Polymorphisms in the lectin pathway of complement activation influence the incidence of acute rejection and graft outcome after kidney transplantation
2016; Elsevier BV; Volume: 89; Issue: 4 Linguagem: Inglês
10.1016/j.kint.2015.11.025
ISSN1523-1755
AutoresDéla Golshayan, Agnieszka Wójtowicz, Stéphanie Bibert, Nitisha Pyndiah, Oriol Manuel, Isabelle Binet, Léo H. Bühler, Uyen Huynh‐Do, Thomas Mueller, Jürg Steiger, Manuel Pascual, Pascal Meylan, Pierre‐Yves Bochud, Rita Achermann, John‐David Aubert, Philippe Baumann, Guido Beldi, Christian Benden, Christoph Berger, Isabelle Binet, Pierre‐Yves Bochud, Elsa Boely, Heiner C. Bucher, Léo H. Bühler, Thierry Carell, Oriol Manuel, Yves Chalandon, Sabina De Geest, Olivier de Rougemont, Michael Dickenmann, Michel Duchosal, Thomas Fehr, Sylvie Ferrari-Lacraz, Christian Garzoni, Yvan Gasche, Paola Gasche Soccal, Emiliano Giostra, Déla Golshayan, Daniel Good, Karine Hadaya, Christoph Hess, Sven Hillinger, Hans H. Hirsch, Günther F.L. Hofbauer, Uyen Huynh‐Do, Franz Immer, Richard Klaghofer, Michael Koller, Thomas Kuntzen, Bettina Laesser, Roger Lehmann, Christian Lovis, Oriol Manuel, Hans‐Peter Marti, Pierre Yves Martin, Pascal Meylan, Paul Mohaçsi, Isabelle Morard, Philippe Morel, Ulrike Mueller, Nicolas J. Mueller, Helen Mueller-McKenna, Thomas Müller, Beat Müllhaupt, David Nadal, Gayathri Nair, Manuel Pascual, Jakob Passweg, Chantal Piot Ziegler, Juliane Rick, Eddy Roosnek, Anne Rosselet, Silvia Rothlin, Frank Ruschitzka, Urs Schanz, Stefan Schaub, Christian Seiler, Nasser Semmo, Susanne Stampf, Jürg Steiger, Christian Toso, Dimitri Tsinalis, Christian van Delden, Jean-Pierre Venetz, Jean Villard, Madeleine Wick, Markus J. Wilhelm, Patrick Yerly,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoThere are conflicting data on the role of the lectin pathway of complement activation and its recognition molecules in acute rejection and outcome after transplantation. To help resolve this we analyzed polymorphisms and serum levels of lectin pathway components in 710 consecutive kidney transplant recipients enrolled in the nationwide Swiss Transplant Cohort Study, together with all biopsy-proven rejection episodes and 1-year graft and patient survival. Functional mannose-binding lectin (MBL) levels were determined in serum samples, and previously described MBL2, ficolin 2, and MBL-associated serine protease 2 polymorphisms were genotyped. Low MBL serum levels and deficient MBL2 diplotypes were associated with a higher incidence of acute cellular rejection during the first year, in particular in recipients of deceased-donor kidneys. This association remained significant (hazard ratio 1.75, 95% confidence interval 1.18–2.60) in a Cox regression model after adjustment for relevant covariates. In contrast, there was no significant association with rates of antibody-mediated rejection, patient death, early graft dysfunction or loss. Thus, results in a prospective multicenter contemporary cohort suggest that MBL2 polymorphisms result in low MBL serum levels and are associated with acute cellular rejection after kidney transplantation. Since MBL deficiency is a relatively frequent trait in the normal population, our findings may lead to individual risk stratification and customized immunosuppression. There are conflicting data on the role of the lectin pathway of complement activation and its recognition molecules in acute rejection and outcome after transplantation. To help resolve this we analyzed polymorphisms and serum levels of lectin pathway components in 710 consecutive kidney transplant recipients enrolled in the nationwide Swiss Transplant Cohort Study, together with all biopsy-proven rejection episodes and 1-year graft and patient survival. Functional mannose-binding lectin (MBL) levels were determined in serum