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• W2953025960 abstract "The clinical significance of non-HLA antibodies on renal allograft survival is a matter of debate, due to differences in reported results and lack of large-scale studies incorporating analysis of multiple non-HLA antibodies simultaneously. We developed a multiplex non-HLA antibody assay against 14 proteins highly expressed in the kidney. In this study, the presence of pretransplant non-HLA antibodies was correlated to renal allograft survival in a nationwide cohort of 4770 recipients transplanted between 1995 and 2006. Autoantibodies against Rho GDP-dissociation inhibitor 2 (ARHGDIB) were significantly associated with graft loss in recipients transplanted with a deceased-donor kidney (N = 3276) but not in recipients of a living-donor kidney (N = 1496). At 10 years after deceased-donor transplantation, recipients with anti-ARHGDIB antibodies (94/3276 = 2.9%) had a 13% lower death-censored covariate-adjusted graft survival compared to the anti-ARHGDIB-negative (3182/3276 = 97.1%) population (hazard ratio 1.82; 95% confidence interval, 1.32-2.53; P = .0003). These antibodies occur independently from donor-specific anti-HLA antibodies (DSA) or other non-HLA antibodies investigated. No significant relations with graft loss were found for the other 13 non-HLA antibodies. We suggest that pretransplant risk assessment can be improved by measuring anti-ARHGDIB antibodies in all patients awaiting deceased-donor transplantation. The clinical significance of non-HLA antibodies on renal allograft survival is a matter of debate, due to differences in reported results and lack of large-scale studies incorporating analysis of multiple non-HLA antibodies simultaneously. We developed a multiplex non-HLA antibody assay against 14 proteins highly expressed in the kidney. In this study, the presence of pretransplant non-HLA antibodies was correlated to renal allograft survival in a nationwide cohort of 4770 recipients transplanted between 1995 and 2006. Autoantibodies against Rho GDP-dissociation inhibitor 2 (ARHGDIB) were significantly associated with graft loss in recipients transplanted with a deceased-donor kidney (N = 3276) but not in recipients of a living-donor kidney (N = 1496). At 10 years after deceased-donor transplantation, recipients with anti-ARHGDIB antibodies (94/3276 = 2.9%) had a 13% lower death-censored covariate-adjusted graft survival compared to the anti-ARHGDIB-negative (3182/3276 = 97.1%) population (hazard ratio 1.82; 95% confidence interval, 1.32-2.53; P = .0003). These antibodies occur independently from donor-specific anti-HLA antibodies (DSA) or other non-HLA antibodies investigated. No significant relations with graft loss were found for the other 13 non-HLA antibodies. We suggest that pretransplant risk assessment can be improved by measuring anti-ARHGDIB antibodies in all patients awaiting deceased-donor transplantation. Chronic kidney disease affects about 10% of the global population.1World Kidney Day. Chronic kidney disease. 2018. https://www.worldkidneyday.org/faqs/chronic-kidney-disease. Accessed September 12, 2018.Google Scholar Over 2 million people worldwide currently receive dialysis treatment, or are recipients of a kidney transplant. Kidney transplantation is the preferred treatment for end-stage renal disease due to superior quality of life and survival rates. However, although short-term renal allograft survival has improved considerably in the last 20 years, antibody-mediated rejection (ABMR) remains one of the major causes of graft loss and of deterioration of graft function in the long-term. Donor-specific anti-HLA antibodies (DSA) are well known to play an important role in this process. Nonetheless, (subclinical) ABMR occurs also in the absence of DSA, which has sparked interest in the short- and long-term clinical relevance of donor-reactive antibodies recognizing proteins other than HLA (ie, non-HLA-antibodies).2Loupy A Vernerey D Tinel C et al.Subclinical rejection phenotypes at 1 year post-transplant and outcome of kidney allografts.J Am Soc Nephrol. 2015; 26: 1721-1731Crossref PubMed Scopus (211) Google Scholar The clinical relevance of non-HLA antibodies on graft survival is not clear.3Tait BD Süsal C Gebel HM et al.Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation.Transplantation. 