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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2011  |  Volume : 22  |  Issue : 1  |  Page : 18-23
Allograft renal rejection and chemokine polymorphism

1 Research Laboratory of Transplantation Immunopathology (LR03SP01), Charles Nicolle Hospital, Tunis, Tunisia
2 Department of Nephrology, Charles Nicolle Hospital, Tunis, Tunisia

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Date of Web Publication30-Dec-2010


Chemokines play a major role in the process by which leukocytes are recruited from the bloodstream into the sites of inflammation. Genes for the chemokine receptors CCR5, CCR2 and MCP-1 are characterized by functional polymorphisms implicated in transplant rejection. To investigate this association, we analyzed polymorphisms of CCR5-∆32, CCR5-59029-A/G, CCR2-V64I and MCP-1 G/A (-2518) in 173 renal transplant recipients and 169 healthy blood donors. The patients were classified in two groups: Group-1 (G-1) included 33 HLA-identical recipients and Group-2 (G-2) included 140 (one or more) mismatched graft recipients. Forty-two patients had developed acute rejection episodes (ARs): seven in G-1 and 35 in G-2. Thirteen G-2 patients developed chronic allograft dysfunction (CAD). The genotypic and allelic frequencies of all polymorphisms studied did not reveal significant differences between patients and controls and among G-1 and G-2 recipients. However, a significant risk of acute renal transplant rejection was found in G-1 patients who possessed the CCR2-64I allele (odds ratio 0.24, 95% confidence inter­val [CI], 0.05-1.06; P = 0.035). There was no significant association of this polymorphism and CAD. In conclusion, the observed association of CCR2-64I with AR should be added to the spectrum of immunogenetic factors known to be involved in allograft renal loss.

How to cite this article:
Gorgi Y, Sfar I, Jendoubi-Ayed S, Makhlouf M, Rhomdhane T B, Bardi R, Aouadi H, Abdallah T B, Abderrahim E, Ayed K. Allograft renal rejection and chemokine polymorphism. Saudi J Kidney Dis Transpl 2011;22:18-23

How to cite this URL:
Gorgi Y, Sfar I, Jendoubi-Ayed S, Makhlouf M, Rhomdhane T B, Bardi R, Aouadi H, Abdallah T B, Abderrahim E, Ayed K. Allograft renal rejection and chemokine polymorphism. Saudi J Kidney Dis Transpl [serial online] 2011 [cited 2021 May 9];22:18-23. Available from: https://www.sjkdt.org/text.asp?2011/22/1/18/74336

   Introduction Top

Genetic polymorphisms, other than those at the HLA locus, are excellent parameters that might explain the clinical heterogeneity in out­come between kidney recipients. Several gene­tic variations have been identified in a number of genes encoding molecules that participate in the recipient's immune response to a renal allo­graft. [1],[2] Among these molecules, chemokines and their respective receptors have been impli­cated in renal transplant rejection. During acute allograft rejection, monocytes and T­effector cells are directed into the transplant kidney and produce a characteristic tubular or vascular infiltrate. [3] The influx of leukocyte sub­sets into the site of tissue injury appears to be mediated by the expression of specific chemo­kines and chemokine receptors. [4] Specifically, the expression of the CC-chemokine (MCP-I) with the corresponding chemokine receptor (CCR2) can be detected in mononuclear cells infiltrating the kidney graft. [5] Furthermore, non­functional CCR5, due to a 32-bp deletion in the CCR5 gene (∆32 mutation), influences the renal allograft survival significantly. [6]

In this study, we analyzed the association of the CCR5-∆32, CCR5-59029-A/G, CCR2-V64I and MCP-I-2518 G/A variants with suscepti­bility of renal allograft Tunisian recipients to acute rejection episodes.