samples, and previously described MBL2, ficolin 2, and MBL-associated serine protease 2 polymorphisms were genotyped. Low MBL serum levels and deficient MBL2 diplotypes were associated with a higher incidence of acute cellular rejection during the first year, in particular in recipients of deceased-donor kidneys. This association remained significant (hazard ratio 1.75, 95% confidence interval 1.18–2.60) in a Cox regression model after adjustment for relevant covariates. In contrast, there was no significant association with rates of antibody-mediated rejection, patient death, early graft dysfunction or loss. Thus, results in a prospective multicenter contemporary cohort suggest that MBL2 polymorphisms result in low MBL serum levels and are associated with acute cellular rejection after kidney transplantation. Since MBL deficiency is a relatively frequent trait in the normal population, our findings may lead to individual risk stratification and customized immunosuppression. The innate immune system is primarily involved in natural immunity against pathogens and tissue injury. In the setting of solid-organ transplantation (SOT), experimental data have highlighted an important role of innate immune activation early after transplantation, in particular following donor brain death and during ischemia–reperfusion injury in the early stages after surgery.1Lin T. Zhou W. Sacks S.H. The role of complement and Toll-like receptors in organ transplantation.Transpl Int. 2007; 20: 481-489Crossref PubMed Scopus (19) Google Scholar, 2Goldstein D.R. Tesar B.M. Akira S. et al.Critical role of the Toll-like receptor signal adaptor protein MyD88 in acute allograft rejection.J Clin Invest. 2003; 111: 1571-1578Crossref PubMed Scopus (305) Google Scholar, 3Baldwin 3rd, W.M. Larsen C.P. Fairchild R.L. Innate immune responses to transplants: a significant variable with cadaver donors.Immunity. 2001; 14: 369-376Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Macrophages and dendritic cells act as immune sentinels in peripheral tissues and secondary lymphoid organs, regularly sampling and processing self-antigens to maintain immune homeostasis in steady-state conditions. These innate immune cells express pattern recognition receptors that specifically detect conserved pathogen-associated or danger-associated molecular patterns, in the context of local infection or sterile tissue damage, respectively. The activation of surface pattern recognition receptors results in the maturation of the cells into competent antigen-presenting cells that produce proinflammatory mediators and can initiate an adaptive immune response.4Palm N.W. Medzhitov R. Pattern recognition receptors and control of adaptive immunity.Immunol Rev. 2009; 227: 221-233Crossref PubMed Scopus (538) Google Scholar, 5Gallucci S. Lolkema M. Matzinger P. Natural adjuvants: endogenous activators of dendritic cells.Nat Med. 1999; 5: 1249-1255Crossref PubMed Scopus (1391) Google Scholar, 6Mellman I. Steinman R.M. Dendritic cells: specialized and regulated antigen processing machines.Cell. 2001; 106: 255-258Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar Besides surface-bound receptors, soluble pattern recognition receptors have been characterized, including complement-activating mannose-binding lectin (MBL) and ficolins (FCNs).7Takahashi K. Ip W.E. Michelow I.C. et al.The mannose-binding lectin: a prototypic pattern recognition molecule.Curr Opin Immunol. 