Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection

TRIM-01035; No of Pages 7 Transplant Immunology xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Transplant Immunology journal homepage: www.elsevier.com/locate/trim

Fas/Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection Fatina I. Fadel a, Manal F. Elshamaa b,⁎, Ahmed Salah a, Marwa Nabhan a, Maha Rasheed c, Solaf Kamel c, Dina Kandil c, Eman H. Thabet c a b c

Pediatric Department, Faculty of Medicine, Cairo University, Cairo, Egypt Pediatric Department, National Research Centre, Cairo, Egypt Clinical & Chemical Pathology Department, National Research Centre, Cairo, Egypt

a r t i c l e

i n f o

Article history: Received 20 February 2016 Received in revised form 28 March 2016 Accepted 20 April 2016 Available online xxxx Keywords: Cellular rejection Fas Ligand Genetic polymorphism Pediatric transplantation

a b s t r a c t Background: An essential milestone in pediatric transplantation is to find noninvasive biomarkers to monitor acute rejection (AR). In this retrospective (Case-control) study, we examined the role of Fas −670A/G and Fas Ligand (FasL) −843C/T gene polymorphisms in allograft nephropathy in pediatric renal transplant recipients. Methods: In 47 pediatric kidney transplant recipients and 20 healthy controls, Fas −670A/G and FasL −843C/T gene polymorphisms as well as serum soluble Fas Ligand level (sFasL) were measured. Results: Serum sFasL levels were significantly higher in transplant recipients children than that in controls (548.25 ± 298.64 pg/ml vs 143.17 ± 44.55 pg/ml, p = 0.0001). There was no significant difference between patients with AR and those without AR in regards to serum sFasL levels (567.70 ± 279.87 pg/ml vs 507.85 ± 342.80 pg/ml, p = 0.56). Fas − 670A/G genotypes or alleles were not significantly different between controls and transplant recipients and among transplant recipients with and without AR. (P N 0.05 for all). FasL −843C/T genotypes were not different between transplant recipients and controls and among transplant recipients with and without AR (P N 0.05 for all). However, Frequency of C allele in transplant patients was significantly higher than that in the control group (44.68% vs 25%, P = 0.03). FasL − 843C/T alleles were significantly different between patients with and without AR (P = 0.03). The percentages of C allele were higher in children with AR (58.82% vs 36.67%). We found that serum FasL and serum creatinine were variables that were independently associated with AR. Conclusion: This study suggests that FasL gene polymorphisms in peripheral blood might be accurate in detecting cellular AR. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Renal transplantation is the treatment of choice for pediatric patients with end-stage l, disease, for the improvement in quality of life when compared to dialysis and the better cardiovascular and mortality outcomes [1,2]. Allograft rejection depends mainly on human leukocyte antigen (HLA) polymorphism between donor and recipient and subsequent T cell recognition, either by direct recognition of allogeneic HLA on donor antigen-presenting cells (APC) or by indirect recognition of donor HLA-derived peptides presented by major histocompatibility complex class II antigens on recipient APCs [3].It is a complex multistage process involving T cells and immune components that are encoded by polymorphic genes [1].

⁎ Corresponding author at: Elbohous street, Dokki, Cairo 12311, Egypt. E-mail addresses: [email protected] (F.I. Fadel), [email protected] (M.F. Elshamaa), [email protected] (A. Salah), [email protected] (M. Nabhan), [email protected] (M. Rasheed), [email protected] (S. Kamel), [email protected] (D. Kandil).

Acute rejection (AR) is expected for the first week after transplantation on and is a cellular rejection involving T cell activation that involves morphologically a mononuclear cell predominant cellular inflammation [4,5]. Although incidence of acute rejection was having a decreasing over the last decade, it is still a main problem in kidney transplantation. Delayed graft function (DGF), which often happens due to deceaseddonor kidney transplantation, may last a few days or weeks. It is accompanied with an increased incidence of AR and produces injurious effects on patient and graft survival [5]. Defining the cooperation between molecular pathways within highly complex biological systems, such as those between immune cell networks and target tissues is certainly a hard task. Recently, there are many investigations in the pathogenesis of renal graft rejection, one of the most evident investigation is the protein molecules that participate in various stages of apoptosis. Fas/Fas Ligand (FasL) interaction seems to play a key role in AR and chronic allograft nephropathy (CAN) [6]. Apo1/Fas, (CD95 and TNFRSF6), is a trans-membrane protein and its major function might be the induction of apoptosis in cells expressing it after ligation by its legend. Its natural ligand, FasL (TNFSF6, CD95L) is a type

http://dx.doi.org/10.1016/j.trim.2016.04.006 0966-3274/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: F.I. Fadel, et al., Fas/Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection, Transpl Immunol (2016), http://dx.doi.org/10.1016/j.trim.2016.04.006