2013; 95: 19-47Crossref PubMed Scopus (596) Google Scholar,4Deltombe C Gillaizeau F Anglicheau D et al.Is pre-transplant sensitization against angiotensin II type 1 receptor still a risk factor of graft and patient outcome in kidney transplantation in the anti-HLA Luminex era? A retrospective study.Transpl Int. 2017; 30: 1150-1160Crossref PubMed Scopus (24) Google Scholar Although it has been reported that non-HLA antibodies against Angiotensin II type 1 receptor (AT1R) are an independent risk factor for long-term graft loss,5Giral 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: 2567-2576Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar others could not replicate these findings,6Gareau AJ Wiebe C Pochinco D et al.Pre-transplant AT1R antibodies correlate with early allograft rejection.Transpl Immunol. 2018; 46: 29-35Crossref PubMed Scopus (38) Google Scholar,7Pinelli DF Friedewald JJ Haarberg KMK et al.Assessing the potential of angiotensin II type 1 receptor and donor specific anti-endothelial cell antibodies to predict long-term kidney graft outcome.Hum Immunol. 2017; 78: 421-427Crossref PubMed Scopus (22) Google Scholar which may be caused by the inclusion or exclusion of patients with pretransplant DSA. However, antibody-mediated rejection of renal allografts from HLA-identical sibling donors and also from deceased donors in the absence of pretransplant DSA has been reported several times.8Kalil J Guilherme L Neumann J et al.Humoral rejection in two HLA identical living related donor kidney transplants.Transplant Proc. 1989; 21: 711-713PubMed Google Scholar,9Amico P Hönger G Mayr M Schaub S Detection of HLA-antibodies prior to renal transplantation: prospects and limitations of new assays.Swiss Med Wkly. 2008; 138: 472-476PubMed Google Scholar And recently it was reported that the development of circulating natural antibodies posttransplant is associated with poorer graft survival, worse graft function, and more microvascular injury.10See SB Aubert O Loupy A et al.Post-transplant natural antibodies associate with kidney allograft injury and reduced long-term survival.J Am Soc Nephrol. 2018; 29: 1761-1770Crossref PubMed Scopus (29) Google Scholar Non-HLA antibodies have been described against a variety of targets, but large-scale studies incorporating analysis of these antibodies simultaneously to assess their clinical relevance in kidney transplantation are lacking. In this retrospective study on a large national cohort of 4770 renal transplant recipients, we assessed the impact on graft survival of IgG autoantibodies against 14 previously identified target proteins.11Kamburova EG Kardol-hoefnagel T Wisse BW Joosten I Wil A Development and validation of a multiplex non-HLA antibody assay for the screening of kidney transplant recipients.Front Immunol. 2018; 9: 3002Crossref PubMed Scopus (23) Google Scholar Between January 1995 and December 2005, 6097 kidney transplantations with a negative complement-dependent cytotoxicity crossmatch were performed in The Netherlands. Clinical data were obtained from the Dutch Organ Transplant Registry. The use of sera and experimental protocols was approved by the Research Ethics Committee for Biobanks and the Medical Ethics Committee of the University Medical Center Utrecht. Experimental protocols were performed in accordance with the Federation of Dutch Medical Scientific Societies Code of Conduct. The study was conducted in accordance with the 2013 Declaration of Helsinki and the 2008 Declaration of Istanbul. Of 4787/6097 (78%) transplantations, pretransplant serum was available. Seventeen transplantations were excluded due to lack of follow-up, and the remaining 4770 transplantations were included in this analysis. Minimal follow-up time was 10 years after transplantation. Data available on request due to privacy/ethical restrictions. We selected 14 non-HLA target proteins from the literature.11Kamburova EG Kardol-hoefnagel T Wisse BW Joosten I Wil A Development and validation of a multiplex non-HLA antibody assay for the screening of kidney transplant recipients.Front Immunol. 2018; 9: 3002Crossref PubMed Scopus (23) Google Scholar Antibodies against the glomerular basement membrane protein agrin have been described in the context of transplant glomerulopathy.12Joosten SA Sijpkens YWJ van Ham V et al.Antibody response against the glomerular basement membrane protein agrin in patients with transplant glomerulopathy.Am J Transplant. 2005; 5: 383-393Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar Antibodies against adipocyte plasma membrane-associated protein (APMAP), Rho GDP-dissociation inhibitor 2 (ARHGDIB), Rho guanine nucleotide exchange factor 6 (ARHGEF6), Lamin B1, BPI fold-containing family B member 1 (LPLUNC1), protein kinase C zeta type (PRKCZ), and tubulin beta-4B (Tubb4B) were all demonstrated either in chronic hemodialysis patients or patients awaiting kidney transplantation. Antibodies directed against targets expressed on the endothelium, that is, AT1R and endothelin type A receptor (ETAR), were reported to be involved directly or indirectly in renal disease. Antibodies against the intracellular proteins vimentin and peroxisomal trans-2-enoyl-CoA reductase (PECR) have been reported to be associated with allograft failure.13Dinavahi R George A Tretin A et al.Antibodies reactive to non-HLA antigens in transplant glomerulopathy.J Am Soc Nephrol. 