2006; 18: 16-23Crossref PubMed Scopus (148) Google Scholar Upon ligation, the downstream MBL-associated serine protease (MASP) signaling activates the complement cascade. MBL and FCN are mainly synthesized in the liver and function as oligomeric molecules assembled with MASP. In circulation, these oligomeric structures bind to surface carbohydrates of microbes and injured cells, leading to C3b-mediated opsonization and phagocytosis. Single-nucleotide polymorphisms (SNPs) in the genes encoding the lectin pathway proteins determine their functional activity and serum levels.8Adamek M. Heyder J. Heinold A. et al.Characterization of mannose-binding lectin (MBL) variants by allele-specific sequencing of MBL2 and determination of serum MBL protein levels.Tissue Antigens. 2013; 82: 410-415Crossref PubMed Scopus (10) Google Scholar Due to prevalent genetic polymorphisms in both regulatory and coding sequences of MBL2, up to 25% of the normal population have reduced serum concentrations of MBL and can be classified as functionally deficient.9Verdu P. Barreiro L.B. Patin E. et al.Evolutionary insights into the high worldwide prevalence of MBL2 deficiency alleles.Hum Mol Genet. 2006; 15: 2650-2658Crossref PubMed Scopus (102) Google Scholar However, MBL functions may be redundant in healthy individuals, as low levels of functional MBL have been associated with susceptibility to infections mainly in immunocompromised individuals.10Koch A. Melbye M. 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Doherty K. et al.Mannose-binding lectin gene polymorphisms are associated with major infection following allogeneic hemopoietic stem cell transplantation.Blood. 2002; 99: 3524-3529Crossref PubMed Scopus (185) Google Scholar As opposed to infectious settings, MBL deficiency has been described to be protective in some glomerular diseases such as IgA nephropathy,14Endo M. Ohi H. Ohsawa I. et al.Glomerular deposition of mannose-binding lectin (MBL) indicates a novel mechanism of complement activation in IgA nephropathy.Nephrol Dial Transplant. 1998; 13: 1984-1990Crossref PubMed Scopus (166) Google Scholar, 15Roos A. Rastaldi M.P. Calvaresi N. et al.Glomerular activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease.J Am Soc Nephrol. 2006; 17: 1724-1734Crossref PubMed Scopus (311) Google Scholar as well as in studies of kidney and heart transplantation where patients with low functional MBL serum levels had a better graft survival.16Berger S.P. Roos A. Mallat M.J. et al.Association between mannose-binding lectin levels and graft survival in kidney transplantation.Am J Transplant. 2005; 5: 1361-1366Crossref PubMed Scopus (97) Google Scholar, 17Berger S.P. Roos A. Mallat M.J. et al.Low pretransplantation mannose-binding lectin levels predict superior patient and graft survival after simultaneous pancreas-kidney transplantation.J Am Soc Nephrol. 2007; 18: 2416-2422Crossref PubMed Scopus (64) Google Scholar, 18Imai N. Nishi S. Alchi B. et al.Immunohistochemical evidence of activated lectin pathway in kidney allografts with peritubular capillary C4d deposition.Nephrol Dial Transplant. 2006; 21: 2589-2595Crossref PubMed Scopus (49) Google Scholar, 19Fildes J.E. Shaw S.M. Walker A.H. et al.Mannose-binding lectin deficiency offers protection from acute graft rejection after heart transplantation.J Heart Lung Transplant. 2008; 27: 1353-1356Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Moreover, complement deficiency or blockade was shown to limit postischemic reperfusion injury in various organs, and protect against acute renal transplant rejection.20Pratt J.R. Basheer S.A. Sacks S.H. Local synthesis of complement component C3 regulates acute renal transplant rejection.Nat Med. 2002; 8: 582-587Crossref PubMed Scopus (426) Google Scholar, 21Pratt J.R. Jones M.E. Dong J. et al.Nontransgenic hyperexpression of a complement regulator in donor kidney modulates transplant ischemia/reperfusion damage, acute rejection, and chronic nephropathy.Am J Pathol. 2003; 163: 1457-1465Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 22Vieyra M.B. Heeger P.S. Novel aspects of complement in kidney injury.Kidney Int. 2010; 77: 495-499Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 23Schwaeble W.J. Lynch N.J. Clark J.E. et al.Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury.Proc Natl Acad Sci U S A. 2011; 108: 7523-7528Crossref PubMed Scopus (145) Google Scholar, 24Asgari E. Farrar C.A. Lynch N. et al.Mannan-binding lectin-associated serine protease 2 is critical for the development of renal ischemia reperfusion injury and mediates tissue injury in the absence of complement C4.FASEB J. 2014; 28: 3996-4003Crossref PubMed Scopus (67) Google Scholar Conflicting data exist on the deleterious versus protective role of the lectin pathway of complement activation in graft rejection, as well as in patient and graft outcomes after SOT.25Fiane A.E. Ueland T. Simonsen S. et al.Low mannose-binding lectin and increased complement activation correlate to allograft vasculopathy, ischaemia, and rejection after human heart transplantation.Eur Heart J. 2005; 26: 1660-1665Crossref PubMed Scopus (36) Google Scholar, 26Bay J.T. Sorensen S.S. Hansen J.M. et al.Low mannose-binding lectin serum levels are associated with reduced kidney graft survival.Kidney Int. 2013; 83: 264-271Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 27Damman J. Seelen M.A. Mannan binding lectin: a two-faced regulator of renal allograft injury?.Kidney Int. 2013; 83: 191-193Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 28Bay J.T. Hein E. Sorensen S.S. et al.Pre-transplant levels of ficolin-3 are associated with kidney graft survival.Clin Immunol. 2013; 146: 240-247Crossref PubMed Scopus (13) Google Scholar Many of the available studies in kidney transplantation are either retrospective, involve small numbers of patients, or have analyzed outcomes under previous eras of immunosuppression and anti-infectious therapies. Therefore, the aim of the present study was to investigate the role of the lectin pathway of complement activation and the possible association between recipient genotype and graft outcomes after kidney transplantation in a large, well-characterized, ongoing prospective cohort. The primary end point was the incidence of biopsy-proven acute rejection episodes at 1 year after transplantation; secondary end points were death-censored graft loss, graft function at 1 year, and patient survival. Donor, recipient, and transplant baseline characteristics are described in Table 1. Among the 710 consecutive Caucasian kidney transplant recipients included in the Swiss Transplant Cohort Study (STCS), 672 (94.6%) underwent a kidney-alone transplantation and 38 also received another organ, mainly pancreas (27/38). One hundred twenty-four (17.5%) patients had a previous transplant, of whom 111 (15.6%) were undergoing kidney transplantation for a second or more time. Deceased donation provided the majority (59.4%) of the organs, with a median deceased donor age of 53 years and median cold ischemia time of 6.5 hours.Table 1Characteristics of the study population at the time of transplantationKidney transplant recipientsN = 710N(% or IQR)Donor age (median years; IQR)5321.0Donor genderaData were missing for 3 patients. M/F345/36248.8/51.2Recipient age (median years; IQR)5420.0Recipient gender M/F473/23766.7/33.4Type of donor Living28840.6 Deceased42259.4Cold ischemia timebConsidered for deceased donors only. (median hours; IQR)6.59.2Multiorgan transplantation385.3HLA mismatch (>3)cData were missing for 158 patients.29252.9 HLA-A mismatchesdData were missing for 6 patients. (0/1/2)99/326/27914.1/46.3/39.6 HLA-B mismatcheseData were missing for 6 patients. (0/1/2)56/297/3517.9/41.8/49.9 HLA-DR mismatchesfData were missing for 5 patients. (0/1/2)81/358/26611.5/50.8/37.7Donor/recipient CMV serostatusgData were missing for 10 patients. D–R–17224.6 D+R–13919.9 D–R+15622.3 D+R+23333.3Induction therapyhData were missing for 43 