2

F.I. Fadel et al. / Transplant Immunology xxx (2016) xxx–xxx

II membrane protein belonging to the TNF family [7,8]. Fas is mainly expressed on tubular cells and may be up-regulated in AR or with injury such as cold ischemia [9,10], while FasL is expressed by graft infiltrating T cells and it has been reported to be a specific graft rejection marker [11]. Tubulitis and death of graft tubular epithelial cells are major components of acute rejection and both Fas and FasL polymorphisms are seemed to play an evident role in tubulitis [3]. However, few previous studies have evaluated the role of the polymorphisms in both of these genes together. Therefore, our study tries to evaluate the role of the polymorphisms of Fas and FasL together in AR in children with renal transplantation. The determination of polymorphisms on the 5′ flanking region on the human Fas gene has introduced useful markers for investigation of graft rejection and survival [12]. The polymorphism may have a potential role in gene regulation as is located on a consensus sequence of the gamma-activated sequence (GAS) [13]. Two single nucleotide polymorphisms located in the promoter region of the. Fas gene had been evaluated by Huang et al. [12]. One of these, a G to A nucleotide substitution at position −671 [Fas −670A/G]), is presented in a putative nuclear transcription element GAS binding site and could affect the expression of the FasL (T or C at position − 843 [FasL −843C/T]) gene [12]. The purpose of the current retrospective (Case-control) study was to investigate distribution of Fas gene −670A/G and FasL gene −843C/T polymorphisms in pediatric renal transplant patients and to investigate the role of these polymorphisms on allograft nephropathy. 2. Methods 2.1. Subjects and study design Forty-seven consecutive children who had received an allograft at Center of Pediatric Nephrology and Transplantation (CPNT), Children's Hospital, Cairo University, Egypt, were evaluated in this study as well as 20 healthy age-matched, unrelated population controls (12 males, mean age 8.7 ± 4.51 years). Healthy children were recruited from the Pediatric Clinic of the National Research Centre (NRC). The study was done from March 2013 to December 2013. The time elapsed from the time of transplantation to the point of the study is 2.39 ± 0.97 years (range 0.5–4.5 years). All patients had received their first transplantation. Age, gender, etiology of end -stage renal disease (ESRD), treatment modality and duration, as well as the donor type, were recorded. Patients were followed at regular intervals in the Nephrology Clinics. Creatinine was measured at least monthly post-transplant. No protocol biopsies were performed, particularly as the patients were pediatric patients where invasive biopsy accrues more cost and risk than in the adult population. Renal biopsies were only performed if there were clinical indications with suspicion for allograft dysfunction. Acute rejection which is cellular rejection due to T cell activation encountered in the first week after post-transplant was defined and graded according to the Banff Criteria [14,15]. It was defined as either borderline/suspicious or acute rejection in patients with stable serum creatinine values at the time of biopsy [16].All of the patients gave informed written consent before participating in the study, which was read and approved by the Ethics Committee of NRC in Egypt. In kidney transplantation, the blood sample was withdrawn after transplantation. In patients having episodes of acute rejection, the samples were taken during the period of rejection and the genotypes and soluble Fas-Ligand assay were performed. Genotype distribution and allele frequencies of Fas/FasL genes were compared among renal transplant recipient and normal healthy children. All patients who had had at least one episode of acute rejection within the first 6 months after transplantation were

classified as having acute rejection [3]. Allele and genotype frequencies of renal transplant patients who had acute rejection were compared with those who did not have acute rejection. Estimated glomerular filtration rate (eGFR) was calculated according to Creatinine-based “Bedside Schwartz” equation [17].An absolute number is used. 2.2. Immunosuppressive (IS) regimens All children received intravenous methylprednisolone (5–10 mg/kg, 150–250 mg/m2 up to 250 mg/dose) on the night before the operation, all the time of induction of anesthesia, all the time of declamping, 6 h post-operative and once on the day following the operation, (20 mg/ m2 mg per day) on the first month of transplantation, and then oral prednisolone was tapered down to 2.5–7.5 mg/day on the first year of transplantation. Immunosuppressive treatment protocol included FK regimen (n = 24) (Prednisolone + FK506 + MMF), cyclosporine (CsA) regimen (n = 18) (Prednisolone + CsA + MMF) and FK/mTORi regimen (n = 2) (Prednisolone + CsA + sirolimus/everolimus). The Initial CsA dose was 10 mg/kg per day by oral route (100– 400 mg/day), and target trough levels were ranged from 66 to154 ng/ml in the CsA based immunosuppression. The initial FK506 dose was 0.16 mg/kg per day by oral route (1.5–6 mg/day), and target trough levels were 3–14 ng/ml in the first 3 months and 4.5 ng/ml in the FK506/everolimus group. The initial dose of MMF was 1200 mg/m2 (800–1800 mg/m2) in 2–3 doses, and the dose was modified based on adverse effects such as diarrhea or leucopenia. IL-2 receptor blocking antibody (anti-IL-2R Ab, Basiliximab) (Simulect, Novartis Pharmaceuticals, Basel, Switzerland), was given to 10 patients (BSX group) (CsA or FK506 based immunosuppression) on day 0 and 3 days after renal transplantation (two 20-mg doses). Anti-thymocyte globulin (ATG) (Thymoglobulin_Genzyme Transplant, Cambridge, MA) was given to 29 patients (THYMO induction) (CsA or FK506 based immunosuppression) from days 0 to 3 (1.5 mg/kg per day, each day),the first dose is given intraoperatively to be completed prior to declamping, Everolimus was administered 2 mg per day and sirolimus was loaded 6 mg per day and then adjusted dose of 2 mg/day was maintained with target trough level of 5–15 ng/ml. 2.3. Peripheral blood sampling A peripheral blood sample was obtained from transplant recipients and healthy controls. An immediate centrifugation was done for 10 min at 5000 rpm at 4 °C. The centrifuged serum was transferred into sterile tubes. All samples were stored at − 70 °C until assay. One ml of venous blood sample was collected in EDTA vials for the extraction of genomic DNA. T lymphocytes count was calculated by measuring CD4 percentage by flow cytometry (Fresh blood samples on EDTA (100 μl) with monoclonal antibodies were incubated 20′ in the room temperature in the dark. Samples were lysed with 0.5 ml lysing solution Optilyse C (Beckman Coulter, Brea, CA, USA) 10′ the room temperature in the dark. Lysing reaction was stopped with 1 ml Cell Wash (optimized PBS) (Becton Dickinson Bioscience, Benelux, Belgium), the pellet suspended in PBS and kept in dark between 2 and 8 °C. Samples were measured on a FC 500 flow cytometer (Beckman Coulter, Brea, CA, USA). Gating strategy: As described before [26,27] cells were gated by side scatter and CD4 expression) then multiplying the results by the absolute lymphocytes counts obtained by the differential pictures of blood samples. 2.4. Soluble Fas-Ligand (sFasL) assay Serum levels of sFasL semi-quantitative measurement were done by in vitro ELISA Kit (Ray Biotech, Inc., USA) in the laboratories of Clinical and Chemical Pathology Unit, of NRC in Egypt.

