (16.7)  6 (18.8)  0.830   Others  13 (21.0)  5 (16.7)  8 (25)  0.421  Immunosuppression, n (%)   IL-2RA  12 (19.4)  4 (13.3)  8 (25)  0.245   Cyclosporine  40 (64.5)  19 (63.3)  21 (65.6)  0.851   Tacrolimus  22 (35.4)  11 (36.7)  11 (34.3)  0.851   MMF  62 (100)  30 (100)  32 (100)  1.000   Prednisolone  62 (100)  30 (100)  32 (100)  1.000  Observation time post-Tx (months), median (IQR)  141 (119–173)  137 (117–162)  147 (121–175)  0.307  CAKUT, congenital anomalies of the kidney and urinary tract; ESRD, end-stage renal disease; IL-2RA, interleukin-2 receptor antagonist; MMF, mycophenolate mofetil; Tx, renal transplantation. Detection of HLA-DSA and AT1R antibodies Patient sera were tested for the presence of AT1R-Ab using a quantitative ELISA (CellTrend, Luckenwalde, Germany). All sera were also analysed for HLA antibodies using LABScreen Luminex kits (One Lambda, Canoga Park, CA, USA). For high-resolution typing of patients and their respective organ donors, polymerase chain reaction-sequence specific primers (SSP) Tray and Sequence kits (CTR, Heidelberg, Germany) and SSP kits (Olerup, Saltsjöbaden, Sweden) were used. DSA was determined against mismatched donor alleles from HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1 and -DPB1 loci and a mean fluorescence intensity (MFI) ≥500 was considered positive [18]. Blood pressure and estimated glomerular filtration rate (eGFR) analysis Systolic and diastolic blood pressures were derived from casual office blood pressure measurements (average of three measurements taken within 5 min) by oscillometry obtained at the time of index biopsy and at three to five outpatient department visits prior to index biopsy and converted to z-scores based on reference data [19]. Arterial hypertension was defined as systolic and/or diastolic blood pressure >1.96 z-score (95th percentile). Graft function was assessed using eGFR according to the Schwartz formula [15]. Immunosuppressive regimen The initial immunosuppressive therapy is depicted in Table 2. None of the patients received anti-thymocyte globulin or rituximab as induction therapy or prior to index biopsy. The immunosuppressive therapy at the time of index biopsy is depicted in Table 1. Three patients received the mammalian target of rapamycin (mTOR) inhibitor sirolimus without a calcineurin inhibitor (CNI) due to severe CNI-induced chronic nephrotoxicity in a previous biopsy. A total of 23 patients (37%) were treated with a corticosteroid-free immunosuppressive regimen. Patients with T-cell-mediated allograft rejection (TCMR) of Banff type I or II were treated with methylprednisolone pulse therapy. Patients with biopsy-proven chronic ABMR received anti-humoral rejection therapy as previously described [20]. None of the patients received intravenous immunoglobulin G during a period of at least 3 months prior to measurement of AT1R-Ab. Histopathology and C4d staining Renal biopsies were carried out as indication biopsies due to an increase of serum creatinine (>20% above baseline without an alternative explanation) and/or de novo persistent proteinuria >100 g protein/mol creatinine. All biopsy specimens were graded using the Banff 2009–15 criteria [21]. Immunohistochemistry for C4d was performed on paraffin sections using a polyclonal antibody (C4dpAb; Bio-medica, Vienna, Austria). Statistical analysis Analyses were performed using Predictive Analytics Software (SPSS) Statistics 22.0 (IBM, Armonk, NY, USA) and Statistical Analysis Software 9.3 (SAS Institute, Cary, NC, USA). Unless stated otherwise, results for continuous variables are presented mean ± SD or as median with interquartile range (IQR). Differences between groups were analysed with one-way analysis of variance (ANOVA), Student’s t-test or, if normality failed, with Kruskal–Wallis or Mann–Whitney U rank-sum test. For categorical data, Pearson chi-square tests were used. Receiver operating characteristic (ROC) plots were generated and area under the curve (AUC) and 95% confidence interval (CI) limits were calculated using the method of Hanley and McNeil [22]. Cox proportional hazards regression analysis and Kaplan–Meier survival analysis were used for time-to-event analyses from index biopsy and tested for significance with the two-sided log-rank test. For multivariable Cox regression models, a forward stepwise selection method was used, with a significance level of P < 0.1 for entering a variable into the model and P ≥ 0.2 for removal of a previously selected variable. P < 0.05 was regarded as statistically significant in a descriptive sense. RESULTS Patient characteristics Patient, donor and transplant characteristics at the time of transplantation are given in Table 2. First, patients were divided into three groups according to index biopsy results: (i) patients with normal biopsy results [n = 19 (30.6%)], (ii) patients with features of TCMR including borderline changes [n = 15 (24.2%)] and (iii) patients with ABMR including those suspicious for ABMR according to Banff 2015 and patients with mixed rejections [n = 28 (45.2%)]. Baseline characteristics at the time of index biopsy were in general comparable among these three groups, with the exception that patients with ABMR less frequently received a tacrolimus (TAC)-based maintenance immunosuppressive regimen than patients with normal biopsy results or patients with TCMR (50.0% versus 84.2% versus 73.3%; P = 0.041) and that the proportion of patients with proteinuria was higher in the ABMR group (67.9% versus 26.3% versus 33.3%; P = 0.010; Table 1). Index biopsies in the ABMR group tended to be performed somewhat later post-transplant (P = 0.067; Table 1). Serum AT1R-Ab stratified according to graft biopsy results Investigating the differences in AT1R-Ab serum concentrations among these three groups revealed significantly higher values in the ABMR group [12.0 U/mL (IQR 10.0–16.8)] compared with the group with normal biopsy results [8.0 U/mL (IQR 6.0–14.0); P = 0.012] or to those with TCMR [9.0 (IQR 7.0–10.0); P = 0.039], while AT1R-Ab was comparable between TCMR and patients without rejection (Figure 1). FIGURE 1 View largeDownload slide Distribution of AT1R-Ab levels (U/mL) based on the outlined index biopsy results. T-cell-mediated rejections include borderline changes and ABMR includes biopsy species suspicious for ABMR according to Banff 2015. FIGURE 1 View largeDownload slide Distribution of AT1R-Ab levels (U/mL) based on the outlined index biopsy results. T-cell-mediated rejections include borderline changes and ABMR includes biopsy species suspicious for ABMR according to Banff 2015. To determine the optimal AT1R-Ab cut-off value discriminating between patients with features of ABMR in the biopsy and those without (no rejection group and TCMR group), ROC curve analyses were performed. An area under the ROC curve value of 0.74 (95% CI 0.61–0.87; P = 0.001) indicated a moderate discriminative capacity (Figure 2). Based on the Youden Index (maximization of sensitivity and specificity), the ideal cut-off value for AT1R-Ab was determined as 9.5 U/mL, resulting in a sensitivity of 79% and a specificity of 71%. Using this cut-off value, 4 of 28 patients (14.3%) with ABMR in the index biopsy were positive only for HLA-DSA, 6 patients (21.4%) were positive only for AT1R-Ab and 16 patients (57.1%) were double positive for both HLA-DSA and AT1R-Ab. FIGURE 2 View largeDownload slide ROC curve analysis to determine the optimal cut-off value differentiating between ABMR and non-ABMR biopsy results. FIGURE 2 View largeDownload slide ROC curve analysis to determine the optimal cut-off value differentiating between ABMR and non-ABMR biopsy results. AT1R-Ab levels were significantly higher in the group with concomitant HLA-DSA positivity compared with HLA-DSA-negative patients [12.0 U/mL (IQR 9.0–16.5) versus 9.0 (IQR 7.0–12.0); P = 0.013]. AT1R-Ab levels did not differ significantly between HLA-DSA-positive patients with or without HLA-DSA C1q positivity (P = 0.407). There was no correlation between AT1R-Ab levels and HLA-DSA MFI values (P = 0.815). Next, we compared the biopsy scores with respect to AT1R-Ab and HLA-DSA status. AT1R-Ab levels were significantly higher in patients with a peritubular capillaritis (PTC) score >1 in the index graft biopsy compared with patients with a PTC score of 0 [9.0 U/mL (IQR 7.0–14.0) versus 12.0 (IQR 11.0–19.0); P = 0.041; Figure 3], while AT1R-Ab levels were not significantly altered in patients with different Banff i (P = 0.521) or t scores (P = 0.503). Patients with AT1R-Ab and HLA-DSA double positivity had a significantly higher microvascular inflammation score [1.0 (IQR 0–2.0)], defined as the sum of glomerulitis and PTC, than DSA-negative patients [0 (IQR 0–0.5); P = 0.004]. C4d positivity was less frequently observed in the cohort that was DSA negative and AT1R-Ab positive (25%; P = 0.154) than in the cohort that was DSA positive and AT1R-Ab negative (44.4%) or in the cohort that was both DSA positive and AT1R-Ab positive (50%; Supplementary data, Table S1), but the numbers were too small for a meaningful comparison. FIGURE 3 View largeDownload slide PTC scores and AT1R-Ab levels (all patients included). AT1R-Ab levels were significantly increased in patients with a PTC score of 2 compared with patients with a PTC score of 0. FIGURE 3 View largeDownload slide PTC scores and AT1R-Ab levels (all patients included). AT1R-Ab levels were significantly increased in patients with a PTC score of 2 compared with patients with a PTC score of 0. Graft survival, graft function and blood pressure according to AT1R-Ab status Patient characteristics at the time of transplantation were comparable between the AT1R-Ab-positive and -negative groups (Table 2). However, at the time of index biopsy, significantly more AT1R-Ab-positive patients showed additional HLA-class II DSA positivity (P = 0.002). AT1R-Ab positivity was associated with a significantly worse graft survival (P = 0.025) and a significantly more rapid decline of graft function (P = 0.004) during an observation period of up to 5 years post-biopsy (Figure 4, upper and lower panels). The 5-year graft survival was only 59% in the AT1R-Ab-positive group compared with 87% in the AT1R-Ab-negative group. Considering the eGFR decline in the time period of 5 years post-biopsy, 61% of AT1R-Ab-positive patients revealed an eGFR decline  ≥50% of baseline compared with only 20% of AT1R-Ab-negative patients. FIGURE 4 View largeDownload slide Association of AT1R-Ab positivity (AT1R-Ab >9.5 U/mL) and graft survival (upper panel) and deterioration of graft function post-biopsy (eGFR <50% of