| کلیدواژههای انگلیسی مقاله |
Kidney transplantation, Receptors, Glucocorticoids, Polymorphism, Genetic, What&,rsquo s Known Glucocorticoids, the mainstay of immunosuppressive therapies, may prevent acute graft rejection and delayed graft function. Some patients show glucocorticoid resistance. Glucocorticoid receptor gene polymorphisms, namely N363S, Bcl1, and ER22/23EK, are associated with glucocorticoid sensitivity in various populations. What&,rsquo s New N363S, Bcl1, and ER22/23EK polymorphisms had significant associations with acute rejection and delayed graft function after renal transplantation, as the clinical outcomes of glucocorticoid resistance. N363S, Bcl1, and ER22/23EK polymorphisms showed no significant associations with the length of hospital stay after transplantation. IntroductionEnd-stage renal disease (ESRD), with its poor prognosis, usually necessitates renal replacement therapy, including dialysis or renal transplantation. 1, Renal transplantation, rather than dialysis, is now deemed the treatment of choice for ESRD, because it improves the quality of life and survival. 2, , 3, The United Network for Organ Sharing (UNOS) recorded 21,167 kidney transplantations in the United States, from January 1, 1998, to August 31, 2019. 4, Iran is the leading country in the Middle East in the field of kidney transplantation. As a case in point, over 2,263 and 2,101 renal transplantations were done in the country in 2018 and 2019, respectively. 5, Acute rejection is a common and critical complication of renal transplantation. 6, In the United States, one year after surgery, 10% of deceased-donor kidney transplantations in 2009 to 2010 led to at least one episode of acute rejection. Nonetheless, this rate of incidence decreased to 8% in the years 2013 to 2014. 7, According to a retrospective study spanning 37 years (1998&,ndash 2015) on 2557 kidney transplantation recipients in the Urmia Kidney Transplantation Center in Iran, graft loss was developed in just 86 (3.36%) patients within the first month of surgery. 8, Delayed graft function (DGF) is an acute kidney injury and a state of renal suboptimal function within the first week after transplantation, which leads to dialysis requirement and may be associated with shorter graft survival. DGF duration implies the number of days before an estimated glomerular filtration rate of 10 mL/min or greater is attained by the transplanted kidney. 9, A research conducted in 2013 in the United Kingdom reported a DGF incidence rate of 24.3% to 70% in brain-death-donor transplantations and 41.2% to 90% in circulatory-death-donor transplantations. 10, Glucocorticoids, the mainstay of immunosuppressive therapy in both induction and maintenance phases after solid organ transplantation, may affect the immune system through different mechanisms. Altering the expression of some genes, either directly or indirectly, and exerting anti-inflammatory and immunomodulatory effects are a number of functions served by glucocorticoids. 11, Resistance to glucocorticoids is a major challenge with unknown molecular mechanisms. N363S, a point mutation in exon 2, causes the substitution of AAT by AGT in codon 363, presenting with asparagine change to serine. 12, Bcl1 mutation consists of C-to-G change at 646 nucleotides downstream from exon 2. 13, Two contiguous codons, 22 and 23, show alterations in sequence as a result of ER22/23EK mutation. The change from GA G ____ A G ____ G to GA A ____ A A ____ G causes the substitution of glutamic acid-arginine (E-R) with glutamic acid-lysine (E-K) in produced proteins. 13, Mutation in codon 22 does not lead to a change in amino acid sequence, whereas mutated codon 23 produces a different amino acid. Generally, the N363S and Bcl1 polymorphisms have been observed to increase glucocorticoid sensitivity, while the ER22/23EK polymorphism is often associated with relative resistance. 