patients. None13119.6 Basiliximab only41462.1 Anti-thymocyte globulin12218.3Initial maintenance immunosuppression Steroids63589.4 Tacrolimus/cyclosporine396/14356.6/20.1 MPA agents/AZA614/286.5/0.3 mTOR inhibitors20.3MBL2 leveliData were missing for 1 patient. (median; IQR)678.21254.9AZA, azathioprine; CMV, cytomegalovirus; D, donor; F, female; HLA, human leukocyte antigen; IQR, interquartile range; M, male; MBL, mannose-binding lectin; MPA, mycophenolic acid; mTOR, mammalian target of rapamycin; R, recipient.a Data were missing for 3 patients.b Considered for deceased donors only.c Data were missing for 158 patients.d Data were missing for 6 patients.e Data were missing for 6 patients.f Data were missing for 5 patients.g Data were missing for 10 patients.h Data were missing for 43 patients.i Data were missing for 1 patient. Open table in a new tab AZA, azathioprine; CMV, cytomegalovirus; D, donor; F, female; HLA, human leukocyte antigen; IQR, interquartile range; M, male; MBL, mannose-binding lectin; MPA, mycophenolic acid; mTOR, mammalian target of rapamycin; R, recipient. Patients were given induction and initial maintenance immunosuppression (prescribed within the first week after transplantation) based on international and local guidelines, according to their immunologic risk status. Basiliximab was used in 62.1% of patients, 18.3% received anti-thymocyte globulin, and in 19.6% no induction was prescribed. Steroids and mycophenolic acid–based agents were used in >86% of the patients. Calcineurin inhibitors were started early in 76.7% of the recipients, with a large preference for tacrolimus (FK) as compared to cyclosporine. The overall median MBL2 serum level measured on the day of transplantation was 678.2 ng/ml (Figure 1). Patients were classified as having "high" (> median) and "low" (≤ median) MBL levels. The latter group was further stratified into "intermediate" (≥10 ng/ml) or "deficient" (<10 ng/ml) MBL levels. As shown in Figure 1, MBL serum levels were largely dependent on the MBL2 genotype in our study population. The YAYA and YAXA diplotypes resulted in the higher serum MBL levels and were the main genotypes found in our population (total 401 patients, 56.6%) as compared to the remaining XAXA, YAYO, and XAYO (total 266 patients, 37.6%) and YOYO (total 41 patients, 5.8%) diplotypes, which were associated with intermediate and deficient MBL serum levels, respectively. These data obtained from pretransplant end-stage renal disease patients were in accordance with previous reports in the general population, describing the correlation between the selected SNPs and functional MBL serum levels.8Adamek M. Heyder J. Heinold A. et al.Characterization of mannose-binding lectin (MBL) variants by allele-specific sequencing of MBL2 and determination of serum MBL protein levels.Tissue Antigens. 2013; 82: 410-415Crossref PubMed Scopus (10) Google Scholar The 12-month cumulative incidence of biopsy-proven acute cellular rejection (ACR) and antibody-mediated rejection (AMR) was 0.20 and 0.05, respectively. A total of 144 ACR episodes were recorded during the first year, of which 122 had occurred during the first 6 months after transplantation. Among the cohort of patients (n = 473) who reached the 3-year follow-up, the incidence of ACR declined over time, with only 16 new ACR episodes reported after the first year. Within the ACR episodes during the first year, 7 were histologically classified as borderline tubulitis. During the first year after transplantation, 32 AMR episodes occurred in our study population. Overall, little modification was made in maintenance immunosuppression during the first year as compared to initial therapy. The majority of patients were still on a combination therapy of calcineurin inhibitors (97%, including 76% under FK) and mycophenolic