|Year : 2013 | Volume
| Issue : 4 | Page : 688-695
|Tumor necrosis factor-alfa and monocyte chemoattractant protein-1 gene polymorphisms in kidney transplant recipients
Ebtesam M El-Gezawy1, Eman Nasr Eldin1, Wafaa S Mohamed1, Maged S Mahmoud1, Seham Ahmed Saied2, Hanan Hareth Abd El-Latif1, Maha Atwa Ibrahim1
1 Clinical Pathology Department, Faculty of Medicine, Assiut University, Assiut, Egypt
2 National Institute of Urology and Nephrology, Cairo, Egypt
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|Date of Web Publication||24-Jun-2013|
| Abstract|| |
Tumor necrosis factor-alfa (TNF-α) gene polymorphism is supposed to have a significant influence on the incidence of acute rejection in renal transplantation. The monocyte chemoattractant protein-1 (MCP-1) is another factor supposed to modulate graft rejection. We studied TNF-α and MCP-1 gene polymorphisms in 84 kidney allograft recipients with polymerase chain reaction and restriction fragment length polymorphism and their serum levels by enzyme-linked immunosorbent assay. The patients were classified into two groups based on their outcomes: Group I (n = 47) recipients with stable graft function as the control group and group II (n=37) recipients who experienced acute graft rejection episodes in the first 30 days post-transplantation. A significantly higher incidence of TNF 2 /TNF 2 genotype was observed among patients with acute graft rejection in comparison with the control group (40.5% and 19.2% respectively, P <0.05), while no statistically significant differences were observed in the TNF 1 /TNF 1 genotype between the groups (59.4% and 80.8%, respectively, P >0.05). A significant elevation of serum TNF-α levels was found in group II than group I and between TNF 2 genotype compared with that of TNF1 genotype within group II recipients. Distribution of MCP-1 genotypes in patients with and without acute rejection episodes was not significantly different (70.2% and 76.6% for MCP-1 A/A and 29.7% and 23.4% for MCP-1 G/G, respectively, P >0.05). The serum MCP-1 levels were not significantly different between the groups and between MCP-1 G/G genotype and MCP-1 A/A genotype in group II recipients. In conclusion, TNF-α gene polymorphism or its serum levels may identify patients at risk of acute rejection, where patients with TNF 2 /TNF 2 genotype or high serum TNF-α levels are more likely to have acute rejection episodes, while there was no relation between MCP-1 genotype or its serum levels and acute rejection.
|How to cite this article:|
El-Gezawy EM, Eldin EN, Mohamed WS, Mahmoud MS, Saied SA, Abd El-Latif HH, Ibrahim MA. Tumor necrosis factor-alfa and monocyte chemoattractant protein-1 gene polymorphisms in kidney transplant recipients. Saudi J Kidney Dis Transpl 2013;24:688-95
|How to cite this URL:|
El-Gezawy EM, Eldin EN, Mohamed WS, Mahmoud MS, Saied SA, Abd El-Latif HH, Ibrahim MA. Tumor necrosis factor-alfa and monocyte chemoattractant protein-1 gene polymorphisms in kidney transplant recipients. Saudi J Kidney Dis Transpl [serial online] 2013 [cited 2020 Jul 3];24:688-95. Available from: http://www.sjkdt.org/text.asp?2013/24/4/688/113855
| Introduction|| |
All patients with terminal renal failure should be considered for transplantation except those at risk from another life-threatening condition. ,,,
The influence of gene polymorphisms on the clinical course post-transplant has become an area of active research as it offers a possible explanation for the heterogeneity in outcomes between individuals.  Observations suggest that genetic variability influence allograft survival beyond that of the major histocompatibility complex (MHC) molecules. 