14, , 15, This study aimed to assess the possible association between the ER22/23EK, N363S, and Bcl1 polymorphisms, and short-term clinical outcomes, including acute rejection and DGF, in kidney transplantation recipients at a referral center in Iran.Patients and MethodsStudy SettingThe present study enrolled 100 patients, who received renal transplantation from living donors or cadavers between April 2017 and September 2018 in Shiraz, Iran. The inclusion criteria were comprised of being at least 18 years old, undergoing first-time renal transplantation, having available data and blood samples, and consenting to participate in the study. The subjects were classified into two groups of control and case, both comprising 50 patients. The former group consisted of patients, who had maintained graft function within the first year after transplantation, with no acute rejection, whereas the latter group encompassed patients, who experienced at least one documented episode of acute rejection (cellular, humoral, or mixed) during the first year following transplantation, regardless of its type and mechanism. The study protocol was approved by the Medical Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1397.1032), and written informed consent forms were taken from each patient at the time of study entry. The parents and grandparents of each patient were of Iranian descent.Data GatheringThe required information from each recruited patient was obtained through a review of medical charts and face-to-face interviews. The data included the recipient&,rsquo s age gender etiology of chronic kidney disease dialysis duration and type prior to transplantation donor type (deceased vs. living), age, and gender cold ischemia time length of hospital stay after transplantation immunosuppressive regimen in induction and maintenance phases other co-administered medications underlying diseases and rejection type (only in the case group). DGF was defined as the requirement for dialysis in the first week after transplantation. 10, Sampling and PreparationBlood samples were provided from the Sample Bank of the Shiraz Transplant Research Center. Through the use of 2 mL of the Ficoll solution for 10 mL of a whole blood sample, followed by centrifuge, buffy coats were extracted. The buffy coats contained monocytes and lymphocytes. Thus, five-to-ten times higher DNA concentrations were obtained. 16, The buffy coats were stored at -20 &,ordm C.DNA ExtractionDNA extraction from 200 &,micro L of the buffy coats was carried out using the FavorPrep Blood Genomic Extraction Mini KIT (Favorgen Biotech, Taiwan) in accordance with the manufacturer&,rsquo s protocol. With a NanoDrop, DNA quantity was measured using 5 &,micro L of DNA and 45 &,micro L of double-distilled water (DDW). Given that the optimum DNA quantity for polymerase chain reaction (PCR) was considered 100 ng, individual volumes of extracted DNA for each sample were calculated.DNA AmplificationThe ER22/23EK, N363S, and Bcl1 polymorphisms were evaluated. The genotypes of both groups were determined via the amplification of genomic fragments through PCR. Particular reaction mixtures were prepared for each polymorphism. The mixtures contained 0.5 &,micro L of 10 &,micro M working solutions of forward and reverse primers (all from 100 &,micro M stock solutions) (Eurofins Scientific, Belgium), 2.5 &,micro L PCR 10X buffer (CinnaGen, Iran), 0.75 &,micro L of 10 mM dNTPs (CinnaGen, Iran), 0.75 &,micro L of 50 mM MgCl2 (CinnaGen, Iran), and 0.5 &,micro L Taq DNA polymerase (CinnaGen, Iran). For the evaluation of the N363S polymorphism, 0.5 &,micro L tween was added in order to eliminate the undesirable bands. The primer sequences were as follows,ER22/23EK forward primer, 5&,rsquo -GATTCGGAGTTAACTAAAAG-3&,rsquo ER22/23EK reverse primer, 5&,rsquo -ATCCCAGGTCATTTCCCATC-3&,rsquo N363S forward primer, 5&,rsquo -AGTACCTCTGGAGGACAGAT-3&,rsquo N363S reverse primer, 5&,rsquo -GTCCATTCTTAAGAAACAGG-3&,rsquo Bcl1forward primer, 5&,rsquo -TGCTGCCTTATTTGTAAATTCGT-3&,rsquo Bcl1 reverse primer, 5&,rsquo -AAGCTTAACAATTTTGGCCATC-3&,rsquo The PCR process, carried out with a thermal cycler (Eppendorf, Germany), involved five minutes of denaturation at 95 &,ordm C, followed by 45 seconds of denaturation at 94 &,ordm C, one minute of annealing at an optimum annealing temperature, and eventually one minute of DNA extension at 72 &,ordm C. The last three steps were repeated for 30 cycles. The annealing temperatures were set at 55.2 &,ordm C, 51.2 &,ordm C, and 55.2 &,ordm C for ER22/23EK, N363S, and Bcl1, respectively. The PCR products were examined randomly via electrophoresis on 1.5% agarose gel and 0.588% buffer.Bcl1 Genotyping with the Bcl1 Restriction EnzymeAs in polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), the amplified fragments were digested using the Bcl1 restriction enzyme (New England Biolabs Inc., USA) in order to determine the patients&,rsquo genotypes. Through the addition of 2.5 &,micro L buffer, 0.5 &,micro L enzyme, and 3 &,micro L DDW to 20 &,micro L PCR product, digestion mixtures were prepared. Digestion was completed in two hours of incubation at 50 &,ordm C.The restriction enzyme causes the fraction of the 335 bp DNA fragment into two fragments of 117 and 222 bp in the case of wild (CC) genotypes. This enzyme results in three fragments (117, 222, and 335 bp) and just one fragment (undigested, 335 bp) for the CG and GG genotypes, respectively. 17, N363S Genotyping with the MluCI Restriction EnzymeFor the determination of the N363S genotypes, MluCI (New England Biolabs Inc., USA) was added to relevant PCR products. Each digestion mixture was prepared by adding 2.5 &,micro L buffer, 0.5 &,micro L endonuclease enzyme, and 3 &,micro L DDW to 20 &,micro L PCR product. The mixtures were incubated at 37 &,ordm C for two hours, while inactivation was not required. MluCI is an isoschizomer of Tsp5091 in other words, it recognizes the same sequence. 18, MluCI acts at its restriction site and produces three fragments (19, 95, and 134 bp), if the wild genotype (NN) is present at codon number 363. Enzyme function on the N363S and S363S genotypes results in two bands (95 and 153 bp) and one band (153 bp), respectively. 19, , 20, ER22/23EK Genotyping with the MnII Restriction EnzymeThe genotypes of the ER22/23EK polymorphism via PCR-RFLP were determined through the use of MnII endonuclease (New England Biolabs Inc., USA).Through the addition of 2.5 &,micro L buffer, 0.5 &,micro L enzyme, and 3 &,micro L DDW to 20 &,micro L PCR product, digestion mixtures were prepared subsequent to 40 minutes of incubation at 50 &,ordm C. The enzymatic compartments of the preparations were inactivated via 20 minutes of incubation at 65 &,ordm C, if immediate electrophoresis was not available. Finally, the bands, which indicated fragments from the three restriction processes, were separated on an agarose gel using electrophoresis. They were observed under ultraviolet light.Statistical AnalysisThe statistical analyses were performed using the SPSS software, version 20, (IBM Company, N.Y., USA). The categorical data were reported in percentage terms. The normality of the distribution of the continuous data was evaluated using the Kolmogorov&,ndash Smirnov test. In the case of normal and abnormal distributions, the data were reported as mean&,plusmn SD and median+interquartile ranges, respectively. A stepwise logistic regression analysis with odds ratios (ORs) and 95% confidence intervals (CI) was applied to determine the associated factors of acute rejection within the first year after transplantation. In the univariate model, each demographic, clinical, and laboratory datum was entered separately. Those with a P value of less than 0.05 were selected and then entered simultaneously into the final multivariate model. The association between the genotypes and the patients&,rsquo length of hospital stay after transplantation was evaluated using the independent-sample t test. The association between the genotypes and DGF as well as rejection types was evaluated using either the Chi square test or Fisher exact test. A P value of less than 0.05 was considered