acid–based agents (87%), and 73% received steroids at the 12-month follow-up. The same trend was observed at subsequent yearly follow-up analysis, with, interestingly, still 58% and 50% of patients maintained on steroid treatment at 2 and 3 years after transplantation, respectively. Low MBL serum levels (≤ median) were significantly associated with the risk of developing ACR during the first year after transplantation (log-rank test = 0.01, Figure 2a). The effect of MBL serum levels on ACR episodes was confirmed when analyzing the data based on MBL2 genotypes (Figure 2b). This was even more striking when we stratified into "high" versus "intermediate" and "deficient" MBL serum levels (Figure 2c) and corresponding "YAYA" or "YAXA" versus "XAYO," "YAYO," or "XAXA" and "YOYO" diplotypes (Figure 2d). In univariate analysis, the other factors negatively influencing the risk of ACR included donor age and donor–recipient human leukocyte antigen (HLA) mismatches (Table 2). Living donation and induction (predominantly anti-thymocyte globulin) as well as steroids and tacrolimus maintenance treatment tended to be protective factors. There was no significant association with donor or recipient gender, cold ischemia time (considered for deceased donors only), cytomegalovirus, or BK-related events. A multivariable analysis was performed to account for patient baseline characteristics in MBL serum levels and genotypes, and to correct for other factors including immunosuppressive treatments. This analysis confirmed that the low MBL status was an independent risk factor for ACR both in stepwise Cox model (hazard ratio [HR] 1.75, 95% confidence interval [CI] 1.18–2.60, P = 0.006; Table 2) and logistic regression model (odds ratio 1.84, 95% CI 1.14–2.98, P = 0.01; Supplementary Table S1 online). Besides low MBL levels, the other factor that remained associated with ACR in the multivariable Cox analysis was donor–recipient HLA mismatches (HR 1.79, 95% CI 1.20–2.66, P = 0.004), a risk factor also identified in the logistic regression analysis together with donor age (odds ratio 1.21, 95% CI 1.02–1.42, P = 0.02) and being on steroids (odds ratio 2.78, 95% CI 1.53–5.06, P < 0.001) (Supplementary Table S1). Data analysis excluding the subgroup of patients with borderline tubulitis yielded similar results (Supplementary Figure S1, Supplementary Tables S2 and S3). There was no association between either MBL serum levels or MBL2 diplotypes and the cumulative incidence of AMR during the first year after transplantation (data not shown).Table 2Risk factors for acute cellular rejection at 1 year after transplantationVariableUnivariate modelStratified multivariable modelfMultivariable analysis was performed by using stepwise regression model. The proportional hazards assumption was tested by using the stphtest command implemented in Stata. No deviation from this assumption was observed for MBLs. However, since 2 covariables in the multivariable model did not verify this hypothesis (basiliximab, P = 0.0001; steroids, P = 0.05), those were accounted for by using a stratified multivariable Cox model. Final global proportional hazards assumption test P value for multivariable Cox model was 0.84. In addition we have performed univariate and multivariable stepwise logistic regression analysis for ACR at 1 year after transplantation to account for those variables (Supplementary Table S1).HR (95% CI)PHR (95% CI)PDonor age (≥ median)1.26 (0.91–1.75)0.17Donor male gender1.15 (0.83–1.60)0.39Recipient age (≥ median)0.88 (0.63–1.22)0.43Recipient male gender1.10 (0.77–1.56)0.61Living donor0.70 (0.50–0.99)0.04Cold ischemia timeaConsidered for deceased donors only. (> median)1.14 (0.79–1.65)0.49HLA mismatch (>3)1.61 (1.09–2.37)0.021.79 (1.20–2.66)0.004Multiorgan transplantation0.87 (0.41–1.87)0.73Donor/recipient