2011; 22: 1168-1178Crossref PubMed Scopus (65) Google Scholar,14Besarani D Cerundolo L Smith JD et al.Role of anti-vimentin antibodies in renal transplantation.Transplantation. 2014; 98 (Jul 15): 72-78Crossref PubMed Scopus (35) Google Scholar Pre- and posttransplant endorepellin antibody levels were increased in patients with vascular rejection15Cardinal H Dieudé M Brassard N et al.Antiperlecan antibodies are novel accelerators of immune-mediated vascular injury.Am J Transplant. 2013; 13 (Apr): 861-874Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar and phospholipase A2 receptor (PLA2R) antibodies are strongly associated with primary membranous nephropathy.16Dähnrich C Komorowski L Probst C et al.Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy.Clin Chim Acta. 2013; 421: 213-218Crossref PubMed Scopus (108) Google Scholar We commercially purchased all proteins with the exception of PLA2R, which was produced in-house, and coupled these proteins directly with carboxylated MagPlex Microspheres (Luminex Corp, Austin, TX) as recommended by Luminex.17De Jager W Velthuis H Prakken BJ et al.Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.Clin Diagn Lab Immunol. 2003; 10: 133-139PubMed Google Scholar In addition, we produced 12 of the 14 proteins with a HaloTag, with the exception of AT1R and PECR. The HaloTag proteins were coupled via a HaloTag Amine (O4) Ligand (Promega, Madison, WI) to the carboxylated MagPlex microspheres. A detailed description of the proteins and the coupling methods is given elsewhere.11Kamburova EG Kardol-hoefnagel T Wisse BW Joosten I Wil A Development and validation of a multiplex non-HLA antibody assay for the screening of kidney transplant recipients.Front Immunol. 2018; 9: 3002Crossref PubMed Scopus (23) Google Scholar A mix of 31 different microspheres was made containing 15 directly coupled proteins (transferrin and 14 target proteins), 13 in-house produced HaloTag-coupled proteins (transferrin and 12 target proteins), an IgG-coupled microsphere as a positive control, and a HaloTag amine-coupled and empty microsphere as additional negative controls. Transferrin (directly- or HaloTag-coupled) also served as a negative control, since it is ubiquitously present and no autoantibodies against transferrin have been reported. Sera (1:25 dilution) were incubated overnight with the microsphere mix. Next, R-phycoerythrin-conjugated goat-anti human antibody was added. After 30 minutes of incubation, wash buffer was added and samples were measured on a Luminex 200 flow analyzer (Luminex Corp). Three-micrometer formalin-fixed paraffin-embedded sections were used. After antigen retrieval in citrate solution pH 6, primary antibody against ARHGDIB (Biobyt, San Francisco, CA) was applied in a 1:2000 solution for 1 hour at room temperature, followed by HRP-labeled polymer anti-rabbit Ig detection antibody (BrightVision, VWR, Duiven). Bound antibody was visualized with Nova Red substrate (Vector labs), and finally nuclei were counterstained with hematoxylin. Because the Kaplan-Meier estimates were biased due to unbalanced distribution of confounders, death-censored graft survival was assessed using the adjusted Kaplan-Meier estimator (AKME) based on inverse probability weighting (IPW). Each observation is weighted by its inverse probability of being in a certain group.18Xie J Liu C Adjusted Kaplan-Meier estimator and log-rank test with inverse probability of treatment weighting for survival data.Stat Med. 2005; 24: 3089-3110Crossref PubMed Scopus (310) Google Scholar Hazard ratios (HRs) and 95% confidence intervals (95% CIs) were derived using multivariable Cox regression. A Bonferroni correction was used to adjust for multiple comparisons when studying the effects of non-HLA antibodies on graft survival and P < .002 (.05 divided by 25 non-HLA antibodies) was considered as statistically significant (applied for analyses in Tables 1, S3, and S4). We adjusted in both the AKME and Cox regression for recipient age (quadratic) and donor age (quadratic), cold ischemia time (for donation after brain death or cardiac death), years on dialysis (quadratic), induction therapy with IL-2 receptor blocker, and the presence of pretransplant single antigen bead-defined DSA against HLA-A/B/DR/DQ. (For more detailed descriptions see Kamburova et al.19Kamburova EG Wisse BW Joosten I et al.Differential effects of donor-specific HLA antibodies in living versus deceased donor transplant.Am J Transplant. 