Please cite this article as: F.I. Fadel, et al., Fas/Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection, Transpl Immunol (2016), http://dx.doi.org/10.1016/j.trim.2016.04.006

F.I. Fadel et al. / Transplant Immunology xxx (2016) xxx–xxx

2.5. Genomic DNA preparation and quantitation Genomic DNA was extracted from EDTA–anticoagulated whole blood samples using the QIAamp DNA Mini isolation kit (QIAGEN, # 51304) following manufacturer's instructions and was stored at −20 °C until the analysis. 2.6. PCR–restriction fragment length polymorphism (PCR-RFLP) genotyping for the −670A/G polymorphism of Fas DNA concentration was determined by Nano Drop 2000c Spectrophotometer (Thermo Fisher) and diluted as 100 ng/μl. Genomic DNA was amplified using polymerase chain reaction (PCR), amplification was carried out on a Veriti thermal cycler Applied Biosystems (USA). in a 25 μl reaction mixture in 0.2 ml thin-wall PCR strip tubes (Axygen Scientific, Inc., CA) containing 200 ng genomic DNA, 12.5 μl master mix using HotStarTaq Plus DNA Polymerase (250 units) Catalog no. 203603. (PE Applied Biosystems), 5 pmol each forward and reverse primers. PCR was performed using: forward primer 5′-CTACCTAAG AGC TAT CTA CCG TTC-3′ and reverse primer 5′-GGC TGTCCA TGT TGT GGC TGC-3′. DNA was initially denatured at 95 °C for 10 min, followed by 35 amplification cycles at 95 °C for 45 s, 62 °C for 45 s, and 72 °C for 45 s, and a final extension at 72 °C for 7 min. Digest conditions: Amplified 331 bp PCR product (3 μl) was digested in a 10 μl final reaction volume using 1 μl of Reaction Buffer 2 and 5 units of MvaI restriction enzyme (New England Biolabs, Beverly, MA), at 37 °C overnight. After digestion allele G yielded three fragments of 99, 188 and 44 bp, whereas allele A yielded two fragments of 99 and 232 bp. digested PCR products were separated by 2.5% agarose gel electrophoresis and visualized after ethidium bromide staining by UV spectrophotometry (Biometra, Germany).

3

polymorphism frequency in the reference population and the expected frequency. Power commonly sets at 80%; however, at that level, a polymorphism would be missed 20% of the time. Data were evaluated between the experimental groups by independent t-test. The HardyWeinberg equilibrium (HWE) assumption was assessed for patients and control groups by comparing the observed numbers of different genotypes with those expected under HWE for the estimated allele frequency and comparing the Pearson goodness-of-fit statistic with a 2 distribution with 1 degree of freedom. A p value of b 0.05 was considered statistically significant.

3. Results 3.1. Subject characteristics The mean age of the 47 transplant children (29 males, 18 females) included in this study was 9.63 ± 3.33 years. Serum sFasL levels were significantly higher in transplant recipients children than that in controls (548.25 ± 298.64 pg/ml vs 143.17 ± 44.55 pg/ml, p = 0.0001) (Table 1). The most common etiologies of chronic renal failure were unknown (n = 8), posterior urethral valve (PUV) (n = 8), PUV with reflux nephropathy (n = 5) and chronic pyelonephritis (44.68% in total). Three (6.38%) patients had received peritoneal dialysis then hemodialysis while 42 (89.36%) had received hemodialysis and two (4.26%) patient had pre-emptive renal transplantation. The mean duration of dialysis was 21.7 ± 2.3 months. The study population included 36 (76.60%) living, related donor organ source (LRD) and 11 (23.40%) living, unrelated donor AR was detected in 17((29.8%), while patients while CAN was detected in 3 (6.38%) patients (Table 2).

2.7. PCR-RFLP genotyping for the −843C/T polymorphism of FasL gene

3.2. Distribution of Fas genotypes

Genomic DNA was amplified using polymerase chain reaction (PCR) carried out on a Veriti thermal cycler Applied Biosystems, the USA in a 25 μl reaction mixture as described above. The cycling conditions comprised initial denaturation at 95 °C for 10 min, followed by 35 amplification cycles at 95 °C for 30 s, 45 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min. An 114-bp fragment containing the promoter polymorphism C − 843 T was amplified using the following primers; sense-5′-CAA TGA AAA TGA ACA CAT TG-3′ and antisense 5′CCCACT TTA GAA ATT AGA TC-3′. Digest conditions: Amplified 114 bp PCR product (7 μl) was digested at 37 °C 3 h in a 20 μl final reaction volume using 2 μl of Reaction Buffer 2 and 5 units of DraIII restriction enzyme (New England Biolabs, Beverly, MA). After digestion allele T yielded two fragments of 98 and 16 bp, whereas allele C was not digested and yielded as 114 bp. Digested PCR samples were subjected to electrophoresis on gels containing a mixture of 1.5% agarose and quantified by UV spectrophotometry (Biometra, Germany).

Independent segregation of alleles for these studied polymorphisms was kept in HWE. Genetic association analyzes with Pearson Chi-square test was performed and data are summarized in Table 3. Comparison of Fas genotype in renal transplant recipients and the controls did not reveal a statistically significant difference. Frequencies of AA, AG and GG genotypes were 31.92, 12.77 and 55.31% of the transplant children respectively while those of the control group were 25, 0 and 75% in the same order (P = 0.16). Further, we have analyzed the data by pooling the AG, GG and AG + GG genotype with AA genotype, respectively, No significant differences were observed. There was no significant difference between patients with AR and those without in regards to serum sFasL levels (567.70 ± 279.87 pg/ml vs 507.85 ± 342.80 pg/ml, p = 0.56) (Table 4). The frequencies of Fas genotypes were also not significantly different between the patients who had AR and those who did not. Frequencies of AA, AG and GG genotypes were 23.53, 17.65 and 58.82% in the rejection group while those in the group without rejection were 36.67, 10 and 53.33% (P = 0.28). Further, we have analyzed the data by pooling the AG, GG and AG + GG genotype with AA genotype, respectively, No significant differences were observed. (Table 5).

2.8. Statistical analysis Statistical analyzes were performed by SPSS 16.0 computer program and Pearson's Chi Square test. Data were summarized as mean ± SD, range or percentage. Histograms and normality plots were used for evaluating the normality of data. For those data with the skewed distribution, log transformation was performed before a t-test. Power analysis was used to calculate the minimum sample size required to accept the outcome of a statistical test with a particular level of confidence. A sample size of 20 will give us approximately 80% power (alpha = 0.05, two-tail) to reject the null hypothesis of zero correlation. We used power calculations performed by the Power and Precision program (Biostat) to determine the number of chromosomes required to detect a significant difference between the

3.3. Frequencies of Fas alleles The frequency of A allele in transplant recipients was insignificantly higher than that in the control group (38.30% vs 25%, P = 0.138). The frequency of the G allele in the transplant children and controls was not significantly different (61.70% and 75%, respectively) (Table 2), although it is higher in the controls than A allele. Comparison of the Fas alleles among transplanted children with and without AR showed no significant difference (P = 0.372) (Table 5).