baseline levels prior to index biopsy) (lower panel). FIGURE 4 View largeDownload slide Association of AT1R-Ab positivity (AT1R-Ab >9.5 U/mL) and graft survival (upper panel) and deterioration of graft function post-biopsy (eGFR <50% of baseline levels prior to index biopsy) (lower panel). In AT1R-Ab-positive patients, systolic but not diastolic blood pressure z-scores averaged over three to five office blood pressure measurements prior to index biopsy were significantly higher than in AT1R-Ab-negative patients (P = 0.046; Table 3). Of note, the total amount of prescribed anti-hypertensive drugs at the time of the index biopsy and the number of patients on an angiotensin II receptor blocker were comparable between these two groups (Table 3). Table 3 Patient and transplant characteristics at the time of indication biopsy according to AT1R-Ab status Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  eGFR (mL/min/1.73 m2), mean ± SDa  41.0 ± 15.3  39.9 ± 14.9  42.0 ± 15.7  0.603  Patients with HLA-DSA Class I, n (%)  12 (19.4)  3 (10.0)  9 (28.1)  0.071  Patients with HLA-DSA Class II, n (%)  27 (43.5)  7 (23.3)  20 (62.5)  0.002  Systolic blood pressure (z-score), mean ± SD  1.18 ± 1.16  0.88 ± 1.02  1.48 ± 1.23  0.046  Diastolic blood pressure (z-score), mean ± SD  0.78 ± 0.89  0.64 ± 0.82  0.92 ± 0.94  0.225  Number of anti-hypertensive drugs per patient, median (IQR)  1.0 (0.75–2.0)  1.0 (0–2.0)  1.0 (1.0–3.0)  0.428  Patients on ARB, n (%)  14 (22.6)  5 (16.7)  9 (28.1)  0.281  Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  eGFR (mL/min/1.73 m2), mean ± SDa  41.0 ± 15.3  39.9 ± 14.9  42.0 ± 15.7  0.603  Patients with HLA-DSA Class I, n (%)  12 (19.4)  3 (10.0)  9 (28.1)  0.071  Patients with HLA-DSA Class II, n (%)  27 (43.5)  7 (23.3)  20 (62.5)  0.002  Systolic blood pressure (z-score), mean ± SD  1.18 ± 1.16  0.88 ± 1.02  1.48 ± 1.23  0.046  Diastolic blood pressure (z-score), mean ± SD  0.78 ± 0.89  0.64 ± 0.82  0.92 ± 0.94  0.225  Number of anti-hypertensive drugs per patient, median (IQR)  1.0 (0.75–2.0)  1.0 (0–2.0)  1.0 (1.0–3.0)  0.428  Patients on ARB, n (%)  14 (22.6)  5 (16.7)  9 (28.1)  0.281  ARB, angiotensin receptor blocker; HLA-DSA, human leucocyte antigen donor-specific antibodies. aeGFR according to Schwartz et al. [15]. Table 3 Patient and transplant characteristics at the time of indication biopsy according to AT1R-Ab status Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  eGFR (mL/min/1.73 m2), mean ± SDa  41.0 ± 15.3  39.9 ± 14.9  42.0 ± 15.7  0.603  Patients with HLA-DSA Class I, n (%)  12 (19.4)  3 (10.0)  9 (28.1)  0.071  Patients with HLA-DSA Class II, n (%)  27 (43.5)  7 (23.3)  20 (62.5)  0.002  Systolic blood pressure (z-score), mean ± SD  1.18 ± 1.16  0.88 ± 1.02  1.48 ± 1.23  0.046  Diastolic blood pressure (z-score), mean ± SD  0.78 ± 0.89  0.64 ± 0.82  0.92 ± 0.94  0.225  Number of anti-hypertensive drugs per patient, median (IQR)  1.0 (0.75–2.0)  1.0 (0–2.0)  1.0 (1.0–3.0)  0.428  Patients on ARB, n (%)  14 (22.6)  5 (16.7)  9 (28.1)  0.281  Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  eGFR (mL/min/1.73 m2), mean ± SDa  41.0 ± 15.3  39.9 ± 14.9  42.0 ± 15.7  0.603  Patients with HLA-DSA Class I, n (%)  12 (19.4)  3 (10.0)  9 (28.1)  0.071  Patients with HLA-DSA Class II, n (%)  27 (43.5)  7 (23.3)  20 (62.5)  0.002  Systolic blood pressure (z-score), mean ± SD  1.18 ± 1.16  0.88 ± 1.02  1.48 ± 1.23  0.046  Diastolic blood pressure (z-score), mean ± SD  0.78 ± 0.89  0.64 ± 0.82  0.92 ± 0.94  0.225  Number of anti-hypertensive drugs per patient, median (IQR)  1.0 (0.75–2.0)  1.0 (0–2.0)  1.0 (1.0–3.0)  0.428  Patients on ARB, n (%)  14 (22.6)  5 (16.7)  9 (28.1)  0.281  ARB, angiotensin receptor blocker; HLA-DSA, human leucocyte antigen donor-specific antibodies. aeGFR according to Schwartz et al. [15]. Graft survival and function according to AT1R-Ab and HLA-DSA status To further investigate the impact of both AT1R-Ab and HLA-DSA on graft survival and function, patients were divided into groups depending on their AT1R-Ab and HLA-DSA status. Baseline patient characteristics at the time of index biopsy were comparable among these three groups: patients without DSA, patients with AT1R-Ab only and patients with double positivity of AT1R-Ab and HLA-DSA (Supplementary data, Table S1). AT1R-Ab-positive (n = 12) as well as AT1R-Ab and HLA-DSA double-positive patients (n = 20) more often experienced an eGFR decline  ≥50% of baseline than DSA-negative patients (n = 21; P = 0.026 and P = 0.001 log-rank test; Figure 5). Patients with AT1R-Ab and HLA-DSA double positivity had a more rapid eGFR decline than patients who were only positive for AT1R-Ab or HLA-DSA (n = 9), but this difference did not reach statistical significance (P = 0.231), most likely due to the small number of observations. FIGURE 5 View largeDownload slide Association of deterioration of graft function (eGFR <50% of baseline prior to index biopsy) stratified according to AT1R-Ab and HLA-DSA status. FIGURE 5 View largeDownload slide Association of deterioration of graft function (eGFR <50% of baseline prior to index biopsy) stratified according to AT1R-Ab and HLA-DSA status. Risk factors for graft function deterioration We analysed non-invasive risk factors such as immunological and biochemical biomarkers for graft function deterioration, defined as an eGFR decline ≥50% of baseline. HLA-DSA (further differentiated according to C1q-complement binding capacity), AT1R-Ab status, eGFR at the time of index biopsy, proteinuria and arterial hypertension were assessed in a multivariate Cox regression model including age at index biopsy and time period from transplantation to index biopsy. In this model, C1q-positive HLA-DSA, eGFR and AT1R-Ab positivity were significantly associated with more rapid graft function decline (Table 4A). When we added histological characteristics such as transplant glomerulopathy and C4d status to this risk factor analysis, only transplant glomerulopathy, C1q-positive HLA-DSA and eGFR at the time of index biopsy remained statistically significant in the multivariate analysis (Table 4B). Table 4 Risk factor analysis for graft deterioration (eGFR <50% of baseline prior to index biopsy) up to 5 years post-biopsy Risk factors  Unadjusted HR (95% CI)  P-value  Adjusted HR (95% CI)  P-value  Serologic risk factors     AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010  2.83 (1.06–7.55)  0.038   HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  5.98 (2.01–17.7)  0.001   Proteinuria (>20 g/mol creatinine)  3.08 (1.37–6.92)  0.007       eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.95 (0.92–0.99)  0.008   Arterial hypertension  2.91 (1.22–6.96)  0.016      Including histopathological risk factors     Transplant glomerulopathy  6.29 (2.83–14.0)  <0.001  4.90 (2.01–12.0)  0.001   C4d positivity  5.10 (2.29–11.4)  <0.001       AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010       HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  4.09 (1.31–12.8)  0.015   eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.96 (0.93–0.99)  0.013  Risk factors  Unadjusted HR (95% CI)  P-value  Adjusted HR (95% CI)  P-value  Serologic risk factors     AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010  2.83 (1.06–7.55)  0.038   HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  5.98 (2.01–17.7)  0.001   Proteinuria (>20 g/mol creatinine)  3.08 (1.37–6.92)  0.007       eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.95 (0.92–0.99)  0.008   Arterial hypertension  2.91 (1.22–6.96)  0.016      Including histopathological risk factors     Transplant glomerulopathy  6.29 (2.83–14.0)  <0.001  4.90 (2.01–12.0)  0.001   C4d positivity  5.10 (2.29–11.4)  <0.001       AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010       HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  4.09 (1.31–12.8)  0.015   eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.96 (0.93–0.99)  0.013  Adjusted HR, each factor has been adjusted for the other factors that have been included in the final model (C1q, C1q HLA-DSA). HLA-DSA, human leucocyte antigen donor-specific antibodies; HR, hazard ratio. aeGFR according to Schwartz et al. [15]. Table 4 Risk factor analysis for graft deterioration (eGFR <50% of baseline prior to index biopsy) up to 5 years post-biopsy Risk factors  Unadjusted HR (95% CI)  P-value  Adjusted HR (95% CI)  P-value  Serologic risk factors     AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010  2.83 (1.06–7.55)  0.038   HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  5.98 (2.01–17.7)  0.001   Proteinuria (>20 g/mol creatinine)  3.08 (1.37–6.92)  0.007       eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.95 (0.92–0.99)  0.008   Arterial hypertension  2.91 (1.22–6.96)  0.016      Including histopathological risk factors     Transplant glomerulopathy  6.29 (2.83–14.0)  <0.001  4.90 (2.01–12.0)  0.001   C4d positivity  5.10 (2.29–11.4)  <0.001       AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010       HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  4.09 (1.31–12.8)  0.015   eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.96 (0.93–0.99)  0.013  Risk factors  Unadjusted HR (95% CI)  P-value  Adjusted HR (95% CI)  P-value  Serologic risk factors     AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010  2.83 (1.06–7.55)  0.038   HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  5.98 (2.01–17.7)  0.001   Proteinuria (>20 g/mol creatinine)  3.08 (1.37–6.92)  0.007       eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.95 (0.92–0.99)  0.008   Arterial hypertension  2.91 (1.22–6.96)  0.016      Including histopathological risk factors     Transplant glomerulopathy  6.29 (2.83–14.0)  <0.001  4.90 (2.01–12.0)  0.001   C4d positivity  5.10 (2.29–11.4)  <0.001       AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010       HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  4.09 (1.31–12.8)  0.015   eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.96 (0.93–0.99)  0.013  Adjusted HR, each factor has been adjusted for the other factors that have been included in the final model (C1q, C1q HLA-DSA). HLA-DSA, human leucocyte antigen donor-specific antibodies; HR, hazard ratio. aeGFR according to Schwartz et al. [15]. DISCUSSION This is the first study in paediatric renal transplant recipients that analysed the prognostic significance of serum AT1R-Ab in conjunction with other known risk factors for graft failure in a carefully phenotyped single-centre cohort. The main result of this study is that the presence of AT1R-Ab in the context of a