statistically significant for all the above analyses. The Hardy&,ndash Weinberg test was applied using the Arlequin software, version 3.11, (Bern, Swiss) to determine the distribution of the alleles. A P value of less than 0.05 in this test indicated no deviation in the distribution of the genotypes and alleles over different generations in a population. ResultsDuring the study period, 250 kidney transplantation recipients were screened primarily. A total of 150 patients were excluded from the study due to incomplete data and medical charts (n=58), lack of access to blood samples for genetic analysis (n=42), second transplantation (n=35), and spontaneous pancreas and kidney transplantation (n=15). Finally, 100 patients were enrolled in the study. The mean&,plusmn SD age of the patients was 41.25&,plusmn 13.52 years. The most common etiology of ESRD was hypertension (39%). Most of the patients (91%) were on hemodialysis before transplantation. The median time of dialysis before transplantation was 12 months. Almost all the patients (97%) received kidney allografts from deceased donors, and 91% of the study population had negative panel-reactive antibodies. The median cold ischemia time of the transplanted kidneys was 3.5 hours. Eighty percent of the patients received anti-thymocyte globulin during the induction phase of immunosuppression early after transplantation. Forty-six percent of the immunosuppressive regimen within the maintenance phase was prednisolone (Nisopred&,reg , Iran Hormone Pharmaceutical Company, Iran)+tacrolimus (Prograf&,reg , Astellas Pharma Inc., Netherlands)+mycophenolate mofetil (Cellcept&,reg , Zahravi Pharmaceutical Company, Iran).According to the univariate analysis, the trough level of tacrolimus (OR, 0.76, 95% CI, 0.61 to 0.95 P=0.01) and the administration of anti-thymocyte globulin to induce immunosuppression (OR, 2.85, 95% CI, 0.99 to 8.17 P=0.05) were selected ( table 1,). Nevertheless, after these variables were fitted into the final model of logistic regression. Only the trough level of tacrolimus was significantly associated with rejection (OR, 0.76, 95% CI, 0.61 to 0.95 P=0.02) ( table 2,). In other words, with each 1 ng/dL increase in the tacrolimus trough level, the average risk of allograft rejection decreased by about 24%.VariablesCase Group (n=50)Control Group (n=50)UnivariateP valueOR (95% confidence interval)Age (year) (mean&,plusmn SD)40.7&,plusmn 13.5841.8&,plusmn 13.560.691.006 (0.97-1.03)Sex (%)Male32 (64)32 (64)-1Female18 (36)18 (36)11 (0.44-2.26)Etiology (%)Others32 (64)29 (58) 1Hypertension18 (36)21 (42)0.540.78 (0.35-1.74)Length of pre-transplantation dialysis (month, mean&,plusmn SD)15.0&,plusmn 1217.5&,plusmn 120.560.99 (0.96-1.02)Donor type (%)Cadaver48 (96)49 (98)-1 Living2 (4)1 (2)0.560.49 (0.04-5.58)Donor age (year) (mean&,plusmn SD)36.38&,plusmn 14.1836.14&,plusmn 15.190.341.01 (0.98-1.04)Donor sex (%)Male41 (82)36 (72)-1Female9 (18)14 (28)0.240.56 (0.22-1.46)Cold ischemia time (hour), median (interquartile range)5.13 (4.5)7.7 (3.0)0.980.99 (0.89-1.11)Panel reactive antibody (%)Negative44 (88)47 (94) 1Positive6 (12)3 (6)0.3032.14 (0.50-9.07)Receiving anti-thymocyte globulin as an induction of immunosuppression (%)Yes44 (88)36 (72)-1No6 (12)14 (28)0.0512.85 (0.995-8.173)Anti-thymocyte globulin dose (mg), median (interquartile range)375 (300)310 (300)0.390.99 (0.99-1.001)Immunosuppressive maintenance regimen (%)Tacrolimus+Mycophenolate+Prednisolone42 (84)4 (72)---1Others8 (16)46 (28)0.1522.042 (0.77-5.42)Glomerular filtration rate at hospital admission (mL/min, median+interquartile range)7.5&,plusmn 0.08.4&,plusmn 0.250.191.06 (0.97-1.16)Cyclosporine trough level (ng/dL, mean&,plusmn SD)711.873&,plusmn 184.1422.41&,plusmn 149.620.370.99 (0.98-1.007)Tacrolimus trough level (ng/dL, mean&,plusmn SD)7.45&,plusmn 3.185.56&,plusmn 2.120.010.76 (0.61-0.95)Delayed graft function (%)Yes7 (14)4 (8)---1No43 (86)46 (92)-0.3431.87 (0.51-6.85)SD, Standard deviation OR, Odds ratio A univariate logistic regression analysis was used. |