Please cite this article as: F.I. Fadel, et al., Fas/Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection, Transpl Immunol (2016), http://dx.doi.org/10.1016/j.trim.2016.04.006

4

F.I. Fadel et al. / Transplant Immunology xxx (2016) xxx–xxx

Table 1 Comparison between kidney transplant recipients and healthy controls as regards to common baseline and clinical characteristics. All kidney transplant recipients (n = 47)

Healthy controls (n = 20)

P-value

General characteristics Recipient gender (males/females) Mean recipient age at transplant (years)

29 (61.70%)/18 (38.30%) 9.63 ± 3.33

12 (60%)/8 (40%) 8.7 ± 4.51

0.55 0.64

Clinical characteristics Serum creatinine at the time of the study (_mol/L) (mean ± SD) e-GFR, ml/min/1.73 m2 T lymphocytes (cells/mm3) Serum sFasL (pg/ml)

0.80 ± 0.24 89.92 ± 35.71 176.52 ± 237.11 548.25 ± 298.64

0.77 ± 0.34 89 ± 8.8 725.33 ± 179.11 143.17 ± 44.55

0.35 0.56 0.04* 0.0001*

sFasL, soluble Fas Ligand, * P was significant if b0.05.

3.4. Distribution of FasL genotypes Independent segregation of alleles for these studied polymorphisms was kept in HWE. Genetic association analyzes with Pearson Chi-square test was performed and data are summarized in Table 3. FasL genotypes were not significantly different between transplant recipients and the control group. CC genotype was present in 44.68 and 25% of the transplantation and control groups, respectively. Frequencies of CT and TT genotypes were 0 and 55.32% in the transplant children while 0 and 75% respectively in the control group (P = 0.13). Further, we have analyzed the data by pooling the CT, TT and CT + TT genotype with CC genotype, respectively, No significant differences were observed. Difference in the frequencies of FasL genotypes was not statistically significant between patients with and without AR but CC genotype was more common in patients with AR (58.82% vs 36.67%) while TT genotype was insignificantly higher in patients without acute rejection (63.33% vs 41.18%) (P = 0.14) Further, we have analyzed the data by pooling the CT, TT and CT + TT genotypes with CC genotype, respectively, No significant differences were observed. (Table 5).

3.5. Frequencies of FasL alleles The frequency of C allele in transplant recipients was significantly higher than that in the control group (44.68% vs 25%, P = 0.032) (Table 3).Also, FasL allele frequencies were significantly different between the patients with AR and the ones without (P = 0.038). The frequency of C allele in children with AR was 58.82% while that in the ones without was 36.67%. Frequencies of the T allele in children with and without AR were 41.18% and 63.33%, respectively (Table 5).

Table 2 Some especial baseline and clinical characteristics of kidney transplant recipients. Parameters

Mean ± SD

Donor age (year, mean ± SD) Donor organ source Deceased Living, related Living, unrelated Number of HLA mismatch Pretransplant-treatment modality Peritoneal then hemodialysis Hemodialysis Pre-emptive Ischemia time (mean ± SD; min) AR (%) Immunosuppression at the time of the study FK506/CsA (%) Sirolimus/everolimus MMF Prednisone

36.49 ± 7.44

FK506, tacrolimus; CsA, cyclosporine, AR, acute rejection.

0 36 (76.60%) 11 (23.40%) 2.28 ± 1.8 3 (6.38%) 42 (89.36%) 2 (4.26%) 44.47 ± 3.49 17 (29.8%) 29 (61.70%)/18 (38.30%) 2 (4.26%) 47 (100%) 47 (100%)

A high significant positive correlation was found between serum FasL level and ischemia time (r = 0.448, P = 0.005). Multiple linear regression analysis demonstrated that the risk factors for acute rejection in patients with renal transplantation were serum FasL (β = 0.464, P = 0.018), and serum creatinine level (β = 0.513, P = 0.003) (Table 6). 4. Discussion Cytokines and the molecules associated with cytolytic effector functions of T lymphocytes have long been studied as surrogates of AR and as possible candidates to the development of a non-invasive diagnostic tool [18–23]. Although with some good initial results, none of the proposed markers studied, until now, are being used routinely in the clinical practice or gained full acceptance. Most of the data reported a low degree of specificity, complicating its wide use in clinical practice. Few studies have revealed the role of immunomodulatory molecules in the pathogenesis of AR in humans, in contra balance of cytotoxic and proinflammatory ones. It is possible to think that both actors share in the immune response, and the end balance between them will be critical for graft outcome. Here, we investigated the role of Fas/FasL polymorphisms in children who had undergone renal transplantation and

Table 3 Distribution of Fas genotypes, Fas −670A/G alleles, FasL genotypes and FasL −840C/T alleles in transplant recipients and healthy controls. Genotype

Transplant recipients (n = 47)

Healthy controls (n = 20)

n

n

%

Fas genotypes AA 15 31.90% AG 6 12.80%

Chi-square P value Adjusted OR (95% CI)

%

5 25.00% 0 0.00% 1.857

0.173

26 55.30% 15 75.00% 0.819

0.366

AG and GG 32 68.10% 15 75.00% 0.320

0.571

Fas −670A/G alleles G 58 61.70% 30 75.00% 2.201 A 36 38.30% 10 25.00%

0.138

GG

FasL genotypes CC 21 44.70% 5 25.00% CT 0 0.00% 0 0.00% – TT 26 55.30% 15 75.00% 2.288

– 0.130

CT and TT

0.130

26 55.30% 15 75.00% 2.288

FasL −840C/T alleles T 52 55.30% 30 75.00% 4.577 C 42 44.70% 10 25.00%

0.032*

1.00 (Reference) 1.333 (1.035–1.717) 0.578 (0.175–1.909) 0.711 (0.218–2.322)

0.537 (0.235–1.229)

1.00 (Reference) – 0.413 (0.129–1.322) 0.413 (0.129–1.322)

0.413(0.181–0.940)

Data was evaluated by the gene counting method. Test for allele frequency difference Chisquare tests were used. Values were presented as percentage. * P was significant if b0.05.

Please cite this article as: F.I. Fadel, et al., Fas/Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection, Transpl Immunol (2016), http://dx.doi.org/10.1016/j.trim.2016.04.006

F.I. Fadel et al. / Transplant Immunology xxx (2016) xxx–xxx Table 4 Comparison of clinical parameters in children with or without acute rejection.