2018; 18: 2274-2284Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) Statistical analyses were performed with R version 3.4.1 and SAS version 9.4 (SAS Institute, Cary, NC). Continuous data were analyzed with the Mann-Whitney U test and categorical data with the chi-square test.TABLE 1Multivariable analyses of the effect of antibodies against ARHGDIB on 10-year death-censored graft failureNo. (%) of transplantations with anti-ARHGDIB antibodiesHazard ratio95% CIP-valueTotal cohort (N = 4770)134 (2.8)1.7011.265-2.288.0004Deceased donors (N = 3276)94 (2.9)1.8201.318-2.531.0003Living donors (N = 1494)40 (2.7)1.2490.587-2.657.5639Note: In this multivariable analysis we evaluated the effect of the presence of pretransplant ARHGDIB on the 10-year death-censored graft failure and adjusted for differences in the following covariates: recipient age (quadratic), donor age (quadratic), donor type (living or deceased, only for the total cohort), cold ischemia time in hours for donation after brain death (DBD) and donation after cardiac death (DCD), time on dialysis in years (quadratic), induction therapy with Interleukin- 2 receptor–blocking antibody and the presence of pretransplant donor-specific anti-HLA antibodies against HLA-A/B/DR/DQ. CI, confidence interval. A Bonferroni correction was used to adjust for multiple comparisons with P < .002 considered as statistically significant. Open table in a new tab Note: In this multivariable analysis we evaluated the effect of the presence of pretransplant ARHGDIB on the 10-year death-censored graft failure and adjusted for differences in the following covariates: recipient age (quadratic), donor age (quadratic), donor type (living or deceased, only for the total cohort), cold ischemia time in hours for donation after brain death (DBD) and donation after cardiac death (DCD), time on dialysis in years (quadratic), induction therapy with Interleukin- 2 receptor–blocking antibody and the presence of pretransplant donor-specific anti-HLA antibodies against HLA-A/B/DR/DQ. CI, confidence interval. A Bonferroni correction was used to adjust for multiple comparisons with P < .002 considered as statistically significant. We analyzed the 4770 pretransplant sera using our multiplex non-HLA assay, and the individual median fluorescence intensitiy (MFI) values with box and whisker plots are shown in Figure S1. As we observed relatively high background signals for some sera (Figure S1A), we decided to use signal-to-background ratios (STBRs) as a parameter to determine non-HLA antibody positivity. The correlation between the MFI of transferrin (directly- or HaloTag-coupled) and the MFIs of target microspheres was stronger than the correlation between MFI of other negative control microspheres and that of target microspheres (data not shown). Therefore, transferrin was selected as the most optimal negative control, and was used correspondingly to calculate the STBRs (Figure S2). To determine the clinically relevant cut-off, we analyzed the impact on 1-, 5-, and 10-year death-censored graft survival of various cut-offs for each antibody in a univariate analysis (Figure 1). For each non-HLA antibody, we selected the ratio and absolute MFI cut-off that resulted in the highest difference in graft survival between the antibody-negative and antibody-positive group. For ARHGDIB, a ratio of 10 in combination with an absolute MFI of 500 was chosen as cut-off values using the directly coupled microspheres, resulting in 134/4770 (2.8%) positive patients (Figure 1). An overview of the selected cut-offs for the other non-HLA antibodies based on the maximal graft survival difference is summarized in Table S1. Depending on the type of non-HLA antibody analyzed, percentages of positive sera ranged from 0.9% to 2.8%, and varying differences were observed in 1-, 5-, and 10-year graft survival. After Bonferroni correction, a significant difference in graft survival was observed between patients with pretransplant antibodies against ARHGDIB compared to patients without antibodies against this target (Table 1). No significant relation with graft loss was observed for the other non-HLA antibodies. A summary of the impact of the other non-HLA antibodies on 10-year graft survival of the total cohort is summarized in Table S2. Because our cohort contained a relatively high proportion of living donors and we previously found that DSA had mainly an impact on deceased-donor transplantations with only a limited effect on living-donor transplantations,19Kamburova EG Wisse BW Joosten I et al.Differential effects of donor-specific HLA antibodies in living versus deceased donor transplant.Am J Transplant. 