Tumor necrosis factor-alfa (TNF-α), produced by macrophages, may play a major part in various chronic inflammatory diseases,  and it has been implicated in the pathogenesis of both acute and chronic transplant rejection.  Many studies have reported an increased concentration of TNF-α in the plasma of recipients during acute rejection of human heart, lung, liver, kidney and other allografts.  In kidney transplantation, TNF 2 is associated with acute rejection in HLA-DR-mismatched allografts, and it is possible that the TNF 2 polymorphism at -308 may alter TNF-α expression levels and, therefore, contributes to aberrant or inappropriate immune function.  Individuals homozygous for the TNF 2 alleles have a higher serum TNF-α level than TNF1 homozygotes. The TNF 2 allele is in linkage disequilibrium with the MHC haplotype A1-B8-DR3.  There is some evidence that the TNF 2 allele is a much stronger transcriptional activator than the TNF1 allele. 
Chemokines and chemokine receptors may affect allograft function not only through acute and chronic allograft rejection but also, perhaps indirectly, through the accelerated cardiovascular co-morbidity of the recipients.  Elevated levels of monocyte chemoattractant protein (MCP-1) were detected in the serum and urine of patients at the time of acute and chronic renal allograft rejection.  A correlation between MCP-1 levels and monocytes in rejecting human kidney allografts had been observed, and normalization of urinary excretion of MCP-1 correlated with a positive response to anti-rejection treatment.  We aimed in this study to determine the influence of TNF-α and MCP-1 gene polymorphism on the occurrence of acute rejection of kidney allografts during the first 30 days post-transplantation.
| Subjects and Methods|| |
The study was conducted on 84 kidney allograft recipients from the National Institute of Urology and Misr International Hospital (Cairo, Egypt). The patients were classified into two groups: 47 kidney transplant recipients with stable graft function (group I) and 37 kidney transplant recipients with acute graft rejection in the first 30 days after transplantation (group 2).
Clinical rejection was identified as an increase in creatinine levels of 15% or more from baseline in the absence of infection, obstruction or evidence of cyclosporine toxicity, associated with a fall to within 10% of the baseline creatinine level after treatment with anti-rejection therapy. Diagnosis was confirmed by allograft biopsy in 18 recipients.
Genotyping of TNF-α and MCP-1 genes Genomic DNA was amplified by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) to detect genotypes.
DNA extraction and thermal cycling An average of 6 μg genomic DNA was extracted using the QIAamp DNA blood mini kit according to the manufacturer's instructions (Qiagen, Hilden-Germany). Thermal cycling was performed for initial denaturation for 5 min at 94°C. This was followed by 35 amplification cycles of denaturation (1 min at 94°C), annealing (1 min at 60°C for TNF-α and 55°C for MCP-1) and extension (1 min at 72°C), followed by a final cycle of primer extension for 5 min at 72°C.
Primer sequences (Bio-Ligo; USA)
TNF-α forward: 5-AGG-CAA-TAG-GTT-TTG-AGG-GCC-AT-3'.
TNF-α reverse: 5-TCC-TCC-CTG-CTC-CGA-TTC-CG-3'. 
MCP-1 forward: 5-CCG-AGA-TGT-TCC-CAG-CAC-AG-3'.
MCP-1 reverse : 5 ' -CTG-CTT-TGC-TTG-TGC-CTC-TT-3'. 
The PCR product was digested by the NcoI restriction enzyme (Amersham-Bioscience, NJ, USA) for TNF-α, which recognizes the polymorphism in the promoter region that involves a guanidine to adenosine transition at position -308, and by the PvuII restriction enzyme (Amersham-Bioscience) for MCP-1, which recognizes the site of polymorphism in the distal 5' regulatory region that involves an adenosine to guanidine transition at position -2518, to detect the genotype. The final product was visualized by ethidium bromide staining after separation on 2% agarose gel.
Serum TNF-α and MCP-1 levels
Three serum samples were collected from the kidney transplant recipients at Days 0, 15 and 30 post-transplantation to measure the pattern of serum levels of TNF-α and MCP-1 with the enzyme-linked immunosorbeny assay technique using the immunoassay kit from Biosource International Inc, CA, USA.
| Statistical Analysis|| |
Statistical analyses were performed using the SPSS version 11 software, using the t-test for numerical data and the chi-square test for categorical data. Measured variables were expressed as mean ± SE. A P-value <0.05 was considered statistically significant and a P-value <0.01 was considered highly significant.
| Results|| |
The demographic and clinical data of the study subjects are summarized in [Table 1].