Recipient gender (males/females) Mean recipient age at transplant (years) Donor organ source Living, related Living, unrelated Number of HLA mismatch Ischemia time (mean ± SD; min) Immunosuppressants FK regimen CsA regimen CsA/mTORi regimen CAN e-GFR, ml/min/1/1.73 m2 T lymphocytes (cells/mm3) Serum sFasL (pg/ml)

Rejection(+) (n = 17)

Rejection(−) (n = 30)

P value

10 (58.82%)/7 (41.18%) 9.37 ± 3.56

19 (63.33%)/11 (36.67%) 10.09 ± 2.95

0.70

14 (82.35%) 3 (17.65%) 2.00 ± 1.65 44.41 ± 3.91

22 (73.33%) 8 (26.67%) 2.43 ± 0.86 44.50 ± 3.31

0.48

12 (70.58%) 2 (11.77%) 0 3 (17.65%) 78.86 ± 29.40 285.77 ± 316.94 567.70 ± 279.87

12 (40%) 16 (53.33%) 2 (6.67%) 0 96.18 ± 37.86 102.85 ± 726.89 507.85 ± 342.80

0.48

0.26 0.94

0.11 0.23 0.56

FK regimen (Prednisolone + FK506 + MMF), CsA regimen (Prednisolone + CsA + MMF) and FK/mTORi regimen (Prednisolone + CsA + sirolimus/everolimus), CAN (chronic allograft nephropathy), sFasL, soluble Fas Ligand, P was significant if b0.05.

compare these polymorphisms in children with and without AR as well as healthy controls. In this study, frequency of C allele of FasL gene polymorphism in pediatric renal transplant recipients was significantly higher than that in the controls. Interaction of Fas and FasL induces a cytolytic pathway leading to caspase- mediated apoptosis [24]. In this view it is a marker of renal damage initiation [24,25]. On the other hand, FasL in renal tubular cells induces removal of antigen- activated CD4 T lymphocytes [24]. Therefore, the occurrence of AR depends on a balance between these mechanisms besides many other factors. Apoptosis via Fas/FasL system has been evidenced to play a role in the pathogenesis of many diseases [26–34].

Table 5 Distribution of Fas genotypes, Fas −670 A/G alleles, FasL genotypes and FasL −840C/T alleles in transplant recipients with and without rejection. Genotype Rejection(+) Rejection(−) Chi-square P (n = 17) (n = 30) value n

Adjusted OR (95% CI)

%

n

%

Fas genotypes AA 4 AG 3

23.50% 17.60%

11 3

36.70% 10.00%

1.050

0.306

GG

10

58.80%

16

53.30%

0.588

0.443

AG and GG

13

76.50%

19

63.30%

0.862

0.353

Fas −670A/G alleles G 23 67.60% A 11 32.40%

35 25

58.30% 41.70%

0.797

0.372

FasL genotypes CC 10 CT 0 TT 7

58.80% 0.00% 41.20%

11 0 19

36.70% 0.00% 63.30%

– 2.155

– 0.142

CT and TT

41.20%

19

63.30%

2.155

0.142

FasL −840C/T alleles T 14 41.20% C 20 58.80%

38 22

63.30% 36.70%

4.310

0.038* 0.405 (0.171–0.959)

7

1.00 (Reference) 2.750 (0.385–19.668) 1.719 (0.428–6.905) 1.882 (0.491–7.217)

1.494 (0.618–3.611)

1.00 (Reference) – 0.405 (0.120–1.370) 0.405 (0.120–1.370)

Data was evaluated by the gene counting method. Test for allele frequency difference Chisquare tests were used. Values were presented as percentage. * P was significant if b0.05.

5

Table 6 Risk factors affecting acute rejection in transplant recipients based on multiple linear regression analysis.

Age (years) Donor age (years) Ischemia time (min) e-GFR (ml/min/1.73 m2) creatinine (_mol/L) T lymphocyte FasL (pg/ml)

β

P-value

0.322 −0.086 0.030 0.020 0.513 0.048 0.464

0.165 0.605 0.952 0.922 0.003* 0.799 0.018*

GFR, glomerular flirtation rate, FasL, Fas Ligand. * P b 0.05 was considered significant.

Along with these, Fas/FasL system has an important role in progressive renal disease and organ rejection in renal, cardiac and liver transplantation [35,36,37]. In a study done by Cappelesso et al., they have revealed that Fas −670GG genotype of the donor was associated with lower level of rejection episodes in renal transplant recipients [3]. Liver transplant recipients with the Fas −670AA genotype showed significantly lower graft survival rate than those with the AG genotype [38]. The present study revealed that sFasL levels were significantly higher in transplant recipients children than that in controls but no significant difference was found between children with AR vs children without. In transplantation, apoptosis is concerned in harmful events such as ischemia, reperfusion injury or graft rejection, but it may also play a key role – by depletion of alloreactive T-cells, apoptosis actively allows transplantation tolerance or decreases the frequency of rejection episodes [39].Seino et al. reported that the recipients of related liver transplants had elevated serum sFas concentrations, but not sFasL levels [40]. Prednisolone, used in this study as an anti-rejection drug, had been reported to induce apoptosis of peripheral T-lymphocytes shortly after intravenous administration [41]. It has also had a stabilizing effect on the plasma membrane, which may inhibit the release of soluble sFas and sFasL molecules from the membranes. In a study of soluble apoptotic markers in liver transplant recipients, serum sFas levels were significantly elevated in acute rejection patients and were brought to normal values by immunosuppressive therapy [42]. AR is under genetic control involving MHC polymorphisms and other different genes [43]. Such single nucleotide polymorphisms may modify the susceptibility of recipient T cells to FasL-mediated apoptosis [43]. In this study, we observed an association between FasL gene polymorphisms and an acute cellular rejection episode. Both in renal transplantation and in animal models of obstruction- induced renal tubular cell apoptosis, mRNA expression of FasL and associated caspases was found to be increased [44]. The FasL expression has been reported to be increased in peripheral blood and urine samples as well as in biopsy specimens from patients with AR [19,20]. Similarly, FasL expression has been detected to be significantly correlated with subacute graft rejection [45]. However, previous studies have failed to find a relationship between Fas gene polymorphisms and AR [43]. Also, Fas gene polymorphisms were not found to be different between transplant recipients and controls [43]. These findings are similar to our results in children that showed similar allele frequencies of Fas gene in controls and transplant children as well as cases with and without AR. Fas expression in renal tubular epithelial cells and accumulation of FasL expressing lymphocytes during reperfusion contributes to the Fas-mediated tissue damage [46]. Therefore, these findings are important in the interpretation of our results that FasL gene polymorphisms are associated with acute cellular rejection episode. Our results were free of genotyping errors/mistakes in data manipulation (“blind” genotyping or validation using different methodologies) and were in accordance with results of others as Ertan et al. [43]. Previously, we demonstrated that expression of CD 25on peripheral blood T cells correlates closely with the presence of acute graft