2018; 18: 2274-2284Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar we also analyzed the impact of non-HLA antibodies on long-term graft survival according to donor status (3276 deceased-donor and 1494 living-donor transplantations). After deceased-donor transplantation, the AKME according to the presence of ARHGDIB antibodies showed a 10-year death-censored graft survival of 61% (95% CI 50%-70%) for the 94 of 3276 patients with and 73% (95% CI 71%-75%) for the 3182/3276 patients without ARHGDIB antibodies (Figure 2A; P = .017). Pretransplant DSA against HLA-A/B/DR/DQ was found in only 10 of 94 patients (10.6%) with ARHGDIB antibodies and 423/3182 patients (13.3%) without (P = .454). A Kaplan-Meier analysis in this small subgroup did not show an indication for a synergistic effect of DSA and anti-ARHGDIB antibodies. In addition, we could find no indication for interaction between the presence of all studied autoantibodies, or an effect of their combined presence on graft survival. There were no significant differences between the patient, donor, and transplant characteristics between the anti-ARHGDIB positive and negative groups transplanted with a deceased-donor kidney, except the cold ischemia time was slightly longer in anti-ARHGDIB-positive patients (23.5 ± 7.7 hours) compared to anti-ARHGDIB-negative patients (21.8 ± 7.2) transplanted with a deceased donor (P = .043, Table 2). The multivariable analysis, adjusted for the same covariates, showed that the presence of ARHGDIB antibodies was associated with a higher risk of 10-year graft failure after transplantation with a deceased-donor kidney (Table 1, HR 1.82, 95% CI 1.32-2.53, P = .0003). At 1 year after transplantation, the HR was 1.620 (95% CI, 0.993-2.643) for the anti-ARHGDIB-positive group compared to the anti-ARHGDIB-negative group (Table S3). Furthermore, the rejection-free survival was comparable for patients with and without antibodies against ARHGDIB (Figure S3). For the living-donor transplantations, the presence of ARHGDIB antibodies was not associated with decreased graft survival (Figure 2B) or increased risk of graft failure in multivariable analysis (Table 1, HR 1.25, 95% CI 0.59-2.66, P = .56). Finally, we also assessed the association between type of donor, anti-ARHGDIB antibodies, and 10-year graft survival in the same model. The hazard ratio for a living donor was 0.563 (95% CI 0.488-0.650, P < .0001) compared to deceased donors, suggesting that that anti-ARHGDIB was significantly associated with graft loss in recipients transplanted with a kidney from a deceased donor but not in recipients of a living-donor kidney.TABLE 2Characteristics of ARHGDIB-positive and ARHGDIB-negative patients transplanted with a deceased-donor kidneyCharacteristicsARHGDIB negative (N = 3182)ARHGDIB positive (N = 94)P-valueDeceased-donor transplantations (N = 3276)PatientAge at transplantation (y, mean ± SD)46.9 ± 14.147.9 ± 13.4.462aMann-Whitney U test for continuous variables.46.1 ± 14.1Female sex - no. (%)1286 (40.4)39 (41.5).834bChi-square test for categorical variables.1325 (40.5)PRA at time of transplantation (%, mean ± SD)6.8 ± 18.79.2 ± 25.1.574aMann-Whitney U test for continuous variables.6.9 ± 18.9Highest PRA (%, mean ± SD)16.4 ± 28.215.4 ± 30.4.300aMann-Whitney U test for continuous variables.16.4 ± 28.2Dialysis, n (%).455bChi-square test for categorical variables.No147 (4.6)3 (3.0)150 (4.6)Yes – hemodialysis1853 (58.2)49 (52.1)1902 (58.1)Yes – peritoneal dialysis1164 (36.6)41 (43.6)1205 (36.8)Unknown18 (0.6)1 (1.1)19 (0.6)Time on dialysis (y, mean ± SD)3.4 ± 2.63.4 ± 2.2.814aMann-Whitney U test for continuous variables.3.4 ± 2.6DonorDonor age (y, mean ± SD)42.7 ± 16.046.0 ± 14.6.062aMann-Whitney U test for continuous variables.42.8 ± 16.0Donor female sex – no. (%)1489 (46.8)46 (48.9).681bChi-square test for categorical variables.1535 (46.9)Cold-ischemia time (hours, mean ± SD)21.8 ± 7.223.5 ± 7.7.043aMann-Whitney U test for continuous variables.21.8 ± 7.2TransplantRepeat transplantation – no. (%)554 (17.4)16 (17.0).922bChi-square test for categorical variables.570 (17.4)Pretransplant DSA against HLA-A/B/DR/DQ – no. (%)423 (13.3)10 (10.6).454bChi-square test for categorical variables.433 (13.2)Induction therapyIL-2 receptor blocker – no. (%)651 (20.5)14 (14.9).186bChi-square test for categorical variables.665 (20.3)T-cell depleting antibodycT cell-depleting antibody therapy: ALG, ATG, OKT3 monoclonal antibodies. no. (%)134 (4.2)0 (0).042bChi-square test for categorical variables.134 (4.1)Initial immunosuppression – no. (%)Steroids3120 (98.1)90 (95.7).117bChi-square test for categorical variables.3210 (98.0)MMF/azathioprine2377 (74.7)63 (67.0).092bChi-square test for categorical variables.244 (74.4)Cyclosporine/tacrolimus3000 (94.3)88 (93.6).785bChi-square test for categorical variables.3088 (94.3)Sirolimus171 (5.4)9 (9.6).078bChi-square test for categorical variables.180 (5.4)Other417 (13.1)11 (11.7).691bChi-square test for categorical variables.428 (13.1)Unknown11 (0.4)1 (1.1).256bChi-square test for categorical variables.12 (0.4)DSA, Donor-specific anti-HLA antibodies; IL, interleukin; MMF, mycophenolate mofetil.a Mann-Whitney U test for continuous variables.b Chi-square test for categorical variables.c T cell-depleting antibody therapy: ALG, ATG, OKT3 monoclonal antibodies. Open table in a new tab DSA, Donor-specific anti-HLA antibodies; IL, interleukin; MMF, mycophenolate mofetil. Finally, we were wondering where in the kidney ARHGDIB is expressed. Therefore, we stained biopsies of a transplanted kidney without histological abnormalities and a transplanted kidney with acute tubular necrosis using an anti-ARHGDIB antibody. In a transplanted kidney without histological abnormalities, weak ARHGDIB expression was seen in endothelial cells of interlobular arteries, endothelial cells of peritubular capillaries, and endothelial cells of glomerular capillaries (Figure 3A). In a transplanted kidney with acute tubular necrosis, strong ARHGDIB expression was seen in endothelial cells of interlobular arteries, endothelial cells of peritubular capillaries, and endothelial cells of glomerular capillaries. In addition, positive staining for ARHGDIB is also seen in some podocytes and lymphocytes (Figure 3B). In the present study, we determined the relation between graft failure and the presence of pretransplant non-HLA antibodies in the sera of 4770 kidney transplantations performed in The Netherlands between 1995 and 2006. The results indicate that pretransplant antibodies against ARHGDIB represent a risk factor for graft loss in deceased-donor transplantations. The 14 non-HLA target proteins included in our assay were selected based on reported antibody specificity in kidney transplant patients or patients with end-stage renal disease, and their expression in the kidney. A number of these non-HLA antibodies have been examined in relation to kidney transplantation. Antibodies against AT1R were associated with rejection and graft survival according to results from several large-scale studies,5Giral 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: 2567-2576Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar,20Taniguchi 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: 2577-2589Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar although a recent large study (n = 940) did not confirm this association.4Deltombe C Gillaizeau F Anglicheau D et al.Is pre-transplant sensitization against angiotensin II type 1 receptor still a risk factor of graft and patient outcome in kidney transplantation in the anti-HLA Luminex era? A retrospective study.Transpl Int. 2017; 30: 1150-1160Crossref PubMed Scopus (24) Google Scholar Pretransplant anti-ETAR antibodies have been associated with higher serum creatinine values at 1 year posttransplantation and with more severe vascular rejection.21Banasik 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: 24-29Crossref PubMed Scopus (57) Google Scholar Antibodies against LG3, a C-terminal fragment of perlecan, were associated with acute tubulointerstitial rejection and long-term renal allograft dysfunction.15Cardinal H Dieudé M Brassard N et al.Antiperlecan antibodies are novel accelerators of immune-mediated vascular injury.Am J Transplant. 2013; 13 (Apr): 861-874Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,22Yang BHK Hénault-Rondeau M Patey N et al.Anti-LG3 antibodies aggravate renal ischemia-reperfusion injury and long-term renal allograft dysfunction.Kidney Int. 2015; 13866: 1-14Google Scholar Antibodies against PLA2R, LPLUNC, APMAP, and PRKCZ were described previously in small case-series and have not been further evaluated.23Sutherland SM Li L Sigdel TK et al.Protein microarrays identify antibodies to protein kinase Czeta that are associated with a greater risk of allograft loss in pediatric renal transplant recipients.Kidney Int. 2009; 76: 1277-1283Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 24Otten HG van Loon M van Ginkel WGJ van de Graaf EA Hene RJ Identification of 3 novel non-HLA target antigens recognized after kidney transplantation.Tissue Antigens. 2007; 69: 375-376Google Scholar, 25Gupta G Fattah H Ayalon R et al.Pre-transplant phospholipase A2 receptor autoantibody concentration is associated with clinically significant recurrence of membranous nephropathy post-kidney transplantation.Clin Transplant. 