Distribution of TNF-α genotypes [Figure 1]
Among the 84 renal transplant recipients, the homozygous TNF 1 allele (TNF 1 /TNF 1 ) genotype was detected in 60/84 recipients, representing 71.4% of the whole group, whereas the homozygous TNF 2 allele (TNF 2 /TNF 2 ) genotype was present in 24/84 recipients, representing 28.6% of the whole group. The heterozygous TNF 1 /TNF 2 genotype was not detected in any of these recipients. In patients with stable graft function (group I), the TNF 1 /TNF 1 genotype was the most common (80.8%) genotype, followed by the TNF 2 /TNF 2 (19.2%) genotype. In patients with acute kidney allograft rejection (group II), the distribution of TNF-ot genotype was as follows: The TNF1/TNF1 genotype was found in 22 patients (59.4%) and the TNF 2 /TNF 2 genotype was found in 15 patients (40.5%). Among patients with acute kidney allograft rejection, a significant prevalence of the TNF 2 /TNF 2 genotype was observed (P <0.05) [Table 2].
|Table 2: Distribution of TNF-α and MCP-1 genotypes in renal transplant recipients.|
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Distribution of MCP-1 genotypes [Figure 2]
The homozygous MCP-1 A/A genotype was detected in 62/84 of the recipients, representing 73.8% of the whole group. The homozygous MCP-1 G/G genotype was found in 22/84 of the recipients, representing 26.2% of the whole group. The heterozygous A/G genotype was not detected in any of these recipients. In patients with stable graft function (group I), the A/A genotype was detected in 36 patients (76.6%) whereas the G/G genotype was detected in 11 patients (23.4%). In patients with acute kidney allograft rejection (group II), the genotype frequency of the MCP-1 was as follows: 26 (70.2%) patients had the A/A genotype, while 11 (29.7%) patients had the G/G genotype. No significant differences were observed when distributions of the (MCP-1) genotypes were compared between the patients with and without acute rejection episodes (P >0.05) [Table 2].
Serum TNF-α levels in the studied groups
Normal TNF-α levels were observed over time in the serum samples of patients in group I on Days 0, 15 and 30 post-transplant (mean ± SE; 11.4 ± 1.57, 10.9 ± 1.43 and 10.3 ± 1.16, respectively), while higher levels were observed in group II (mean ± SE; 18.5 ± 1.55, 23.0 ± 2.67 and 24.7 ± 1.79, respectively), and these differences between the groups were statistically significant [Table 3].
Among group II recipients, there was a highly significant elevation of serum TNF-α levels in the TNF-2 (-308 A/A) genotype (15 recipients) compared with those of the TNF-1 (-308 G/G) genotype (P <0.01), and this significance continued overtime [Table 4].
Serum MCP-1 levels in the studied groups
No differences were observed in MCP-1 levels over time in group I on the Days 0, 15 and 30 post-transplant serum samples (mean ± SE; 151.2 ± 30.4, 152.2 ± 30.27 and 154.7 ± 30.41, respectively), while slightly higher levels were observed in group II (mean ± SE; 155.8 ± 38.85, 152.0 ± 36.72 and 162.6 ± 34.24, respectively), with no statistically significant differences between the groups [Table 3].
Among group II recipients, there were increased levels of serum MCP-1 expressed by carriers of the G allele of the -2518 polymorphism (MCP-1 G/G genotype) than the MCP-1 A/A genotype serum MCP-1 level, but this elevation was not statistically significant [Table 5].
| Discussion|| |
The majority of cases of acute rejection occur in the first few weeks post-transplantation,  and typing of some genetic determinants may help in identifying factors predisposing for the same.  The positive associations found between the polymorphism and the adverse clinical events suggest that they may be utilized in the identification of kidney allograft recipients predisposed for acute rejection episodes and thus potentially benefiting from individually tailored immunosuppression. 