Please cite this article as: F.I. Fadel, et al., Fas/Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection, Transpl Immunol (2016), http://dx.doi.org/10.1016/j.trim.2016.04.006

6

F.I. Fadel et al. / Transplant Immunology xxx (2016) xxx–xxx

rejection in renal allograft pediatric recipients. Measurement of this surface marker may provide a rapid, noninvasive, and accurate means by which graft rejection can be identified [47]. A high significant positive correlation was found between serum FasL level and ischemia time. Tubular and/or glomerular Fas is elevated in progressive tubular atrophy, ischemia–reperfusion, experimental endotoxemia [24], and remnant renal glomerulosclerosis. In humans, Fas is elevated in proliferative glomerulonephritis. Fas initiates tubular injury during ischemia—reperfusion, as the tubular damage was reduced in B6 LPR mice when compared to B6 mice [24]. Also, an intact FasL/Fas system is required to restrict certain inflammatory responses, and constancy of systemic inflammation, including renal inflammation, follows murine CMV infection, in spite of clearance of the virus, in B6 LPR/LPR mice but not in control B6 mice [48]. On correlating acute rejection to different risk markers by multiple linear regression analysis, we found that serum FasL and serum creatinine were variables that were independently associated with AR. Carstens et al., reported that significant differences were present between acute rejection and zero-hour samples and acute rejection and non-rejection samples for FasL measured transcripts. No significant difference was found between acute borderline rejection (n = 16) and non-rejection samples [49]. Also, the curative antirejection-therapy resistance of overt, acute-rejection episode was significantly linked with higher Fas Ligand gene expression (AUC = 0.764, P b 0.01, sensitivity [71%], specificity [99.5%]).They demonstrated that the coinciding measurement of the mRNA up-regulation of Fas Ligand might clarify an efficient good maker for the prediction of the pejorative outcome of acute rejection [19]. Post-transplant monitoring currently relies on surveillance of allograft function (e.g. serum creatinine levels and GFR, in kidney transplantation). An acute alteration in the functional parameter suggests acute rejection occurrence, but confirmation requires the invasive procedure of allograft biopsy. Moreover, this approach recognizes rejection at a relatively advanced stage of the immune process and tissue injury and fails to diagnose subclinical acute rejection: histological rejection in the absence of graft dysfunction. Protocol or scheduled biopsies have been executed mainly in recipients of kidney or heart allografts to detect acute rejection prior to graft dysfunction [50]. Moreover, the diagnosis of subclinical rejection requires multiple biopsies; the biopsy procedures are invasive, complications occur, and sampling errors may bias the histological diagnosis. The costs and timing of this procedure need to be considered as well. Gene/protein expression patterns of noninvasively collected biologic specimens have been investigated as biomarkers of allograft status [51]. A noninvasive tool has a number of potential advantages including frequent and sequential assessments of recipient's immune status. Molecular perturbations may precede not only graft dysfunction but also histological changes. Importantly, molecular parameters may serve to guide minimization of immunosuppression and individualization of immunosuppressive therapy. Clinicians with a more refined knowledge of the recipient's immune status may be able to adjust the immunosuppressive drug therapy that is often responsible for numerous side effects [50]. A molecular-based approach that incorporates a set of markers could function not only as a surrogate for the invasive biopsy procedure, but could also provide predictive, diagnostic, and prognostic information as well as provide mechanistic insights into the pathobiology of allograft dysfunction. Kinetic studies in three patients with AR revealed that increased perforin, granzyme B and FasL mRNA levels could precede or were concomitant with increased serum creatinine levels [52]. Limitations of this study include moderate sample size, but it can suitable as we search on the pediatric population. Furthermore, the results represent a single center experience of a racially homogeneous cohort and may not be generalizable to other populations. We found no significant relations between acute rejection and genotypes except for the FasL alleles. This might be attributed to type 1 inflation due to the great number of comparisons that were done. Further large- scale