CMV serostatus D–R–reference D+R–0.89 (0.56–1.43)0.64 D–R+0.77 (0.48–1.23)0.28 D+R+0.72 (0.47–1.10)0.13CMV viremia/disease0.99 (0.59–1.67)0.98BK viremia/disease0.75 (0.34–1.64)0.47Induction therapy Nonereference Basiliximab only0.73 (0.49–1.08)0.12 Anti-thymocyte globulin0.44 (0.24–0.79)0.006Maintenance immunosuppressionbTime-dependent covariates. Steroids0.68 (0.40–1.15)0.15 Cyclosporine1.24 (0.87–1.79)0.24 Tacrolimus0.78 (0.55–1.11)0.17 MPA agents1.51 (0.74–3.09)0.26 mTOR inhibitors0.69 (0.25–1.86)0.45MBL2 level High (>678 ng/ml)reference Low (≤678 ng/ml)1.54 (1.10–2.14)0.011.75 (1.18–2.60)0.006MBL2 diplotypescMBL genotype O consists of rare allele D (R52C, rs5030737), B (G54D, rs1800450), and C (G57E, rs1800451), respectively, and the wild-type allele of each is referred to as genotype A. MBL genotype Y refers to presence of rare variant C of promoter SNP G221C (rs7096206) and genotype X as wild-type allele G of that polymorphism. YAYA or YAXAreference XAYO, YAYO, or XAXA1.25 (0.88–1.76)0.21 YOYO2.05 (1.16–3.64)0.01dP value after correction for multiple testing (N = 10) is 0.1.FCN2 genotypes rs7851696 TT or TG versus GG1.39 (0.97–2.00)0.07eP value after correction for multiple testing (N = 10) is 0.7. rs17514136 GG or GA versus AA1.22 (0.88–1.69)0.24 rs3124953 AA or AG versus GG1.01 (0.72–1.41)0.96 rs3124952 GG or GA versus AA0.96 (0.67–1.39)0.85MASP2 genotypes rs56392418 AA or AG versus GG0.95 (0.35–2.56)0.91 rs12085877 TT or TC versus CC1.14 (0.56–2.33)0.72 rs1033638 AA versus GA or GG1.03 (0.72–1.45)0.89CI, confidence interval; CMV, cytomegalovirus; D, donor; FCN, ficolin; HLA, human leukocyte antigen; HR, hazard ratio; MASP, mannose-binding lectin–associated serine protease; MBL, mannose-binding lectin; MPA, mycophenolic acid; mTOR; mammalian target of rapamycin; R, recipient.Bold P values are significant.a Considered for deceased donors only.b Time-dependent covariates.c MBL genotype O consists of rare allele D (R52C, rs5030737), B (G54D, rs1800450), and C (G57E, rs1800451), respectively, and the wild-type allele of each is referred to as genotype A. MBL genotype Y refers to presence of rare variant C of promoter SNP G221C (rs7096206) and genotype X as wild-type allele G of that polymorphism.d P value after correction for multiple testing (N = 10) is 0.1.e P value after correction for multiple testing (N = 10) is 0.7.f Multivariable analysis was performed by using stepwise regression model. The proportional hazards assumption was tested by using the stphtest command implemented in Stata. No deviation from this assumption was observed for MBLs. However, since 2 covariables in the multivariable model did not verify this hypothesis (basiliximab, P = 0.0001; steroids, P = 0.05), those were accounted for by using a stratified multivariable Cox model. Final global proportional hazards assumption test P value for multivariable Cox model was 0.84. In addition we have performed univariate and multivariable stepwise logistic regression analysis for ACR at 1 year after transplantation to account for those variables (Supplementary Table S1). Open table in a new tab CI, confidence interval; CMV, cytomegalovirus; D, donor; FCN, ficolin; HLA, human leukocyte antigen; HR, hazard ratio; MASP, mannose-binding lectin–associated serine protease; MBL, mannose-binding lectin; MPA, mycophenolic acid; mTOR; mammalian target of rapamycin; R, recipient. Bold P values are significant. Ficolins and MASP are other important components of the lectin pathway, and their serum concentrations were previously shown to correspond to polymorphisms in the respective FCN2 and MASP2 genes.29Garred P. Honore C. Ma Y.J. et al.MBL2, FCN1, FCN2 and FCN3-The genes behind the initiation of the lectin pathway of complement.Mol Immunol. 