late (>1 year post-transplant) indication biopsy identifies a subgroup of patients with a more rapid subsequent eGFR decline. In the multivariate Cox regression model of non-invasive risk factors associated with enhanced graft function deterioration, AT1R-Ab positivity [adjusted hazard ratio (HR) 2.83] was the second strongest independent predictor of adverse graft outcome after the parameter C1q-positive DSA (HR 5.98). We also observed that serum AT1R-Ab concentrations were significantly higher in patients with biopsy-proven ABMR than in patients with TCMR or no rejection, consistent with recent findings in adult renal transplant recipients [23]. It is noteworthy that in our study a relevant subset of patients (21.4%) with features of ABMR in the index biopsy was only positive for AT1R-Ab and not for HLA-DSA. Hence, from these and other data it appears advisable to test patients with ABMR not only for the presence of HLA-DSA, but also for non-HLA antibodies against endothelial targets such as AT1R-Ab. The question arises whether AT1R-Ab directly induces or promotes ABMR or is an epiphenomenon of endothelial injury. While these two possibilities cannot easily be differentiated in a clinical study, experimental data indicate a direct effect of AT1R-Ab on endothelial injury, inflammation and allograft dysfunction [24, 25]. AT1R, the target for AT1R-Ab, is expressed at high levels on endothelial cells, and activation of this receptor in human microvascular endothelial cells in the presence of AT1R-Ab causes endothelial cell dysfunction and neutrophil migration; these effects were reduced in the presence of an angiotensin receptor blocker [24]. Therefore, AT1R-Ab is thought to contribute to graft dysfunction by inducing activation of signalling pathways in a similar fashion as the endogenous ligand for this receptor, angiotensin II [24–26]. In addition, experimental data indicate that activation of AT1R leads to an upregulation of HLA class II antigens on endothelial cells [27]. This experimental finding corresponds with our observation that patients with AT1R-Ab and HLA-DSA double positivity had a higher vascular micro-inflammation score and more rapid graft function deterioration than DSA-negative patients. Our study extends the observations of Philogene et al. [23], because we also investigated the prognostic significance of AT1R-Ab positivity regarding graft function and survival. The 5-year graft survival was only 59% in the AT1R-Ab-positive group compared with 87% in the AT1R-Ab-negative group. In the time period of 5 years post-biopsy, 61% of AT1R-Ab-positive patients revealed an eGFR decline  ≥50% of baseline compared with only 20% of AT1R-Ab-negative patients. Furthermore, patients with AT1R-Ab and HLA-DSA double positivity tended to have a more pronounced decline of graft function than patients who were only positive for AT1R-Ab or HLA-DSA. As outlined above, double positivity for AT1R-Ab and HLA-DSA appears to enhance the histopathological lesions of ABMR associated with more rapid graft function decline, but further studies in larger patient populations are required to confirm this hypothesis. We determined an optimal AT1R-Ab cut-off value of 9.5 U/mL discriminating between patients with or without features of ABMR in the biopsy. Three previous studies investigating AT1R-Ab levels in renal transplant recipients have suggested a similar threshold for positivity [6, 7, 28]. Giral et al. [7] observed that pre-transplant sensitization against AT1R is a risk factor for acute rejection and graft loss in adult patients and determined, by use of the same assay as in our study, a threshold of AT1R-Ab levels at 10 U/mL based on a statistical analysis using the Hothorn and Zeileis method [29]. Lee et al. [8] calculated an optimal cut-off value for pre-transplant AT1R-Ab of 9.05 U/mL for the risk prediction of acute rejection in the first year post-transplant in Asian adult renal transplant recipients. While it is interesting that these cut-off values are well comparable among different patient populations and age groups, further studies of AT1R-Ab both in the pre- and post-transplant setting are required to firmly establish valid cut-off values for AT1R-Ab positivity. Only two previous studies [12, 13] and one case report [14] have reported on AT1R-Ab serum concentrations in paediatric renal transplant recipients. A small study (n = 29) reported that 40% of patients developed AT1R-Ab in the first year post-transplant, but AT1R-Ab did not correlate with worse clinical outcomes [12]. Bjerre et al. [13] reported in a cross-sectional study of 30 children that AT1R-Ab levels were significantly higher in stable paediatric versus adult renal transplant recipients and higher compared with controls of comparable age groups, but they did not analyse the relationship of serum AT1R-Ab with outcome measures such as graft histology, function or survival. One case report described a girl with accelerated acute C4d-positive kidney transplant rejection, malignant hypertension, encephalopathy and the presence of both AT1R-Ab and HLA class II antibodies [14]. The strength of our study is that it was based on a prospective protocol investigating a paediatric patient cohort at an increased risk of graft deterioration reflected by a clinically indicated graft biopsy >1 year post-transplant. A limitation is the relatively small number of patients investigated, but this is an inherent problem for all studies in the paediatric renal transplant population. Furthermore, AT1R-Ab was measured only at a single time point post-transplant. Future studies will have to evaluate whether longitudinal analyses of AT1R-Ab in an unselected patient cohort of paediatric kidney allograft recipients is a surrogate marker for increased immunological risk requiring more intense immunosuppressive therapy. It will also be important to investigate the effect of potential treatment modalities on AT1R-Ab and if they improve outcomes for these patients. In conclusion, the presence of AT1R-Ab in the context of an indication biopsy >1 year post-transplant is associated with the histopathology of ABMR and is a non-invasive risk factor for subsequent accelerated graft function decline. Hence the determination of serum AT1R-Ab may enhance the predictive value of HLA-DSA and/or C1q-binding DSA. Screening for AT1R-Ab in conjunction with HLA-DSA at regular intervals post-transplant may have the potential to identify patients at high risk for ABMR, in whom targeted enhancement of immunosuppressive therapy may improve transplant outcome. This is especially relevant for the paediatric kidney transplant population, in whom, in the light of their long life expectancy, preservation of good graft function is of utmost importance. AUTHORS’ CONTRIBUTIONS A.F, C.S. and B.T. participated in research design. A.F, C.S., B.H., S.R., R.W., J.H.W., A.S., D.D. and B.T. participated in performance of the research, data analysis and in writing the article. All authors reviewed the manuscript, believe it represents valid work and approved it for submission. ACKNOWLEDGEMENTS We wish to thank Marzena Kirschke, Manuela Schneidenbach, Fatma Karci and our HLA and DNA laboratory teams for excellent technical assistance. We gratefully acknowledge the support of this study by a grant from the Peter-Stiftung für die Nierenwissenschaft, Münster, Germany. SUPPLEMENTARY DATA Supplementary data are available at ndt online. CONFLICT OF INTEREST STATEMENT None declared. This work is original and submitted solely to NDT for consideration. The results presented in this article have not been published previously in whole or part, except in abstract form. REFERENCES 1 Einecke G, Sis B, Reeve J. Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure. Am J Transplant  2009; 9: 2520– 2531 Google Scholar CrossRef Search ADS PubMed  2 Lee PC, Zhu L, Terasaki PI et al.  . HLA-specific antibodies developed in the first year posttransplant are predictive of chronic rejection and renal graft loss. Transplantation  2009; 88: 568– 574 Google Scholar CrossRef Search ADS PubMed  3 Süsal C, Fichtner A, Tönshoff B et al.  . Clinical relevance of HLA antibodies in kidney transplantation: recent data from the Heidelberg Transplant Center and the Collaborative Transplant Study. J Immunol Res  2017; 2017: 1– 19402 Google Scholar CrossRef Search ADS   4 Chaudhuri A, Ozawa M, Everly MJ et al.  . The clinical impact of humoral immunity in pediatric renal transplantation. J Am Soc Nephrol  2013; 24: 655– 664 Google Scholar CrossRef Search ADS PubMed  5 Dragun D, Catar R, Philippe A. Non-HLA antibodies against endothelial targets bridging allo- and autoimmunity. Kidney Int  2016; 90: 280– 288 Google Scholar CrossRef Search ADS PubMed  6 Reinsmoen NL, Lai CH, Heidecke H et al.  . Anti-angiotensin type 1 receptor antibodies associated with antibody-mediated rejection in donor HLA antibody negative patients. Transplantation  2010; 90: 1473– 1477 Google Scholar CrossRef Search ADS PubMed  7 Giral M, Foucher Y, Dufay A et al.  . Pretransplant sensitization against angiotensin II type 1 receptor is a risk factor for acute rejection and graft loss. Am J Transplant  2013; 13: 2567– 2576 Google Scholar CrossRef Search ADS PubMed  8 Lee J, Huh KH, Park Y et al.  . The clinicopathological relevance of pretransplant anti-angiotensin II type 1 receptor antibodies in renal transplantation. Nephrol Dial Transpl  2015; 0: 1– 7 9 Banasik M, Boratyńska M, Kościelska-Kasprzak K et al.  . The impact of non-HLA antibodies directed against endothelin-1 type A receptors (ETAR) on early renal transplant outcomes. Transpl Immunol  2014; 30: 24– 29. Google Scholar CrossRef Search ADS PubMed  10 Fuss A, Hope CM, Deayton S et al.  . C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies. Nephrology (Carlton)  2015; 20: 467– 473 Google Scholar CrossRef Search ADS PubMed  11 Dharnidharka VR, Fiorina P, Harmon WE. Kidney transplantation in children. N Engl J Med  2014; 371: 549– 558 Google Scholar CrossRef Search ADS PubMed  12 Hesemann LE, Subramanian V, Mohanakumar T et al.  . De novo development of antibodies to kidney-associated self-antigens angiotensin II receptor type I, collagen IV, and fibronectin occurs at early time points after kidney transplantation in children. Pediatr Transplant  2015; 19: 499– 503 Google Scholar CrossRef Search ADS PubMed  13 Bjerre A, Tangeraas T, Heidecke H et al.  . Angiotensin II type 1 receptor antibodies in childhood kidney transplantation. Pediatr Transplant  2016; 20: 627– 632 Google Scholar CrossRef Search ADS PubMed  14 Kelsch R, Everding AS, Kuwertz-Bröking E et al.  . Accelerated kidney transplant rejection and hypertensive encephalopathy in a pediatric patient associated with antibodies against angiotensin type 1 receptor and HLA class II. Transplantation  2011; 92: e57– e59 Google Scholar CrossRef Search ADS PubMed  15 Schwartz GJ, Munoz A, Schneider MF et al.  . New equations to estimate GFR in children with CKD. J Am Soc Nephrol  2009; 20: 629– 637 Google Scholar CrossRef Search ADS PubMed  16 Fichtner A, Süsal C, Höcker B et al.  . Association of C1q-fixing DSA with late graft failure in pediatric renal transplant recipients. Pediatr Nephrol  2016; 31: 1157– 1166 Google Scholar CrossRef Search ADS PubMed  17 Vandenbroucke JP, von Elm E, Altman DG et al.  . Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): explanation and elaboration. Epidemiology  2007; 18: 805– 835 Google Scholar CrossRef Search ADS PubMed  18 Süsal C, Wettstein D, Döhler B et al.  . Association of kidney graft loss with de novo produced donor-specific and non-donor-specific HLA antibodies detected by single antigen testing. Transplantation  2015; 99: 1976– 1980 Google Scholar CrossRef Search ADS PubMed  19 National High Blood Pressure Education Program Working Group on High Blood Pressure in Children and Adolescents. The fourth report on the diagnosis, evaluation, and treatment of high blood pressure in children and adolescents. Pediatrics  2004; 114: 555– 576 CrossRef Search ADS PubMed  20 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: 1165– 1173 Google Scholar CrossRef Search ADS PubMed  21 Sis B, Mengel M, Haas M et al.  . Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups. 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J Cell Physiol  2013; 228: 142– 148 Google Scholar CrossRef Search ADS PubMed  26 Kill A, Tabeling C, Undeutsch R et al.  . Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis. Arthritis Res Ther  2014; 16: R29. Google Scholar CrossRef Search ADS PubMed  27 Hiemann NE, Meyer R, Wellnhofer E et al.  . Non-HLA antibodies targeting vascular receptors enhance alloimmune response and microvasculopathy after heart transplantation. Transplantation  2012; 94: 919– 924 Google Scholar CrossRef Search ADS PubMed  28 Taniguchi M, Rebellato LM, Cai J et al.  . Higher risk of kidney graft failure in the presence of anti-angiotensin II type-1 receptor antibodies. Am J Transplant  2013; 13: 2577– 2589 Google Scholar CrossRef Search ADS PubMed  29 Hothorn T, Zeileis A. Generalized maximally selected statistics. Biometrics  2008; 64: 1263– 1269 Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nephrology Dialysis Transplantation Oxford University Press

Association of angiotensin II type 1 receptor antibodies with graft histology, function and survival in paediatric renal transplant recipients

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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0931-0509
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1460-2385
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10.1093/ndt/gfy008
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Abstract

ABSTRACT Background We analysed in a carefully phenotyped cohort of paediatric patients the association of serum angiotensin II type 1 receptor antibodies (AT1R-Ab) with specific histological lesions and with graft function and survival in conjunction with overall and complement-binding donor-specific human leucocyte antigen donor-specific antibodies (HLA-DSA). Methods Sera of 62 patients at the time of renal graft biopsy for clinical indication >1 year post-transplant were assessed for AT1R-Ab by enzyme-linked immunosorbent assay (ELISA) and for DSA and C1q-fixing DSA by single-antigen bead technology. Results Serum AT1R-Ab concentration was significantly higher in antibody-mediated rejection (ABMR) than in T-cell-mediated rejection or control. By receiver operating characteristic (ROC) curve analysis, the optimal AT1R-Ab cut-off value discriminating between patients with features of ABMR and those without was 9.5 U/mL. A total of 6 of 28 patients (21.4%) with ABMR were only positive for AT1R-Ab. Patients with AT1R-Ab and HLA-DSA double positivity had a significantly higher vascular micro-inflammation score than DSA-negative patients. The 5-year graft survival was only 59% in the AT1R-Ab-positive group compared with 87% in the AT1R-Ab-negative group. Patients with AT1R-Ab and HLA-DSA double positivity tended to have a more rapid decline of estimated glomerular filtration rate (eGFR) than patients who were only positive for AT1R-Ab or HLA-DSA. In a multivariate Cox regression model of non-invasive factors, C1q-positive HLA-DSA, eGFR and AT1R-Ab positivity were significantly associated with accelerated graft function decline. Conclusions Serum AT1R-Ab positivity in the context of an indication biopsy >1 year post-transplant is associated with the histopathology of ABMR and is an independent non-invasive risk factor for adverse graft outcome. angiotensin II type 1 receptor antibodies, C1q-binding donor-specific antibodies, donor-specific HLA antibodies, graft failure, indication biopsy, paediatric renal transplantation INTRODUCTION Antibody-mediated rejection (ABMR) is the major cause of graft loss in both adult [1–3] and paediatric renal transplant recipients [3, 4]. The majority of these rejections are caused by preformed and/or de novo donor-specific antibodies against human leucocyte antigen donor-specific antibodies (HLA-DSA). However, there is a significant subset of patients with histological features of ABMR in the graft biopsy in whom HLA-DSA cannot be detected in the circulation despite the use of highly sensitive assays such as the single-antigen bead (SAB) technology. In recent years, therefore, there have been increasing efforts directed towards the detection and biological characterization of antibodies against other endothelial targets beside HLA [5]. Newly developed solid-phase assays enable the detection of functional non-HLA antibodies targeting G protein-coupled receptors such as the angiotensin II type 1 receptor. In one study of 63 HLA-DSA-negative adult patients, the presence of angiotensin II type 1 receptor antibodies (AT1R-Ab) was strongly associated with ABMR [6]. In a large prospective study in 599 kidney transplant recipients, the pre-transplant positivity for AT1R-Ab was an independent risk factor for allograft loss [7]. More recent smaller studies have confirmed the increased rejection risk in kidney transplant recipients with preformed AT1R-Ab [8, 9] and its link to a C4d-negative ABMR phenotype [10]. However, findings from adult populations cannot necessarily be extrapolated to paediatric patients. There are unique differences in the immune system in children, namely the naivety of their immune system and differences in alloimmunological reactivity, as reflected by small numbers of antigen-experienced T cells, mature dendritic cells or macrophages and HLA-alloreactive B cells [11]. Up to now, data on AT1R-Ab in paediatric renal transplant recipients have been scarce, from small descriptive case series [12–14]. The aims of this study were to investigate the association of serum AT1R-Ab with specific histological lesions and with graft function and survival in paediatric patients undergoing graft biopsies >1 year post-transplant for clinical indication. Targets of AT1R-Ab are constitutively expressed on the vascular endothelium and expression may be induced or increased during inflammatory events. Analysis of the phenotypic characteristics of biopsies in the presence of AT1R-Ab could provide further evidence linking them to allograft dysfunction. Because AT1R-Ab may exert pathophysiologic effects alone or in synergy with HLA antibodies, we analysed serum AT1R-Ab in conjunction with overall and complement-binding donor-specific HLA-DSA. MATERIALS AND METHODS Study design and patient population This is a retrospective single-centre cohort analysis based on prospectively collected serum samples of all paediatric patients ≤18 years of age at the date of transplantation who were non-presensitized [HLA antibodies negative on complement-dependent cytotoxicity (CDC) and enzyme-linked immunosorbent assay (ELISA) testing], received a kidney transplant at our institution between January 1999 and January 2010 and underwent a kidney graft biopsy for clinical indication (functional impairment or severe de novo proteinuria) >1 year post-transplant (index biopsy). Only patients with index biopsies after January 2004 were included, when routine C4d staining was introduced in our institution. T- or B-cell crossmatch-positive patients and recipients of ABO-incompatible or combined organ transplants were excluded from the analysis. In all, eight patients who met the inclusion criteria were secondarily excluded due to insufficient amounts of serum available or loss of follow-up. Therefore 62 of 70 eligible patients were analysed. Patient and transplant characteristics at the time of index biopsy are depicted in Table 1 and parameters at the time of engrafting are shown in Table 2. Serum HLA-DSA and AT1R-Ab were measured at the time of index biopsy. Serum was collected in the framework of the ongoing prospective Collaborative Transplant Study (CTS) Serum Project. Data on C1q-fixing DSA in this cohort have been published previously [16]. Ethics committee approval was obtained and investigations were performed in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines. Written informed consent was obtained by all parents or guardians and patients when appropriate for their age. This study was designed, analysed and reported according to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines [17]. Table 1 Patient and transplant characteristics at the time of index biopsy (Banff 2015) Characteristics  Entire cohort  No rejection  T-cell-mediated rejectiona  Antibody-mediated rejectionb  P-value    (n = 62)  (n = 19)  (n = 15)  (n = 28)    Age (years), mean ± SD  13.7 ± 5.5  13.3 ± 5.18  12.5 ± 5.36  14.5 ± 5.72  0.489  eGFR (mL/min/1.73 m2), mean ± SDc  41.0 ± 15.3  44.6 ± 16.8  42.8 ± 12.0  37.5 ± 15.5  0.260  Proteinuria (>100 g/mol creatinine), n (%)  8 (12.9)  2 (10.5)  0  6 (21.4)  0.127  Proteinuria (>20 g/mol creatinine), n (%)  29 (46.8)  5 (26.3)  5 (33.3)  19 (67.9)  0.010  Number of pre-biopsies per patient, median (IQR)  2.0 (0–4)  2.0 (1.0–3.0)  2.0 (1.0–2.0)  2.0 (0.25–3.0)  0.943  Patients with BPAR (≥Banff IA) in pre-biopsies, n (%)  17 (27.4)  4.0 (21.1)  3 (20.0)  10 (35.7)  0.413  Patients with treated AR in pre-biopsies, n (%)  32 (51.6)  13 (68.4)  6 (40.0)  14 (50.0)  0.231  Major biopsy features, n (%)   C4d positivity  21 (33.9)  2 (10.5)  0  19 (67.9)  <0.001   TG  19 (30.6)  1 (5.3)  0  18 (64.3)  <0.001  Immunosuppression, n (%)   Tacrolimus  41 (66.1)  16 (84.2)  11 (73.3)  14 (50.0)  0.041   Cyclosporine  18 (29.0)  3 (15.8)  4 (26.7)  11 (39.3)  0.214   CNI-free/SRL  3 (4.8)  0  0  3 (10.7)  0.147   Prednisolone  39 (62.9)  12 (63.2)  13 (86.7)  14 (50.0)  0.060   MMF  52 (83.9)  16 (84.2)  11 (73.3)  25 (89.3)  0.399  A-B-DR-DQ mismatches/8  2.68 ± 1.21  2.58 ± 1.71  2.47 ± 0.83  2.86 ± 0.97  0.558   A-B  1.33 ± 0.70  1.47 ± 0.91  1.33 ± 0.49  1.25 ± 0.65  0.568   DR  0.76 ± 0.53  0.58 ± 0.69  0.73 ± 0.46  0.89 ± 0.42  0.138   DQ  0.58 ± 0.62  0.53 ± 0.70  0.40 ± 0.63  0.71 ± 0.53  0.255  Patients with HLA-DSA, n (%)  29 (46.8)  4 (21.1)  5 (33.3)  20 (71.4)  0.002   Class I  12 (19.4)  1 (5.3)  0  11 (29.3)  0.001   Class II  27 (43.5)  3 (15.8)  5 (33.3)  19 (67.9)  0.001  Time Tx to biopsy (months), median (IQR)  53.5 (33.8–75.0)  51.0 (27.0–63.0)  35.0 (24.0–60.0)  62.0 (39.3–109)  0.067  Follow-up post-biopsy (months), median (IQR)  79.5 (65.0–99.0)  73.0 (62.0–88.0)  90.0 (69.0–105)  80.5 (63.5–115)  0.390  Characteristics  Entire cohort  No rejection  T-cell-mediated rejectiona  Antibody-mediated rejectionb  P-value    (n = 62)  (n = 19)  (n = 15)  (n = 28)    Age (years), mean ± SD  13.7 ± 5.5  13.3 ± 5.18  12.5 ± 5.36  14.5 ± 5.72  0.489  eGFR (mL/min/1.73 m2), mean ± SDc  41.0 ± 15.3  44.6 ± 16.8  42.8 ± 12.0  37.5 ± 15.5  0.260  Proteinuria (>100 g/mol creatinine), n (%)  8 (12.9)  2 (10.5)  0  6 (21.4)  0.127  Proteinuria (>20 g/mol creatinine), n (%)  29 (46.8)  5 (26.3)  5 (33.3)  19 (67.9)  0.010  Number of pre-biopsies per patient, median (IQR)  2.0 (0–4)  2.0 (1.0–3.0)  2.0 (1.0–2.0)  2.0 (0.25–3.0)  0.943  Patients with BPAR (≥Banff IA) in pre-biopsies, n (%)  17 (27.4)  4.0 (21.1)  3 (20.0)  10 (35.7)  0.413  Patients with treated AR in pre-biopsies, n (%)  32 (51.6)  13 (68.4)  6 (40.0)  14 (50.0)  0.231  Major biopsy features, n (%)   C4d positivity  21 (33.9)  2 (10.5)  0  19 (67.9)  <0.001   TG  19 (30.6)  1 (5.3)  0  18 (64.3)  <0.001  Immunosuppression, n (%)   Tacrolimus  41 (66.1)  16 (84.2)  11 (73.3)  14 (50.0)  0.041   Cyclosporine  18 (29.0)  3 (15.8)  4 (26.7)  11 (39.3)  0.214   CNI-free/SRL  3 (4.8)  0  0  3 (10.7)  0.147   Prednisolone  39 (62.9)  12 (63.2)  13 (86.7)  14 (50.0)  0.060   MMF  52 (83.9)  16 (84.2)  11 (73.3)  25 (89.3)  0.399  A-B-DR-DQ mismatches/8  2.68 ± 1.21  2.58 ± 1.71  2.47 ± 0.83  2.86 ± 0.97  0.558   A-B  1.33 ± 0.70  1.47 ± 0.91  1.33 ± 0.49  1.25 ± 0.65  0.568   DR  0.76 ± 0.53  0.58 ± 0.69  0.73 ± 0.46  0.89 ± 0.42  0.138   DQ  0.58 ± 0.62  0.53 ± 0.70  0.40 ± 0.63  0.71 ± 0.53  0.255  Patients with HLA-DSA, n (%)  29 (46.8)  4 (21.1)  5 (33.3)  20 (71.4)  0.002   Class I  12 (19.4)  1 (5.3)  0  11 (29.3)  0.001   Class II  27 (43.5)  3 (15.8)  5 (33.3)  19 (67.9)  0.001  Time Tx to biopsy (months), median (IQR)  53.5 (33.8–75.0)  51.0 (27.0–63.0)  35.0 (24.0–60.0)  62.0 (39.3–109)  0.067  Follow-up post-biopsy (months), median (IQR)  79.5 (65.0–99.0)  73.0 (62.0–88.0)  90.0 (69.0–105)  80.5 (63.5–115)  0.390  AR, acute rejection; BPAR, biopsy-proven acute rejection; CNI, calcineurin inhibitors; HLA-DSA, human leucocyte antigen donor-specific antibodies; MMF, mycophenolate mofetil; SRL, sirolimus; TG, transplant glomerulopathy; Tx, renal transplantation. a Including borderline changes. b Including suspicious for ABMR. c eGFR according to Schwartz et al. [15]. Table 1 Patient and transplant characteristics at the time of index biopsy (Banff 2015) Characteristics  Entire cohort  No rejection  T-cell-mediated rejectiona  Antibody-mediated rejectionb  P-value    (n = 62)  (n = 19)  (n = 15)  (n = 28)    Age (years), mean ± SD  13.7 ± 5.5  13.3 ± 5.18  12.5 ± 5.36  14.5 ± 5.72  0.489  eGFR (mL/min/1.73 m2), mean ± SDc  41.0 ± 15.3  44.6 ± 16.8  42.8 ± 12.0  37.5 ± 15.5  0.260  Proteinuria (>100 g/mol creatinine), n (%)  8 (12.9)  2 (10.5)  0  6 (21.4)  0.127  Proteinuria (>20 g/mol creatinine), n (%)  29 (46.8)  5 (26.3)  5 (33.3)  19 (67.9)  0.010  Number of pre-biopsies per patient, median (IQR)  2.0 (0–4)  2.0 (1.0–3.0)  2.0 (1.0–2.0)  2.0 (0.25–3.0)  0.943  Patients with BPAR (≥Banff IA) in pre-biopsies, n (%)  17 (27.4)  4.0 (21.1)  3 (20.0)  10 (35.7)  0.413  Patients with treated AR in pre-biopsies, n (%)  32 (51.6)  13 (68.4)  6 (40.0)  14 (50.0)  0.231  Major biopsy features, n (%)   C4d positivity  21 (33.9)  2 (10.5)  0  19 (67.9)  <0.001   TG  19 (30.6)  1 (5.3)  0  18 (64.3)  <0.001  Immunosuppression, n (%)   Tacrolimus  41 (66.1)  16 (84.2)  11 (73.3)  14 (50.0)  0.041   Cyclosporine  18 (29.0)  3 (15.8)  4 (26.7)  11 (39.3)  0.214   CNI-free/SRL  3 (4.8)  0  0  3 (10.7)  0.147   Prednisolone  39 (62.9)  12 (63.2)  13 (86.7)  14 (50.0)  0.060   MMF  52 (83.9)  16 (84.2)  11 (73.3)  25 (89.3)  0.399  A-B-DR-DQ mismatches/8  2.68 ± 1.21  2.58 ± 1.71  2.47 ± 0.83  2.86 ± 0.97  0.558   A-B  1.33 ± 0.70  1.47 ± 0.91  1.33 ± 0.49  1.25 ± 0.65  0.568   DR  0.76 ± 0.53  0.58 ± 0.69  0.73 ± 0.46  0.89 ± 0.42  0.138   DQ  0.58 ± 0.62  0.53 ± 0.70  0.40 ± 0.63  0.71 ± 0.53  0.255  Patients with HLA-DSA, n (%)  29 (46.8)  4 (21.1)  5 (33.3)  20 (71.4)  0.002   Class I  12 (19.4)  1 (5.3)  0  11 (29.3)  0.001   Class II  27 (43.5)  3 (15.8)  5 (33.3)  19 (67.9)  0.001  Time Tx to biopsy (months), median (IQR)  53.5 (33.8–75.0)  51.0 (27.0–63.0)  35.0 (24.0–60.0)  62.0 (39.3–109)  0.067  Follow-up post-biopsy (months), median (IQR)  79.5 (65.0–99.0)  73.0 (62.0–88.0)  90.0 (69.0–105)  80.5 (63.5–115)  0.390  Characteristics  Entire cohort  No rejection  T-cell-mediated rejectiona  Antibody-mediated rejectionb  P-value    (n = 62)  (n = 19)  (n = 15)  (n = 28)    Age (years), mean ± SD  13.7 ± 5.5  13.3 ± 5.18  12.5 ± 5.36  14.5 ± 5.72  0.489  eGFR (mL/min/1.73 m2), mean ± SDc  41.0 ± 15.3  44.6 ± 16.8  42.8 ± 12.0  37.5 ± 15.5  0.260  Proteinuria (>100 g/mol creatinine), n (%)  8 (12.9)  2 (10.5)  0  6 (21.4)  0.127  Proteinuria (>20 g/mol creatinine), n (%)  29 (46.8)  5 (26.3)  5 (33.3)  19 (67.9)  0.010  Number of pre-biopsies per patient, median (IQR)  2.0 (0–4)  2.0 (1.0–3.0)  2.0 (1.0–2.0)  2.0 (0.25–3.0)  0.943  Patients with BPAR (≥Banff IA) in pre-biopsies, n (%)  17 (27.4)  4.0 (21.1)  3 (20.0)  10 (35.7)  0.413  Patients with treated AR in pre-biopsies, n (%)  32 (51.6)  13 (68.4)  6 (40.0)  14 (50.0)  0.231  Major biopsy features, n (%)   C4d positivity  21 (33.9)  2 (10.5)  0  19 (67.9)  <0.001   TG  19 (30.6)  1 (5.3)  0  18 (64.3)  <0.001  Immunosuppression, n (%)   Tacrolimus  41 (66.1)  16 (84.2)  11 (73.3)  14 (50.0)  0.041   Cyclosporine  18 (29.0)  3 (15.8)  4 (26.7)  11 (39.3)  0.214   CNI-free/SRL  3 (4.8)  0  0  3 (10.7)  0.147   Prednisolone  39 (62.9)  12 (63.2)  13 (86.7)  14 (50.0)  0.060   MMF  52 (83.9)  16 (84.2)  11 (73.3)  25 (89.3)  0.399  A-B-DR-DQ mismatches/8  2.68 ± 1.21  2.58 ± 1.71  2.47 ± 0.83  2.86 ± 0.97  0.558   A-B  1.33 ± 0.70  1.47 ± 0.91  1.33 ± 0.49  1.25 ± 0.65  0.568   DR  0.76 ± 0.53  0.58 ± 0.69  0.73 ± 0.46  0.89 ± 0.42  0.138   DQ  0.58 ± 0.62  0.53 ± 0.70  0.40 ± 0.63  0.71 ± 0.53  0.255  Patients with HLA-DSA, n (%)  29 (46.8)  4 (21.1)  5 (33.3)  20 (71.4)  0.002   Class I  12 (19.4)  1 (5.3)  0  11 (29.3)  0.001   Class II  27 (43.5)  3 (15.8)  5 (33.3)  19 (67.9)  0.001  Time Tx to biopsy (months), median (IQR)  53.5 (33.8–75.0)  51.0 (27.0–63.0)  35.0 (24.0–60.0)  62.0 (39.3–109)  0.067  Follow-up post-biopsy (months), median (IQR)  79.5 (65.0–99.0)  73.0 (62.0–88.0)  90.0 (69.0–105)  80.5 (63.5–115)  0.390  AR, acute rejection; BPAR, biopsy-proven acute rejection; CNI, calcineurin inhibitors; HLA-DSA, human leucocyte antigen donor-specific antibodies; MMF, mycophenolate mofetil; SRL, sirolimus; TG, transplant glomerulopathy; Tx, renal transplantation. a Including borderline changes. b Including suspicious for ABMR. c eGFR according to Schwartz et al. [15]. Table 2 Patient and transplant characteristics at time of transplantation according to AT1R-Ab status Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  Age (years), mean ± SD  8.6 ± 5.0  9.03 ± 4.80  8.30 ± 5.22  0.570  Male gender, n (%)  46 (74.2)  21 (70)  25 (78.1)  0.465  Donor age (years), mean ± SD  36.2 ± 12.9  36.2 ± 14.1  36.2 ± 11.9  0.992  Deceased donor, n (%)  40 (64.5)  21 (70)  19 (59.4)  0.533  Cold ischaemia time (h), mean ± SD  13.9 ± 6.4  13.3 ± 6.76  14.7 ± 5.83  0.576  Delayed graft function, n (%)  6 (9.7)  4 (13.3)  2 (6.3)  0.346  A-B-DR-DQ mismatch/8  2.68 ± 1.21  2.70 ± 1.15  2.66 ± 1.29  0.888   A-B  1.33 ± 0.70  1.50 ± 0.68  1.19 ± 0.70  0.079   DR  0.76 ± 0.53  0.67 ± 0.55  0.84 ± 0.52  0.194   DQ  0.58 ± 0.62  0.53 ± 0.63  0.63 ± 0.61  0.562  Cause of ESRD, n (%)   CAKUT  33 (53.2)  18 (60)  15 (46.9)  0.301   Nephronophthisis  5 (8.1)  2 (6.7)  3 (9.4)  0.696   Glomerular diseases  11 (17.7)  5 (16.7)  6 (18.8)  0.830   Others  13 (21.0)  5 (16.7)  8 (25)  0.421  Immunosuppression, n (%)   IL-2RA  12 (19.4)  4 (13.3)  8 (25)  0.245   Cyclosporine  40 (64.5)  19 (63.3)  21 (65.6)  0.851   Tacrolimus  22 (35.4)  11 (36.7)  11 (34.3)  0.851   MMF  62 (100)  30 (100)  32 (100)  1.000   Prednisolone  62 (100)  30 (100)  32 (100)  1.000  Observation time post-Tx (months), median (IQR)  141 (119–173)  137 (117–162)  147 (121–175)  0.307  Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  Age (years), mean ± SD  8.6 ± 5.0  9.03 ± 4.80  8.30 ± 5.22  0.570  Male gender, n (%)  46 (74.2)  21 (70)  25 (78.1)  0.465  Donor age (years), mean ± SD  36.2 ± 12.9  36.2 ± 14.1  36.2 ± 11.9  0.992  Deceased donor, n (%)  40 (64.5)  21 (70)  19 (59.4)  0.533  Cold ischaemia time (h), mean ± SD  13.9 ± 6.4  13.3 ± 6.76  14.7 ± 5.83  0.576  Delayed graft function, n (%)  6 (9.7)  4 (13.3)  2 (6.3)  0.346  A-B-DR-DQ mismatch/8  2.68 ± 1.21  2.70 ± 1.15  2.66 ± 1.29  0.888   A-B  1.33 ± 0.70  1.50 ± 0.68  1.19 ± 0.70  0.079   DR  0.76 ± 0.53  0.67 ± 0.55  0.84 ± 0.52  0.194   DQ  0.58 ± 0.62  0.53 ± 0.63  0.63 ± 0.61  0.562  Cause of ESRD, n (%)   CAKUT  33 (53.2)  18 (60)  15 (46.9)  0.301   Nephronophthisis  5 (8.1)  2 (6.7)  3 (9.4)  0.696   Glomerular diseases  11 (17.7)  5 (16.7)  6 (18.8)  0.830   Others  13 (21.0)  5 (16.7)  8 (25)  0.421  Immunosuppression, n (%)   IL-2RA  12 (19.4)  4 (13.3)  8 (25)  0.245   Cyclosporine  40 (64.5)  19 (63.3)  21 (65.6)  0.851   Tacrolimus  22 (35.4)  11 (36.7)  11 (34.3)  0.851   MMF  62 (100)  30 (100)  32 (100)  1.000   Prednisolone  62 (100)  30 (100)  32 (100)  1.000  Observation time post-Tx (months), median (IQR)  141 (119–173)  137 (117–162)  147 (121–175)  0.307  CAKUT, congenital anomalies of the kidney and