studies allow more information about the issue and appropriate targeted therapy and surveillance could be performed. In conclusion, FasL gene polymorphisms might play a key role in AR while Fas polymorphisms have not been found to be different between children with and without acute renal graft rejection. We found that serum FasL and serum creatinine were variables that were independently associated with AR. It could provide predictive information into the pathobiology of AR. We can hypothesize that a comprehensive analysis of this relationship in the pediatric recipients could predict the risk of AR and influence therapeutic strategies. Conflict of interest The authors declare that they have no competing interests References [1] V.H. Karam, I. Gasquet, V. Delvart, C. Hiesse, R. Dorent, C. Danet, et al., Quality of life in adult survivors beyond 10 years after liver, kidney, and heart transplantation, Transplantation 76 (12) (2003) 1699–1704. [2] J.J. Curtis, End-stage renal disease patients: referral for transplantation, J. Am. Soc. Nephrol. 9 (12 Suppl.) (1998) S137–S140. [3] S. Cappellesso, J.F. Valentin, B. Giraudeau, et al., Association of donor TNFRSF6 (FAS) gene polymorphism with acute rejection in renal transplant patients: a case control study, Nephrol. Dial. Transplant. 19 (2) (2004) 439–443. [4] Y. Vanneterghem, Impact of acute rejection on the longterm outcome after renal transplantation, Graft 3 (1) (2000) 31–33. [5] B.D. Kahan, C. Ponticelli, A. Tarantino, Rejection and other renal complications, in: Morris PJ (Ed.), Kidney Transplantation, Principles and Practice, fifth ed.Saunders, Philadelphia 2001, pp. 207–216. [6] F. Strutz, Pathogenesis of tubulointerstitial fibrosis in chronic allograft dysfunction, Clin. Transpl. 23 (Suppl. 21) (2009) 26–32. [7] Ş. Kobak, A. Berdeli, Fas/FasL promoter gene polymorphism in patients with rheumatoid arthritis, Reumatismo 64 (6) (Dec 20 2012) 374–379. [8] A. Krueger, S. Baumann, P.H. Krammer, S. Kirchhoff, FLICE-inhibitory proteins: regulators of death receptor-mediated apoptosis, Mol. Cell. Biol. 21 (24) (2001) 8247–8254. [9] J. Ho, C. Wiebe, I.W. Gibson, D.N. Rush, P.W. Nickerson, Immune monitoring of kidney allografts, Am. J. Kidney Dis. 60 (4) (2012 Oct) 629–640. [10] Y. Zhang, G. Cheng, Z.W. Xu, Z.L. Li, C.J. Song, Q. Li, L.H. Chen, K. Yang, A.G. Yang, B.Q. Jin, Down regulation of TRAIL and FasL on NK cells by Cyclosporin A in renal transplantation patients, Immunol. Lett. 152 (1) (2013 Apr) 1–7. [11] R.J. Plenter, T.J. Grazia, D.P. Nelson, M.R. Zamora, R.G. Gill, Pietra BA Ectopic expression of Fas Ligand on cardiomyocytes renders cardiac allografts resistant to CD4(+) T-cell mediated rejection, Cell Immunol. 293 (1) (Jan 2015) 30–33. [12] Q.R. Huang, D. Morris, N. Manolios, Identification and characterization of polymorphisms in the promoter region of the human Apo-1/Fas (CD95) gene, Mol. Immunol. 34 (8–9) (1997) 577–582. [13] J. Cheng, C. Liu, W.J. Koopman, et al., Characterization of human Fas gene. Exon/intron organization and promoter region, J. Immunol. 154 (3) (1995) 1239–1245. [14] G. Stallone, B. Infante, A. Schena, et al., Rapamycin for treatment of chronic allograft nephropathy in renal transplant patients, J. Am. Soc. Nephrol. 16 (2005) 3755. [15] 23. W. Land, F. Vincenti, Toxicity-sparing protocols using mycophenolate mofetil in renal transplantation, Transplantation 80 (2005) S221. [16] 24. K. Solez, R.B. Colvin, L.C. Racusen, M. Haas, B. Sis, M. Mengel, P.F. Halloran, W. Baldwin, G. Banfi, A.B. Collins, F. Cosio, D.S. David, C. Drachenberg, G. Einecke, A.B. Fogo, I.W. Gibson, D. Glotz, S.S. Iskandar, E. Kraus, E. Lerut, R.B. Mannon, M. Mihatsch, B.J. Nankivell, V. Nickeleit, Banff '07 classification of renal allograft pathology: updates and future directions, Am. J. Transplant. 8 (2008) 753–760. [17] 17. G.J. Schwartz, A. Muñoz, M.F. Schneider, R.H. Mak, F. Kaskel, B.A. Warady, S.L. Furth, New equations to estimate GFR in children with CKD, J. Am. Soc. Nephrol. 20 (3) (2009 Mar) 629–637. [18] T. Simon, G. Opelz, M. Wiesel, R.C. Ott, C. Susal, Serial peripheral blood perforin and granzyme B gene expression measurements for prediction of acute rejection in kidney graft recipients, Am. J. Transplant. 3 (9) (2003) 1121–1127. [19] D. Desvaux, M. Schwarzinger, M. Pastural, C. Baron, M. Abtahi, F. Berrehar, et al., Molecular diagnosis of renal allograft rejection: correlation with histopathologic evaluation and antirejection therapy resistance, Transplantation 78 (50) (2004) 647–653. [20] M. Yannaraki, J.M. Rebibou, D. Ducloux, et al., Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection, Transpl. Int. 19 (9) (2006) 759–768. [21] M.J. Ross, S. Martinka, V.D. D'Agati, et al., NFkappaB regulates Fas-mediated apoptosis in HIV-associated nephropathy, J. Am. Soc. Nephrol. 16 (8) (2005) 2403–2411. [22] V.N. Carpio, E.C. Aquino-Dias, T.A. Prochnow, et al., Evaluation of apoptosis in peripheral blood lymphocytes of renal transplant patients, Transplant. Proc. 38 (6) (2006) 1898–1900. [23] R. Graziotto, D. Del Prete, P. Rigotti, et al., Perforin, Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft rejection: analyses on serial biopsies suggest methodological issues, Transplantation 81 (8) (2006) 1125–1132.

Please cite this article as: F.I. Fadel, et al., Fas/Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection, Transpl Immunol (2016), http://dx.doi.org/10.1016/j.trim.2016.04.006