2016; 30: 461-469Crossref PubMed Scopus (37) Google Scholar During the development of our non-HLA antibody assay we found that MFI results on a specific bead was comparable between singleplex and multiplex measurement, indicating no occurrence of compromise by multiplex analysis. In general, non-HLA antibodies frequently occur within the healthy population and for many autoantibodies a serum is considered positive when exceeding a cut-off yielding a limited (<5%) frequency of positive results within the healthy population. This definition does not necessarily provide the most clinically relevant cut-off value with regard to prognosis. To this end, we chose to define a clinically relevant cut-off as it was previously described for HLA antibodies using a combination of STBR combined with a minimal MFI level.26Wisse BW Kamburova EG Joosten I et al.Toward a sensible single antigen bead cut-off based on kidney graft survival.Transplantation. 2019; 103: 789-797Crossref PubMed Scopus (23) Google Scholar Using individual cut-offs for each non-HLA antibody, optimally discriminating patients with long-term functioning grafts vs graft loss, we did not find associations between the presence of any of the abovementioned previously studied non-HLA antibodies and graft loss, function, or rejection. We did not analyze the autoantibody MFI levels as continuous variables, because in the field of anti-HLA antibodies it is well accepted that MFI is not an indication of antibody titer and is influenced by several factors such as (1) the antigen density, (2) the affinity of the antibody to the antigen, and (3) the amount/titer of the antibody (also dependent on the serum dilution used in the assay). At present, no studies have been reported showing the association between anti-ARHGDIB antibodies and graft loss. Here, we found significant associations between graft loss and the pretransplant presence of antibodies against ARHGDIB. The effect of these antibodies is observed predominantly in patients transplanted with a deceased-donor kidney. This suggests an interaction between the presence of anti-ARHGDIB antibodies and ischemia reperfusion injury, which is less prominent in living-donor kidney transplantation.27Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney transplantation.Lancet. 2004; 364: 1814-1827Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar ARHGDIB is widely expressed, including in the renal pelvis and glomeruli. Increased expression of ARHGDIB has been reported in several solid tumors and hematological malignancies.28Ma L Xu G Sotnikova A et al.Loss of expression of LyGDI (ARHGDIB), a rho GDP-dissociation inhibitor, in Hodgkin lymphoma.Br J Haematol. 2007; 139: 217-223Crossref PubMed Scopus (42) Google Scholar, 29Cho HJ Baek KE Yoo J RhoGDI2 as a therapeutic target in cancer.Expert Opin Ther Targets. 2010; 14: 67-75Crossref PubMed Scopus (40) Google Scholar, 30Pont MJ Hobo W Honders MW et al.LB-ARHGDIB-1R as a novel minor histocompatibility antigen for therapeutic application.Haematologica. 2015; 100: e419-e422Crossref PubMed Scopus (11) Google Scholar, 31Von Klot CA Dubrowinskaja N Peters I et al.Rho GDP dissociation inhibitor-β in renal cell carcinoma.Oncol Lett. 2017; 14: 8190-8196PubMed Google Scholar Bilalic et al32Bilalic S Veitinger M Ahrer K-H et al.Identification of non-HLA antigens targeted by alloreactive antibodies in patients undergoing chronic hemodialysis.J Proteome Res. 2010; 9: 1041-1049Crossref PubMed Scopus (24) Google Scholar first described that dialysis patients can have autoreactive antibodies against ARHGDIB. Further analysis in kidney biopsies showed cytoplasmic expression in endothelial cells of interlobular arteries and peritubular capillaries, and in podocytes. We consider it possible that cellular damage caused by ischemia reperfusion results in accessibility of ARHGDIB in endothelial cells to circulating auto-antibodies. After binding, anti-ARHGDIB antibodies may initiate complement activation causing local inflammation, which may explain why a relation between their presence and graft loss was observed only after deceased-donor transplantation. In our study, non-HLA proteins were coated directly or indirectly (via HaloTag) to the microspheres. This strategy was chosen because the coupling process may influence the conformation and accessibility of epitopes predominantly recognized on non-HLA beads.11Kamburova EG Kardol-hoefnagel T Wisse BW Joosten I Wil A Development and validation of a multiplex non-HLA antibody assay for the screening of kidney transplant recipients.Front Immunol. 