urinary tract; ESRD, end-stage renal disease; IL-2RA, interleukin-2 receptor antagonist; MMF, mycophenolate mofetil; Tx, renal transplantation. Table 2 Patient and transplant characteristics at time of transplantation according to AT1R-Ab status Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  Age (years), mean ± SD  8.6 ± 5.0  9.03 ± 4.80  8.30 ± 5.22  0.570  Male gender, n (%)  46 (74.2)  21 (70)  25 (78.1)  0.465  Donor age (years), mean ± SD  36.2 ± 12.9  36.2 ± 14.1  36.2 ± 11.9  0.992  Deceased donor, n (%)  40 (64.5)  21 (70)  19 (59.4)  0.533  Cold ischaemia time (h), mean ± SD  13.9 ± 6.4  13.3 ± 6.76  14.7 ± 5.83  0.576  Delayed graft function, n (%)  6 (9.7)  4 (13.3)  2 (6.3)  0.346  A-B-DR-DQ mismatch/8  2.68 ± 1.21  2.70 ± 1.15  2.66 ± 1.29  0.888   A-B  1.33 ± 0.70  1.50 ± 0.68  1.19 ± 0.70  0.079   DR  0.76 ± 0.53  0.67 ± 0.55  0.84 ± 0.52  0.194   DQ  0.58 ± 0.62  0.53 ± 0.63  0.63 ± 0.61  0.562  Cause of ESRD, n (%)   CAKUT  33 (53.2)  18 (60)  15 (46.9)  0.301   Nephronophthisis  5 (8.1)  2 (6.7)  3 (9.4)  0.696   Glomerular diseases  11 (17.7)  5 (16.7)  6 (18.8)  0.830   Others  13 (21.0)  5 (16.7)  8 (25)  0.421  Immunosuppression, n (%)   IL-2RA  12 (19.4)  4 (13.3)  8 (25)  0.245   Cyclosporine  40 (64.5)  19 (63.3)  21 (65.6)  0.851   Tacrolimus  22 (35.4)  11 (36.7)  11 (34.3)  0.851   MMF  62 (100)  30 (100)  32 (100)  1.000   Prednisolone  62 (100)  30 (100)  32 (100)  1.000  Observation time post-Tx (months), median (IQR)  141 (119–173)  137 (117–162)  147 (121–175)  0.307  Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  Age (years), mean ± SD  8.6 ± 5.0  9.03 ± 4.80  8.30 ± 5.22  0.570  Male gender, n (%)  46 (74.2)  21 (70)  25 (78.1)  0.465  Donor age (years), mean ± SD  36.2 ± 12.9  36.2 ± 14.1  36.2 ± 11.9  0.992  Deceased donor, n (%)  40 (64.5)  21 (70)  19 (59.4)  0.533  Cold ischaemia time (h), mean ± SD  13.9 ± 6.4  13.3 ± 6.76  14.7 ± 5.83  0.576  Delayed graft function, n (%)  6 (9.7)  4 (13.3)  2 (6.3)  0.346  A-B-DR-DQ mismatch/8  2.68 ± 1.21  2.70 ± 1.15  2.66 ± 1.29  0.888   A-B  1.33 ± 0.70  1.50 ± 0.68  1.19 ± 0.70  0.079   DR  0.76 ± 0.53  0.67 ± 0.55  0.84 ± 0.52  0.194   DQ  0.58 ± 0.62  0.53 ± 0.63  0.63 ± 0.61  0.562  Cause of ESRD, n (%)   CAKUT  33 (53.2)  18 (60)  15 (46.9)  0.301   Nephronophthisis  5 (8.1)  2 (6.7)  3 (9.4)  0.696   Glomerular diseases  11 (17.7)  5 (16.7)  6 (18.8)  0.830   Others  13 (21.0)  5 (16.7)  8 (25)  0.421  Immunosuppression, n (%)   IL-2RA  12 (19.4)  4 (13.3)  8 (25)  0.245   Cyclosporine  40 (64.5)  19 (63.3)  21 (65.6)  0.851   Tacrolimus  22 (35.4)  11 (36.7)  11 (34.3)  0.851   MMF  62 (100)  30 (100)  32 (100)  1.000   Prednisolone  62 (100)  30 (100)  32 (100)  1.000  Observation time post-Tx (months), median (IQR)  141 (119–173)  137 (117–162)  147 (121–175)  0.307  CAKUT, congenital anomalies of the kidney and urinary tract; ESRD, end-stage renal disease; IL-2RA, interleukin-2 receptor antagonist; MMF, mycophenolate mofetil; Tx, renal transplantation. Detection of HLA-DSA and AT1R antibodies Patient sera were tested for the presence of AT1R-Ab using a quantitative ELISA (CellTrend, Luckenwalde, Germany). All sera were also analysed for HLA antibodies using LABScreen Luminex kits (One Lambda, Canoga Park, CA, USA). For high-resolution typing of patients and their respective organ donors, polymerase chain reaction-sequence specific primers (SSP) Tray and Sequence kits (CTR, Heidelberg, Germany) and SSP kits (Olerup, Saltsjöbaden, Sweden) were used. DSA was determined against mismatched donor alleles from HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1 and -DPB1 loci and a mean fluorescence intensity (MFI) ≥500 was considered positive [18]. Blood pressure and estimated glomerular filtration rate (eGFR) analysis Systolic and diastolic blood pressures were derived from casual office blood pressure measurements (average of three measurements taken within 5 min) by oscillometry obtained at the time of index biopsy and at three to five outpatient department visits prior to index biopsy and converted to z-scores based on reference data [19]. Arterial hypertension was defined as systolic and/or diastolic blood pressure >1.96 z-score (95th percentile). Graft function was assessed using eGFR according to the Schwartz formula [15]. Immunosuppressive regimen The initial immunosuppressive therapy is depicted in Table 2. None of the patients received anti-thymocyte globulin or rituximab as induction therapy or prior to index biopsy. The immunosuppressive therapy at the time of index biopsy is depicted in Table 1. Three patients received the mammalian target of rapamycin (mTOR) inhibitor sirolimus without a calcineurin inhibitor (CNI) due to severe CNI-induced chronic nephrotoxicity in a previous biopsy. A total of 23 patients (37%) were treated with a corticosteroid-free immunosuppressive regimen. Patients with T-cell-mediated allograft rejection (TCMR) of Banff type I or II were treated with methylprednisolone pulse therapy. Patients with biopsy-proven chronic ABMR received anti-humoral rejection therapy as previously described [20]. None of the patients received intravenous immunoglobulin G during a period of at least 3 months prior to measurement of AT1R-Ab. Histopathology and C4d staining Renal biopsies were carried out as indication biopsies due to an increase of serum creatinine (>20% above baseline without an alternative explanation) and/or de novo persistent proteinuria >100 g protein/mol creatinine. All biopsy specimens were graded using the Banff 2009–15 criteria [21]. Immunohistochemistry for C4d was performed on paraffin sections using a polyclonal antibody (C4dpAb; Bio-medica, Vienna, Austria). Statistical analysis Analyses were performed using Predictive Analytics Software (SPSS) Statistics 22.0 (IBM, Armonk, NY, USA) and Statistical Analysis Software 9.3 (SAS Institute, Cary, NC, USA). Unless stated otherwise, results for continuous variables are presented mean ± SD or as median with interquartile range (IQR). Differences between groups were analysed with one-way analysis of variance (ANOVA), Student’s t-test or, if normality failed, with Kruskal–Wallis or Mann–Whitney U rank-sum test. For categorical data, Pearson chi-square tests were used. Receiver operating characteristic (ROC) plots were generated and area under the curve (AUC) and 95% confidence interval (CI) limits were calculated using the method of Hanley and McNeil [22]. Cox proportional hazards regression analysis and Kaplan–Meier survival analysis were used for time-to-event analyses from index biopsy and tested for significance with the two-sided log-rank test. For multivariable Cox regression models, a forward stepwise selection method was used, with a significance level of P < 0.1 for entering a variable into the model and P ≥ 0.2 for removal of a previously selected variable. P < 0.05 was regarded as statistically significant in a descriptive sense. RESULTS Patient characteristics Patient, donor and transplant characteristics at the time of transplantation are given in Table 2. First, patients were divided into three groups according to index biopsy results: (i) patients with normal biopsy results [n = 19 (30.6%)], (ii) patients with features of TCMR including borderline changes [n = 15 (24.2%)] and (iii) patients with ABMR including those suspicious for ABMR according to Banff 2015 and patients with mixed rejections [n = 28 (45.2%)]. Baseline characteristics at the time of index biopsy were in general comparable among these three groups, with the exception that patients with ABMR less frequently received a tacrolimus (TAC)-based maintenance immunosuppressive regimen than patients with normal biopsy results or patients with TCMR (50.0% versus 84.2% versus 73.3%; P = 0.041) and that the proportion of patients with proteinuria was higher in the ABMR group (67.9% versus 26.3% versus 33.3%; P = 0.010; Table 1). Index biopsies in the ABMR group tended to be performed somewhat later post-transplant (P = 0.067; Table 1). Serum AT1R-Ab stratified according to graft biopsy results Investigating the differences in AT1R-Ab serum concentrations among these three groups revealed significantly higher values in the ABMR group [12.0 U/mL (IQR 10.0–16.8)] compared with the group with normal biopsy results [8.0 U/mL (IQR 6.0–14.0); P = 0.012] or to those with TCMR [9.0 (IQR 7.0–10.0); P = 0.039], while AT1R-Ab was comparable between TCMR and patients without rejection (Figure 1). FIGURE 1 View largeDownload slide Distribution of AT1R-Ab levels (U/mL) based on the outlined index biopsy results. T-cell-mediated rejections include borderline changes and ABMR includes biopsy species suspicious for ABMR according to Banff 2015. FIGURE 1 View largeDownload slide Distribution of AT1R-Ab levels (U/mL) based on the outlined index biopsy results. T-cell-mediated rejections include borderline changes and ABMR includes biopsy species suspicious for ABMR according to Banff 2015. To determine the optimal AT1R-Ab cut-off value discriminating between patients with features of ABMR in the biopsy and those without (no rejection group and TCMR group), ROC curve analyses were performed. An area under the ROC curve value of 0.74 (95% CI 0.61–0.87; P = 0.001) indicated a moderate discriminative capacity (Figure 2). Based on the Youden Index (maximization of sensitivity and specificity), the ideal cut-off value for AT1R-Ab was determined as 9.5 U/mL, resulting in a sensitivity of 79% and a specificity of 71%. Using this cut-off value, 4 of 28 patients (14.3%) with ABMR in the index biopsy were positive only for HLA-DSA, 6 patients (21.4%) were positive only for AT1R-Ab and 16 patients (57.1%) were double positive for both HLA-DSA and AT1R-Ab. FIGURE 2 View largeDownload slide ROC curve analysis to determine the optimal cut-off value differentiating between ABMR and non-ABMR biopsy results. FIGURE 2 View largeDownload slide ROC curve analysis to determine the optimal cut-off value differentiating between ABMR and non-ABMR biopsy results. AT1R-Ab levels were significantly higher in the group with concomitant HLA-DSA positivity compared with HLA-DSA-negative patients [12.0 U/mL (IQR 9.0–16.5) versus 9.0 (IQR 7.0–12.0); P = 0.013]. AT1R-Ab levels did not differ significantly between HLA-DSA-positive patients with or without HLA-DSA C1q positivity (P = 0.407). There was no correlation between AT1R-Ab levels and HLA-DSA MFI values (P = 0.815). Next, we compared the biopsy scores with respect to AT1R-Ab and HLA-DSA status. AT1R-Ab levels were significantly higher in patients with a peritubular capillaritis (PTC) score >1 in the