   Materials and Methods Top

Patients and Controls

The study included DNA samples from 169 sex-matched healthy controls and 173 renal transplant recipients (108 males and 65 fe­males; mean age 31 ± 10.61 years). The allo­graft donors were living donors in 139 cases (134 related and five unrelated donors) and de­ceased donors in 34 cases. Among the 173 re­nal recipients, 33 patients received HLA iden­tical kidney transplant (G-1) and 140 received one or more mismatched grafts (G-2). Forty­two patients (24.27%) had developed acute rejection (AR) episodes within the first six months post-transplantation: seven in Group-1 (21.2%) and 35 in Group-2 (25%). Among the 42 cases of AR, 37 recipients developed only one episode (6/7 in G-1 and 31/35 in G-2) and five patients developed two AR episodes (one case in G-1 and four in G-2). The diagnosis of AR was made by clinical, histological and bio­chemical standard assessment (Banff criteria). Thirteen G-2 patients developed chronic allo­graft dysfunction (CAD).

The study was approved by the local ethics committee and informed consent was obtained from all subjects.

   Methods Top

DNA Extraction

Genomic DNA was isolated from EDTA-anti­coagulated peripheral blood samples of unre­lated healthy blood donors and renal recipients and was extracted by a standard salting-out procedure.

Determination of CCR5-∆32

The CCR5-∆35 genotype was determined by sizing PCR amplicons that include the entire region, as previously published. [7] The 15 ul PCR contained 50 ng DNA, 5 pmol of each primers, 0.5U of Taq polymerase (Promega Corpora­tion, Madison, WI, USA), 1.5 mM MgCl2 and 0.175 mM of each deoxynucleotide triphos­phates (dNTPs). The following primers were used: forward (5'-TgTTTgCgTCTCTCCCAg­3') and reverse (5'-CACAgCCCTgTgCCTCTT­3'). Thermo cycling was performed with an initial denaturation at 94°C for four mins fol­lowed by 35 cycles at 94°C for 30 seconds, 60°C for 45 seconds and 72°C for one min and a final extension at 72°C for seven min. Am­plicons were visualized by ultraviolet in 4% agarose gel with ethidium bromide, which re­sulted in a 233-bp product for the wild-type amplicon (CCR5 +/+) and 201-bp for the dele­tion product (CCR5-∆32).

Determination of CCR5-59029 G/A genes genotype

Genotyping was performed using the restric­tion fragment length polymorphism-polyme­rase chain reaction (RFLP-PCR) method. [7] The forward primer, 5'-CCCgTgAgCCCATAgTT­AAAACTC-3' and the reverse primer 5'­TACCAgggCTTTTCAACAgTAAgg-3' were used with PCR conditions identical to those used for CCR5-∆32, except for an annealing temperature of 66°C. A total of 5 gL of PCR product was digested with three units of Bsp­12861 (Promega). The presence of the G nuc­leotide at position 59029 of the CCR5 gene creates a recognition site for the Bsp12861 enzyme at 37°C overnight. Cut amplicons from homozygous patients for 59029 G appear as a single, 130-bp band in agarose gel electropho­resis at 2%, homozygous for 59029 A appear as a 258 bp band and heterozygous have both bands.

Determination of MCP-1 A/G promoter geno­type

The identification of this polymorphism was carried out using PCR-RFLP assay as des­cribed by Steinmetz OM et al. [8] The regulatory region of the MCP-1 gene (-1817 to -2746) was amplified by PCR, resulting in 930 bp fragment. Genomic DNA: 100 ng were added to 20 gL of amplification buffer containing 2 mM of MgCl2, 0.2 mM of dNTPs, 5 pmol of each primers, and 0.5U of Taq polymerase (Promega). Primers used were: forward 5'­CCgAgATgTTCCCAgCACAg-3' and reverse 5'-CTgCTTTgCTTgTgCCTCTT-3'. PCR was run for 40 cycles using the following tem­perature profile: denaturation at 94°C for 60 seconds, annealing at 60°C for 60 seconds, ex­tension at 72°C for one min 30 seconds, fol­lowed by a single final extension at 72°C for 10 min. Ten microliters of the PCR products were digested with five units of PvuII (Pro­mega) in 10x buffer and H2O up to a final vo­lume of 20 gL at 37°C overnight. The resul­ting products were separated by gel electropho­ resis in 1.5% agarose gel containing ethidium bromide. Samples showing only a 930 bp band were assigned as A/A, samples showing two bands of 708 and 222 bp were considered G/G and samples showing three bands at 930, 708 and 222 bp were typed A/G.