2009; 46: 2737-2744Crossref PubMed Scopus (124) Google Scholar We did not find any significant association between the investigated SNPs and ACR in our study population, except for a trend toward a deleterious effect of the FCN2 rs7851696 SNP (HR 1.39, 95% CI 0.97–2.00, P = 0.07). This, however, did not reach statistical significance in the multivariable analysis. From our starting cohort of 710 patients, 666 patients were followed for at least 1 year, 638 patients for up to 24 months after transplantation, and 473 patients reached the 3-year follow-up time. At 3 years, a total of 22 patients had lost their kidney graft (13 during the first year), and 42 died with a functioning graft (23 during the first year). Table 3 summarizes the risk factors for death at 1 year after transplantation. The MBL status at the time of transplantation did not have a significant impact on short-term patient survival, nor FCN2 and MASP2 genotypes. In the multivariable analysis, donor male gender was significantly associated with death (HR 3.76, 95% CI 1.24–11.4, P = 0.02) as well as recipient age (HR 2.22, 95% CI 1.42–3.47, P < 0.001), while immunosuppressive regimens with calcineurin inhibitors or mycophenolic acid–based agents were protective. We next analyzed death-censored graft loss after transplantation (Table 4) and found a strong association with rejection episodes during the first year (HR 6.21, 95% CI 2.71–14.2, P < 0.002), as well as with recipient age (HR 2.04, 95% CI 1.48–2.81, P < 0.001), and a trend toward association with the MASP2 rs12085877 SNP (HR 2.90, 95% CI 0.85–9.95, P = 0.09), but not with MBL2 or FCN2 polymorphisms. Overall, while the low MBL status was associated with ACR, there was no direct association with patient death or graft loss at 1 year in the uni- and multivariable analysis. Analysis of the cohort of patients that reached 3 years follow-up corroborated the results on patient and graft survival obtained at 1 year (data not shown).Table 3Predictors of patients' death at 1 year after transplantationVariableUnivariate modelStratified multivariable modeldMultivariable analysis was performed by using stepwise regression model. The proportional hazards assumption was tested by using the stphtest command implemented in Stata. No deviation from this assumption was observed for MBLs. However, since 2 covariables in the multivariable model did not verify this hypothesis (rejection episodes, P = 0.003; steroids, P = 0.04), those were accounted for by using a stratified multivariable Cox model. Final global proportional hazards assumption test P value for multivariable Cox model was 0.56.HR (95% CI)PHR (95% CI)PDonor age (per 10-year increase)1.25 (0.94–1.67)0.12Donor male gender1.62 (0.70–3.74)0.263.76 (1.24–11.4)0.02Recipient age (per 10-year increase)1.71 (1.18–2.50)0.0052.22 (1.42–3.47) median)1.56 (0.58–4.21)0.38HLA mismatch (>3)0.54 (0.20–1.50)0.24Multiorgan transplantation0.77 (0.10–5.72)0.80Rejection episodes (regrouping ACR and AMR)3.37 (1.46–7.75)0.004Donor/recipient CMV serostatus D–R–reference D+R–1.53 (0.47–5.01)0.48 D–R+0.91 (0.24–3.39)0.89 D+R+1.23 (0.40–4.91)0.59CMV viremia/disease1.92 (0.78–4.70)0.16BK viremia/disease1.41 (0.40–4.91)0.59Induction therapy Nonereference Basiliximab only0.69 (0.24–1.99)0.50 Anti-thymocyte globulin1.53 (0.48–4.81)0.47Maintenance immunosuppressionbTime-dependent covariates. Steroids0.07 (0.03–0.17)<0.001 Cyclosporine0.33 (0.08–1.40)0.130.09 (0.02–0.50)0.006 Tacrolimus0.12 (0.05–0.31)<0.0010.08 (0.02–0.29)<0.001 MPA agents0.03 (0.01–0.08)<0.0010.09 (0.02–0.28) 678 ng/ml)reference Low (≤678 ng/ml)0.90 (0.40–2.04)0.80MBL2 diplotypescMBL genotype O consists of rare allele D (R52C, rs5030737), B (G54D, rs1800450), and C (G57E, rs1800451), respectively, and the wild-type allele of each is referred to
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