index graft biopsy compared with patients with a PTC score of 0 [9.0 U/mL (IQR 7.0–14.0) versus 12.0 (IQR 11.0–19.0); P = 0.041; Figure 3], while AT1R-Ab levels were not significantly altered in patients with different Banff i (P = 0.521) or t scores (P = 0.503). Patients with AT1R-Ab and HLA-DSA double positivity had a significantly higher microvascular inflammation score [1.0 (IQR 0–2.0)], defined as the sum of glomerulitis and PTC, than DSA-negative patients [0 (IQR 0–0.5); P = 0.004]. C4d positivity was less frequently observed in the cohort that was DSA negative and AT1R-Ab positive (25%; P = 0.154) than in the cohort that was DSA positive and AT1R-Ab negative (44.4%) or in the cohort that was both DSA positive and AT1R-Ab positive (50%; Supplementary data, Table S1), but the numbers were too small for a meaningful comparison. FIGURE 3 View largeDownload slide PTC scores and AT1R-Ab levels (all patients included). AT1R-Ab levels were significantly increased in patients with a PTC score of 2 compared with patients with a PTC score of 0. FIGURE 3 View largeDownload slide PTC scores and AT1R-Ab levels (all patients included). AT1R-Ab levels were significantly increased in patients with a PTC score of 2 compared with patients with a PTC score of 0. Graft survival, graft function and blood pressure according to AT1R-Ab status Patient characteristics at the time of transplantation were comparable between the AT1R-Ab-positive and -negative groups (Table 2). However, at the time of index biopsy, significantly more AT1R-Ab-positive patients showed additional HLA-class II DSA positivity (P = 0.002). AT1R-Ab positivity was associated with a significantly worse graft survival (P = 0.025) and a significantly more rapid decline of graft function (P = 0.004) during an observation period of up to 5 years post-biopsy (Figure 4, upper and lower panels). The 5-year graft survival was only 59% in the AT1R-Ab-positive group compared with 87% in the AT1R-Ab-negative group. Considering the eGFR decline in the time period of 5 years post-biopsy, 61% of AT1R-Ab-positive patients revealed an eGFR decline  ≥50% of baseline compared with only 20% of AT1R-Ab-negative patients. FIGURE 4 View largeDownload slide Association of AT1R-Ab positivity (AT1R-Ab >9.5 U/mL) and graft survival (upper panel) and deterioration of graft function post-biopsy (eGFR <50% of baseline levels prior to index biopsy) (lower panel). FIGURE 4 View largeDownload slide Association of AT1R-Ab positivity (AT1R-Ab >9.5 U/mL) and graft survival (upper panel) and deterioration of graft function post-biopsy (eGFR <50% of baseline levels prior to index biopsy) (lower panel). In AT1R-Ab-positive patients, systolic but not diastolic blood pressure z-scores averaged over three to five office blood pressure measurements prior to index biopsy were significantly higher than in AT1R-Ab-negative patients (P = 0.046; Table 3). Of note, the total amount of prescribed anti-hypertensive drugs at the time of the index biopsy and the number of patients on an angiotensin II receptor blocker were comparable between these two groups (Table 3). Table 3 Patient and transplant characteristics at the time of indication biopsy according to AT1R-Ab status Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  eGFR (mL/min/1.73 m2), mean ± SDa  41.0 ± 15.3  39.9 ± 14.9  42.0 ± 15.7  0.603  Patients with HLA-DSA Class I, n (%)  12 (19.4)  3 (10.0)  9 (28.1)  0.071  Patients with HLA-DSA Class II, n (%)  27 (43.5)  7 (23.3)  20 (62.5)  0.002  Systolic blood pressure (z-score), mean ± SD  1.18 ± 1.16  0.88 ± 1.02  1.48 ± 1.23  0.046  Diastolic blood pressure (z-score), mean ± SD  0.78 ± 0.89  0.64 ± 0.82  0.92 ± 0.94  0.225  Number of anti-hypertensive drugs per patient, median (IQR)  1.0 (0.75–2.0)  1.0 (0–2.0)  1.0 (1.0–3.0)  0.428  Patients on ARB, n (%)  14 (22.6)  5 (16.7)  9 (28.1)  0.281  Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  eGFR (mL/min/1.73 m2), mean ± SDa  41.0 ± 15.3  39.9 ± 14.9  42.0 ± 15.7  0.603  Patients with HLA-DSA Class I, n (%)  12 (19.4)  3 (10.0)  9 (28.1)  0.071  Patients with HLA-DSA Class II, n (%)  27 (43.5)  7 (23.3)  20 (62.5)  0.002  Systolic blood pressure (z-score), mean ± SD  1.18 ± 1.16  0.88 ± 1.02  1.48 ± 1.23  0.046  Diastolic blood pressure (z-score), mean ± SD  0.78 ± 0.89  0.64 ± 0.82  0.92 ± 0.94  0.225  Number of anti-hypertensive drugs per patient, median (IQR)  1.0 (0.75–2.0)  1.0 (0–2.0)  1.0 (1.0–3.0)  0.428  Patients on ARB, n (%)  14 (22.6)  5 (16.7)  9 (28.1)  0.281  ARB, angiotensin receptor blocker; HLA-DSA, human leucocyte antigen donor-specific antibodies. aeGFR according to Schwartz et al. [15]. Table 3 Patient and transplant characteristics at the time of indication biopsy according to AT1R-Ab status Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  eGFR (mL/min/1.73 m2), mean ± SDa  41.0 ± 15.3  39.9 ± 14.9  42.0 ± 15.7  0.603  Patients with HLA-DSA Class I, n (%)  12 (19.4)  3 (10.0)  9 (28.1)  0.071  Patients with HLA-DSA Class II, n (%)  27 (43.5)  7 (23.3)  20 (62.5)  0.002  Systolic blood pressure (z-score), mean ± SD  1.18 ± 1.16  0.88 ± 1.02  1.48 ± 1.23  0.046  Diastolic blood pressure (z-score), mean ± SD  0.78 ± 0.89  0.64 ± 0.82  0.92 ± 0.94  0.225  Number of anti-hypertensive drugs per patient, median (IQR)  1.0 (0.75–2.0)  1.0 (0–2.0)  1.0 (1.0–3.0)  0.428  Patients on ARB, n (%)  14 (22.6)  5 (16.7)  9 (28.1)  0.281  Characteristics  Entire cohort (n = 62)  AT1R-Ab-negative cohort (n = 30)  AT1R-Ab-positive cohort (n = 32)  P-value  eGFR (mL/min/1.73 m2), mean ± SDa  41.0 ± 15.3  39.9 ± 14.9  42.0 ± 15.7  0.603  Patients with HLA-DSA Class I, n (%)  12 (19.4)  3 (10.0)  9 (28.1)  0.071  Patients with HLA-DSA Class II, n (%)  27 (43.5)  7 (23.3)  20 (62.5)  0.002  Systolic blood pressure (z-score), mean ± SD  1.18 ± 1.16  0.88 ± 1.02  1.48 ± 1.23  0.046  Diastolic blood pressure (z-score), mean ± SD  0.78 ± 0.89  0.64 ± 0.82  0.92 ± 0.94  0.225  Number of anti-hypertensive drugs per patient, median (IQR)  1.0 (0.75–2.0)  1.0 (0–2.0)  1.0 (1.0–3.0)  0.428  Patients on ARB, n (%)  14 (22.6)  5 (16.7)  9 (28.1)  0.281  ARB, angiotensin receptor blocker; HLA-DSA, human leucocyte antigen donor-specific antibodies. aeGFR according to Schwartz et al. [15]. Graft survival and function according to AT1R-Ab and HLA-DSA status To further investigate the impact of both AT1R-Ab and HLA-DSA on graft survival and function, patients were divided into groups depending on their AT1R-Ab and HLA-DSA status. Baseline patient characteristics at the time of index biopsy were comparable among these three groups: patients without DSA, patients with AT1R-Ab only and patients with double positivity of AT1R-Ab and HLA-DSA (Supplementary data, Table S1). AT1R-Ab-positive (n = 12) as well as AT1R-Ab and HLA-DSA double-positive patients (n = 20) more often experienced an eGFR decline  ≥50% of baseline than DSA-negative patients (n = 21; P = 0.026 and P = 0.001 log-rank test; Figure 5). Patients with AT1R-Ab and HLA-DSA double positivity had a more rapid eGFR decline than patients who were only positive for AT1R-Ab or HLA-DSA (n = 9), but this difference did not reach statistical significance (P = 0.231), most likely due to the small number of observations. FIGURE 5 View largeDownload slide Association of deterioration of graft function (eGFR <50% of baseline prior to index biopsy) stratified according to AT1R-Ab and HLA-DSA status. FIGURE 5 View largeDownload slide Association of deterioration of graft function (eGFR <50% of baseline prior to index biopsy) stratified according to AT1R-Ab and HLA-DSA status. Risk factors for graft function deterioration We analysed non-invasive risk factors such as immunological and biochemical biomarkers for graft function deterioration, defined as an eGFR decline ≥50% of baseline. HLA-DSA (further differentiated according to C1q-complement binding capacity), AT1R-Ab status, eGFR at the time of index biopsy, proteinuria and arterial hypertension were assessed in a multivariate Cox regression model including age at index biopsy and time period from transplantation to index biopsy. In this model, C1q-positive HLA-DSA, eGFR and AT1R-Ab positivity were significantly associated with more rapid graft function decline (Table 4A). When we added histological characteristics such as transplant glomerulopathy and C4d status to this risk factor analysis, only transplant glomerulopathy, C1q-positive HLA-DSA and eGFR at the time of index biopsy remained statistically significant in the multivariate analysis (Table 4B). Table 4 Risk factor analysis for graft deterioration (eGFR <50% of baseline prior to index biopsy) up to 5 years post-biopsy Risk factors  Unadjusted HR (95% CI)  P-value  Adjusted HR (95% CI)  P-value  Serologic risk factors     AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010  2.83 (1.06–7.55)  0.038   HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  5.98 (2.01–17.7)  0.001   Proteinuria (>20 g/mol creatinine)  3.08 (1.37–6.92)  0.007       eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.95 (0.92–0.99)  0.008   Arterial hypertension  2.91 (1.22–6.96)  0.016      Including histopathological risk factors     Transplant glomerulopathy  6.29 (2.83–14.0)  <0.001  4.90 (2.01–12.0)  0.001   C4d positivity  5.10 (2.29–11.4)  <0.001       AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010       HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  4.09 (1.31–12.8)  0.015   eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.96 (0.93–0.99)  0.013  Risk factors  Unadjusted HR (95% CI)  P-value  Adjusted HR (95% CI)  P-value  Serologic risk factors     AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010  2.83 (1.06–7.55)  0.038   HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  5.98 (2.01–17.7)  0.001   Proteinuria (>20 g/mol creatinine)  3.08 (1.37–6.92)  0.007       eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.95 (0.92–0.99)  0.008   Arterial hypertension  2.91 (1.22–6.96)  0.016      Including histopathological risk factors     Transplant glomerulopathy  6.29 (2.83–14.0)  <0.001  4.90 (2.01–12.0)  0.001   C4d positivity  5.10 (2.29–11.4)  <0.001       AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010       HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  4.09 (1.31–12.8)  0.015   eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.96 (0.93–0.99)  0.013  Adjusted HR, each factor has been adjusted for the other factors that have been included in the final model (C1q, C1q HLA-DSA). HLA-DSA, human leucocyte antigen donor-specific antibodies; HR, hazard ratio. aeGFR according to Schwartz et