2018; 9: 3002Crossref PubMed Scopus (23) Google Scholar Previously we found that the correlation between results obtained with our test and with commercial assays depended on the coupling method of the proteins. This demonstrates the necessity of complete transparency and detailed description of methods when comparing the effects of non-HLA antibodies in patient groups. Because there are no commercially available reference sera against the non-HLA antibodies we selected, we were not able to properly compare our Luminex assay to other commercially available ELISA or Luminex autoantibody assays. In the sera used in our multiplex assay, we measured non-HLA IgG antibodies. It is possible that some non-HLA antibodies relevant to prognosis are of another isotype and were not detected here. In some autoimmune diseases, autoantibodies are of other isotypes, such as IgM-RF or IgA-anti-tTG. However, most clinically relevant autoantibodies are of the IgG isotype. We also examined whether a reactivity pattern between non-HLA antibodies can be distinguished, for instance due to cross-reactivity, but we did not find significant associations between each of the 14 non-HLA antibodies investigated and there was no relationship with pretransplant DSA. In a large cohort studied by Opelz, a 9% difference in 10-year graft survival was observed between recipients of HLA-identical sibling donors with or without panel-reactive HLA antibodies, which led to the conclusion that a high immunization grade against HLA may indicate an increased immunity against non-HLA.33Opelz G Non-HLA transplantation immunity revealed by lymphocytotoxic antibodies.Lancet. 2005; 365: 1570-1576Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar In our study, the observed adverse effects of non-HLA antibodies were independent of the presence of DSA as we adjusted for this covariable. Because ARHGDIB is considered to be a minor histocompatibility antigen, we examined the relation between antibody levels and potentially immunizing events, but found no link with repeat transplantation, female sex, pregnancies, or potentially confounding factors such as diabetes type 1, or several primary renal diseases. Therefore, mechanisms may be involved in the induction of non-HLA antibody formation other than the well-known sensitizing events stimulating HLA antibody production. We were not able to include posttransplant samples (sera and/or biopsies) in our study, thereby limiting the use of results in pretransplant risk stratification. Future studies have to be performed to evaluate whether posttransplant monitoring of anti-ARHGDIB is useful. Another limitation of the study was that we did not have detailed clinical information, such as autoimmune diseases or hypertension, to potentially link the presence of anti-ARHGDIB antibodies to a clinical parameter. Due to lack of detailed rejection and histology information, we cannot directly link anti-ARHGDIB antibodies with the rejection phenotype. In conclusion, our study demonstrates that pretransplant non-HLA antibodies against ARHGDIB are a significant risk factor in deceased-donor transplantation. These antibodies occur independently from DSA or other non-HLA antibodies investigated. It is currently unknown whether the presence of these antibodies is a biomarker, as is the case in many autoimmune diseases, or whether they play a role in the pathogenesis of graft loss. Although validation of our findings in independent cohorts is necessary, based on these results, we suggest that pretransplant risk assessment can be improved by measuring these antibodies in all patients awaiting deceased-donor transplantation. The authors thank Dr. Tri Nguyen (Department of Pathology, University Medical Center Utrecht, The Netherlands) for his assistance in performing and interpreting the anti-ARGHDIB staining on kidney tissue. This study was supported by research funding from the Dutch Kidney Foundation Project code CP12.23 Risk assessment of kidney graft failure by HLA antibody profiling. The authors of this manuscript have no conflicts of interests to disclose as described by the American Journal of Transplantation. Data available on request due to privacy/ethical restrictions. Additional supporting information may be found online in the Supporting Information section at the end of the article. Download .pdf (.91 MB) Help with pdf files" @default.
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• W2953025960 date "2019-12-01" @default.
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• W2953025960 title "Antibodies against ARHGDIB are associated with long-term kidney graft loss" @default.
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