F.I. Fadel et al. / Transplant Immunology xxx (2016) xxx–xxx [24] B. Yang, K.P. Harris, S. Jain, et al., Caspase-7, Fas and FasL in long-term renal ischaemia/reperfusion and immunosup-pressive injuries in rats, Am. J. Nephrol. 27 (4) (2007) 397–408. [25] C. Mitchell, M. Mahrouf-Yorgov, A. Mayeuf, M.A. Robin, A. Mansouri, B. Fromenty, H. Gilgenkrantz, Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease, Lab. Investig. 91 (2) (2011 Feb) 273–282. [26] M. Perl, C.S. Chung, U. Perl, J. Lomas-Neira, M. de Paepe, W.G. Cioffi, A. Ayala, Fasinduced pulmonary apoptosis and inflammation during indirect acute lung injury, Am. J. Respir. Crit. Care Med. 176 (6) (2007 Sep 15) 591–601. [27] L.L. Bi, G. Pan, T.P. Atkinson, et al., Dominant inhibition of Fas ligand-mediated apoptosis due to a heterozygous mutation associated with autoimmune lymphoproliferative syndrome (ALPS) Type Ib, BMC Med. Genet. 8 (2007) 41. [28] E. Hatano, Tumor necrosis factor signaling in hepatocyte apoptosis, J. Gastroenterol. Hepatol. 22 (Suppl. 1) (Jun 2007) S43–S44. [29] S. Fountoulakis, G. Vartholomatos, N. Kolaitis, S. Frillingos, G. Philippou, A. Tsatsoulis, Differential expression of Fas system apoptotic molecules in peripheral lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis, Eur. J. Endocrinol. 158 (6) (2008 Jun) 853–859. [30] S.G. Duan, L. Cheng, D.J. Li, J. Zhu, Y. Xiong, X.W. Li, S.G. Wang, The role of MAPK-ERK pathway in 67-kDa laminin receptor-induced FasL expression in human cholangiocarcinoma cells, Dig. Dis. Sci. 55 (10) (Oct 2010) 2844–2852. [31] Y.J. Kim, H.Y. Kim, J.S. Kim, J.H. Lee, J.H. Yoon, C.Y. Kim, B.L. Park, H.S. Cheong, J.S. Bae, S. Kim, H.D. Shin, H.S. Lee, Putative association of transforming growth factor-alpha polymorphisms with clearance of hepatitis B virus and occurrence of hepatocellular carcinoma in patients with chronic hepatitis B virus infection, J. Viral Hepat. 17 (7) (Jul 2010) 518–526. [32] B. Zhang, T. Sun, L. Xue, et al., Functional polymorphisms in FAS and FASL contribute to increased apoptosis of tumor infiltration lymphocytes and risk of breast cancer, Carcinogenesis 28 (5) (2007) 1067–1073. [33] S. Bousserouel, H. Kauntz, F. Gossé, et al., Identification of gene expression profiles correlated to tumor progression in a preclinical model of colon carcinogenesis, Int. J. Oncol. 36 (6) (2010) 1485–1490. [34] J. Aguilar-Reina, M. Ruiz-Ferrer, M.A. Pizarro, et al., The −670ANG polymorphism in the promoter region of the FAS gene is associated with necrosis in periportal areas in patients with chronic hepatitis C, J. Viral Hepat. 12 (6) (2005) 568–573. [35] R. Kettritz, S. Wilke, S. von Vietinghoff, F. Luft, W. Schneider, Apoptosis, proliferation and inflammatory infiltration in ANCA-positive glomerulonephritis, Clin. Nephrol. 65 (5) (May 2006) 309–316. [36] E.C. Perez, N. Shulzhenko, A. Morgun, et al., Expression of Fas, FasL, and soluble Fas mRNA in endomyocardial biopsies of human cardiac allografts, Hum. Immunol. 67 (1–2) (2006) 22–26. [37] W. Liu, Z.F. Huang, Q.F. Ye, S.J. Ye, Transfusion of endothelial cells with lentivirusmediated expression of fas ligand prolonged survival of rat liver allograft, Transplant. Proc. 44 (5) (Jun 2012) 1399–1403.

7

[38] L.A. Marin, M. Muro, M.R. Moya-Quiles, et al., Study of Fas (CD95) and FasL (CD178) polymorphisms in liver transplant recipients, Tissue Antigens 67 (2) (2006) 117–126. [39] J. Hökl, J. Černý, J. Ondrášek, H. Bedáňová, J. Vespalec, A. Sirotková, Soluble Fas and Fas-ligand plasma levels in heart transplant recipients treated with neoral or tacrolimus, Scr. Med. (Brno) 79 (3) (July 2006) 163–168. [40] K. Seino, N. Kayagaki, N. Yamaguchi, et al., Soluble forms of CD95 and CD95 ligands after living related liver transplantation, Transplantation 67 (1999) 634–636. [41] N. Bonnefoy-Berard, L. Genestier, X. Preville, J.-P. Revillard, TNFα and CD95-L contribute to apoptosis of activated lymphocytes triggered by ATGs, Transplant. Proc. 31 (1999) 775–777. [42] M. Rivero, J. Crespo, M. Mayorga, et al., Soluble Fas in acute rejection in patients with liver transplantation. Response to treatment, Gastroenterol. Hepatol. 23 (2000) 215–218. [43] P. Ertan, S. Mir, N. Ozkayin, A. Berdeli, Association of FAS −670A/G and FASL −843C/T Gene polymorphisms on allograft nephropathy in pediatric renal transplant patients Iran, J. Pediatr. 20 (4) (December 2010) 442–450. [44] Matthew T. Campbell, Pierre Dagher, Karen L. Hile, Hongji Zhang, Daniel R. Meldrum, Richard C. Rink, Kirstan K. Meldrum, Tumor necrosis factor-α induces intrinsic apoptotic signaling during renal obstruction through truncated bid activation, J. Urol. 180 (6) (Dec 2008) 2694–2700. [45] R. Madhu, Sophia Y. Cleland, Anthony C. Cruz, Richard M. Siegel, Many checkpoints on the road to cell death: regulation of Fas-FasL interactions and Fas signaling in peripheral immune responses, Results Probl. Cell Differ. 49 (2009) 17–47. [46] B. Yang, K.P. Harris, S. Jain, et al., Caspase-7, Fas and FasL in long-term renal ischemia/reperfusion and immunosuppressive injuries in rats, Am. J. Nephrol. 27 (4) (2007) 397–408. [47] F.I. Fadel, E.A. Elghoroury, M.F. Elshamaa, H.M. Bazaraa, D.M. Salah, N.M. Kassem, M.H. Ibrahim, G.S. El-Saaid, S.A. Nasr, H.M. Koura, Lymphocyte activation markers in pediatric kidney transplant recipients, Int. J. Biomed. Sci. 11 (3) (2015 Sep) 121–130. [48] M. Fleck, E.R. Kern, T. Zhou, et al., Apoptosis mediated by fas but not tumor necrosis factor receptor 1 prevents chronic disease in mice infected with murine cytomegalovirus, J. Clin. Invest. 102 (1998) 1431–1443. [49] J. Carstens, A. Ozbay, C. Tørring, H.E. Hansen, Intragraft mRNA cytotoxic molecule expression in renal allograft recipients, Transpl. Immunol. 20 (4) (2009 Mar) 212–217. [50] D. Anglicheau, M. Suthanthiran, Noninvasive prediction of organ graft rejection and outcome using gene expression patterns, Transplantation 86 (2) (Jul 27 2008) 192–199. [51] B. Merhi, G. Bayliss, Gohh RY, Role for urinary biomarkers in diagnosis of acute rejection in the transplanted kidney, World J. Transplant. 5 (4) (2015 Dec 24) 251–260. [52] M. Yannaraki, J.M. Rebibou, D. Ducloux, et al., Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection, Transpl. Int. 19 (9) (2006) 759–768.

Please cite this article as: F.I. Fadel, et al., Fas/Fas Ligand pathways gene polymorphisms in pediatric renal allograft rejection, Transpl Immunol (2016), http://dx.doi.org/10.1016/j.trim.2016.04.006