al. [15]. Table 4 Risk factor analysis for graft deterioration (eGFR <50% of baseline prior to index biopsy) up to 5 years post-biopsy Risk factors  Unadjusted HR (95% CI)  P-value  Adjusted HR (95% CI)  P-value  Serologic risk factors     AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010  2.83 (1.06–7.55)  0.038   HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  5.98 (2.01–17.7)  0.001   Proteinuria (>20 g/mol creatinine)  3.08 (1.37–6.92)  0.007       eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.95 (0.92–0.99)  0.008   Arterial hypertension  2.91 (1.22–6.96)  0.016      Including histopathological risk factors     Transplant glomerulopathy  6.29 (2.83–14.0)  <0.001  4.90 (2.01–12.0)  0.001   C4d positivity  5.10 (2.29–11.4)  <0.001       AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010       HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  4.09 (1.31–12.8)  0.015   eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.96 (0.93–0.99)  0.013  Risk factors  Unadjusted HR (95% CI)  P-value  Adjusted HR (95% CI)  P-value  Serologic risk factors     AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010  2.83 (1.06–7.55)  0.038   HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  5.98 (2.01–17.7)  0.001   Proteinuria (>20 g/mol creatinine)  3.08 (1.37–6.92)  0.007       eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.95 (0.92–0.99)  0.008   Arterial hypertension  2.91 (1.22–6.96)  0.016      Including histopathological risk factors     Transplant glomerulopathy  6.29 (2.83–14.0)  <0.001  4.90 (2.01–12.0)  0.001   C4d positivity  5.10 (2.29–11.4)  <0.001       AT1R-Ab >9.5 U/mL  3.14 (1.31–7.52)  0.010       HLA-DSA positive      C1q negative  1.75 (0.69–4.40)  0.237        C1q positive  6.23 (2.31–16.8)  <0.001  4.09 (1.31–12.8)  0.015   eGFR at time of biopsya  0.96 (0.94–0.99)  0.017  0.96 (0.93–0.99)  0.013  Adjusted HR, each factor has been adjusted for the other factors that have been included in the final model (C1q, C1q HLA-DSA). HLA-DSA, human leucocyte antigen donor-specific antibodies; HR, hazard ratio. aeGFR according to Schwartz et al. [15]. DISCUSSION This is the first study in paediatric renal transplant recipients that analysed the prognostic significance of serum AT1R-Ab in conjunction with other known risk factors for graft failure in a carefully phenotyped single-centre cohort. The main result of this study is that the presence of AT1R-Ab in the context of a late (>1 year post-transplant) indication biopsy identifies a subgroup of patients with a more rapid subsequent eGFR decline. In the multivariate Cox regression model of non-invasive risk factors associated with enhanced graft function deterioration, AT1R-Ab positivity [adjusted hazard ratio (HR) 2.83] was the second strongest independent predictor of adverse graft outcome after the parameter C1q-positive DSA (HR 5.98). We also observed that serum AT1R-Ab concentrations were significantly higher in patients with biopsy-proven ABMR than in patients with TCMR or no rejection, consistent with recent findings in adult renal transplant recipients [23]. It is noteworthy that in our study a relevant subset of patients (21.4%) with features of ABMR in the index biopsy was only positive for AT1R-Ab and not for HLA-DSA. Hence, from these and other data it appears advisable to test patients with ABMR not only for the presence of HLA-DSA, but also for non-HLA antibodies against endothelial targets such as AT1R-Ab. The question arises whether AT1R-Ab directly induces or promotes ABMR or is an epiphenomenon of endothelial injury. While these two possibilities cannot easily be differentiated in a clinical study, experimental data indicate a direct effect of AT1R-Ab on endothelial injury, inflammation and allograft dysfunction [24, 25]. AT1R, the target for AT1R-Ab, is expressed at high levels on endothelial cells, and activation of this receptor in human microvascular endothelial cells in the presence of AT1R-Ab causes endothelial cell dysfunction and neutrophil migration; these effects were reduced in the presence of an angiotensin receptor blocker [24]. Therefore, AT1R-Ab is thought to contribute to graft dysfunction by inducing activation of signalling pathways in a similar fashion as the endogenous ligand for this receptor, angiotensin II [24–26]. In addition, experimental data indicate that activation of AT1R leads to an upregulation of HLA class II antigens on endothelial cells [27]. This experimental finding corresponds with our observation that patients with AT1R-Ab and HLA-DSA double positivity had a higher vascular micro-inflammation score and more rapid graft function deterioration than DSA-negative patients. Our study extends the observations of Philogene et al. [23], because we also investigated the prognostic significance of AT1R-Ab positivity regarding graft function and survival. The 5-year graft survival was only 59% in the AT1R-Ab-positive group compared with 87% in the AT1R-Ab-negative group. In the time period of 5 years post-biopsy, 61% of AT1R-Ab-positive patients revealed an eGFR decline  ≥50% of baseline compared with only 20% of AT1R-Ab-negative patients. Furthermore, patients with AT1R-Ab and HLA-DSA double positivity tended to have a more pronounced decline of graft function than patients who were only positive for AT1R-Ab or HLA-DSA. As outlined above, double positivity for AT1R-Ab and HLA-DSA appears to enhance the histopathological lesions of ABMR associated with more rapid graft function decline, but further studies in larger patient populations are required to confirm this hypothesis. We determined an optimal AT1R-Ab cut-off value of 9.5 U/mL discriminating between patients with or without features of ABMR in the biopsy. Three previous studies investigating AT1R-Ab levels in renal transplant recipients have suggested a similar threshold for positivity [6, 7, 28]. Giral et al. [7] observed that pre-transplant sensitization against AT1R is a risk factor for acute rejection and graft loss in adult patients and determined, by use of the same assay as in our study, a threshold of AT1R-Ab levels at 10 U/mL based on a statistical analysis using the Hothorn and Zeileis method [29]. Lee et al. [8] calculated an optimal cut-off value for pre-transplant AT1R-Ab of 9.05 U/mL for the risk prediction of acute rejection in the first year post-transplant in Asian adult renal transplant recipients. While it is interesting that these cut-off values are well comparable among different patient populations and age groups, further studies of AT1R-Ab both in the pre- and post-transplant setting are required to firmly establish valid cut-off values for AT1R-Ab positivity. Only two previous studies [12, 13] and one case report [14] have reported on AT1R-Ab serum concentrations in paediatric renal transplant recipients. A small study (n = 29) reported that 40% of patients developed AT1R-Ab in the first year post-transplant, but AT1R-Ab did not correlate with worse clinical outcomes [12]. Bjerre et al. [13] reported in a cross-sectional study of 30 children that AT1R-Ab levels were significantly higher in stable paediatric versus adult renal transplant recipients and higher compared with controls of comparable age groups, but they did not analyse the relationship of serum AT1R-Ab with outcome measures such as graft histology, function or survival. One case report described a girl with accelerated acute C4d-positive kidney transplant rejection, malignant hypertension, encephalopathy and the presence of both AT1R-Ab and HLA class II antibodies [14]. The strength of our study is that it was based on a prospective protocol investigating a paediatric patient cohort at an increased risk of graft deterioration reflected by a clinically indicated graft biopsy >1 year post-transplant. A limitation is the relatively small number of patients investigated, but this is an inherent problem for all studies in the paediatric renal transplant population. Furthermore, AT1R-Ab was measured only at a single time point post-transplant. Future studies will have to evaluate whether longitudinal analyses of AT1R-Ab in an unselected patient cohort of paediatric kidney allograft recipients is a surrogate marker for increased immunological risk requiring more intense immunosuppressive therapy. It will also be important to investigate the effect of potential treatment modalities on AT1R-Ab and if they improve outcomes for these patients. In conclusion, the presence of AT1R-Ab in the context of an indication biopsy >1 year post-transplant is associated with the histopathology of ABMR and is a non-invasive risk factor for subsequent accelerated graft function decline. Hence the determination of serum AT1R-Ab may enhance the predictive value of HLA-DSA and/or C1q-binding DSA. Screening for AT1R-Ab in conjunction with HLA-DSA at regular intervals post-transplant may have the potential to identify patients at high risk for ABMR, in whom targeted enhancement of immunosuppressive therapy may improve transplant outcome. This is especially relevant for the paediatric kidney transplant population, in whom, in the light of their long life expectancy, preservation of good graft function is of utmost importance. AUTHORS’ CONTRIBUTIONS A.F, C.S. and B.T. participated in research design. A.F, C.S., B.H., S.R., R.W., J.H.W., A.S., D.D. and B.T. participated in performance of the research, data analysis and in writing the article. All authors reviewed the manuscript, believe it represents valid work and approved it for submission. ACKNOWLEDGEMENTS We wish to thank Marzena Kirschke, Manuela Schneidenbach, Fatma Karci and our HLA and DNA laboratory teams for excellent technical assistance. We gratefully acknowledge the support of this study by a grant from the Peter-Stiftung für die Nierenwissenschaft, Münster, Germany. SUPPLEMENTARY DATA Supplementary data are available at ndt online. CONFLICT OF INTEREST STATEMENT None declared. This work is original and submitted solely to NDT for consideration. The results presented in this article have not been published previously in whole or part, except in abstract form. REFERENCES 1 Einecke G, Sis B, Reeve J. Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure. Am J Transplant  2009; 9: 2520– 2531 Google Scholar CrossRef Search ADS PubMed  2 Lee PC, Zhu L, Terasaki PI et al.  . 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Biometrics  2008; 64: 1263– 1269 Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Nephrology Dialysis TransplantationOxford University Press

Published: Feb 12, 2018

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