In our study, the TNF 2 /TNF 2 genotype was present in 19.2% of the stable patients and in 40.5% of those with acute rejection. Similar findings were previously reported by other investigators.  However, other studies have reported that the distribution of TNF-ot genotypes in patients with acute kidney allograft rejection was TNFi/TNFi genotype 56% patients, TNFJ/TNF2 40% patients and TNF 2 / TNF 2 4% patients.  There are several factors that may explain the discrepancies in genotype distribution among the different studies. Some of the studies encompass patient's ethnic background, in which the risk factors may be different, with uniform genetic groups being relatively small in number.  Furthermore, in some studies, the genotype distribution and the frequencies of the gene polymorphisms in recipient populations were not reported, making comparison of results between investigators difficult. 
Our study revealed a highly significant prevalence of the TNF 2 /TNF 2 genotype among patients with acute kidney allograft rejection. This was attributed to the elevated serum levels of TNF-α in recipients with the AA allele (TNF 2 genotype) compared with that of the GG allele TNF] genotype. Other studies had similar conclusions.  " 
In our study, no significant MCP-1 polymorphism differences were observed when the distributions of-2518 MCP-1 genotypes in patients with and without acute rejection episodes were compared. The gene frequency of MCP-1 (-2518 G allele) was 29.7% for the acute rejection group and 23.4% for the control group. There is some controversy about the role of this chemokine in renal allograft rejection. There is some evidence that supports the influence of the recipient -2518 MCP-1 gene polymorphism on the outcome of renal human allograft.  This difference could be explained by the fact that the G/A polymorphism demonstrates racial heterogeneity. The G allele frequency is increased in Asian and Mexican populations compared with Caucasian and African American populations.  However, the present study is similar to the result reported by Kruger et al,  who did not find an association between (-2518 A/G) MCP-1 gene polymorphism and the incidence of acute renal allograft rejection. Also, they showed that MCP-1 had a primary role in the development of transplant vasculopathy, while playing a secondary role in acute rejection. Marshall et al  suggested a similar statement. On the other hand, a study carried out by Grandaliano et al  on 20 kidney transplant recipients with acute graft dysfunction reported that in patients with acute tubular damage, the MCP-1 expression was significantly higher than in controls. Moreover, Kruger et al  found that renal allograft recipients homozygous for MCP-1 (-2518G allele) experience a significantly shor-tened graft survival without an association with the incidence of acute rejection.
In our study, the MCP-1 G/G genotype carriers showed elevated levels of serum MCP-1 compared with the MCP-1 A/A genotype carriers, but this elevation was statistically not significant probably due to the small number of recipients carrying the G/G genotype. Another explanation is that the elevated levels of MCP-1 from G carriers, which was observed in some studies, was induced by interleukin (IL)-1β.  Therefore, monocytes from individuals carrying the G allele at -2518 produce more MCP-1 after treatment with IL-1β than monocytes from A/A homozygous subjects. The effect of the G allele appears to be dose dependent as cells from individuals homozygous for G at -2518 produced more MCP-1 than cells from G/A heterozygotes. 
In conclusion, in this study, the TNF 2 /TNF 2 genotype shows a high prevalence in recipients with acute graft rejection, and TNF-α gene polymorphism may identify patients at risk of rejection episodes before kidney transplantation, defining a subgroup of patients who will take advantage of a better HLA-matched organ allocation. After transplantation, knowledge of this genotype may permit better monitoring of patients at a greater risk of rejection. Serial assays of serum TNF-α and serum creatinine have a greater predictive value than either test alone for the early diagnosis of acute rejection. No association was found between either MCP-1 gene polymorphism or its serum levels and acute rejection.
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Eman Nasr Eldin
Department of Clinical Pathology, Assiut University Hospital, Assiut
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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