Determination of CCR2-V64I genotype

The CCR2 wild-type (CCR2 +/+) and mutant (CCR2-64I) alleles were typed by polymerase chain reaction using sequence-specific primers (PCR-SSP) as previously described. [9]

The primer sequences were 5'-gTgggCAA­CATgCTggTCA-3' primer for the wild-type allele, 5'-gTgggCAACATgCTggTCG-3' primer for the mutant allele and 5'-CCCAAAgACC­CACTC-ATTTg-3' for the common primer. For characterization of the CCR2 polymor­phism, two amplification reactions were used: the first with specific primers for the wild-type allele sequence and the second with specific primers for the sequence of the mutant allele. In the case of homozygous wild-type indivi­dual, the product (173 bp band) was observed only in the first reaction; in the case of homo­zygous mutant individual, the product was de­tected only in the second reaction; and in the case of heterozygous individual, the products were detected in both reactions. Genomic DNA: 100 ng was added to 15 tL of amplification buffer containing 2 mM of MgCl2, 0.2 mM of dNTPs, 5 pmol of each primers and 0.25U of Taq polymerase (Promega). We performed the PCR amplification sequentially in four steps: a pre-heating step to activate the Taq polyme­rase and to denature the DNA at 96°C for 60 seconds, an initial five cycles (96°C 25 seconds, 70°C 45 seconds, 72°C 45 seconds) followed by 21 cycles (96°C 25 seconds, 65°C 50 se­conds, 72°C 45 seconds) and a final four cycles (96°C 25 seconds, 55°C 60 seconds, 72°C 2 min). We loaded the PCR products directly on a 2% agarose gel containing 0.5 μg/mL ethi­dium bromide and carried out the electropho­resis.

   Statistical Analysis Top

Allelic and genotypic frequencies were eva­luated by direct counting. Statistical compa­risons were performed between patients and controls by the K 2 test calculated on 2 × 2 con­tingency tables using the Statcalc program (Epi Info version 20010; Centers for Disease Control and Prevention, Atlanta, GA, USA). Fisher exact test was used when an expected cell value was less than five. P-value <0.05 was considered to be statistically significant. Odds ratio (OR), with 95% confidence intervals (95% CI), was calculated using the same soft­ware.

   Results Top

The allelic frequencies of CCR5-∆32, CCR5­59029 (A/G) and MCP-1 (G/A) among pa­tients and controls did not reveal significant differences [Table 1]. However, the CCR2-64I allele was more frequent in G-1 than in G-2 patients: 0.212 versus 0.145 and 0.139 respec­tively, but the difference was not significant.
Table 1: Genotype and allele frequencies of chemokines and their receptors studied in controls and patients.

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Furthermore, a significant risk of acute renal transplant rejection was found in G-1 patients who possessed the CCR2-64I allele [Table 2] (OR 0.24, 95% CI 0.05-1.06; P = 0.035), but this allele was not associated with the number of acute rejection episodes. In G-2, there was no significant association between this poly­morphism and the 13 cases with chronic allo­graft dysfunction.
Table 2: Genotype and allele frequencies of CCR2 gene polymorphism in G-1 patients with acute rejection.

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The allelic distribution of the CC-chemokine MCP-1-2518G in combination with the corres­ponding chemokine receptor CCR2-64I did not differ in patients compared with controls. The G-1 recipients with this allelic association had a higher risk of acute rejection [Figure 1], but the difference was not significant (OR 4.13, 95% CI 0.78-21.77; P = 0.06). On the other hand, acute rejection was significantly more frequent among G-1 patients possessing the CCR2-64I allele in association with the CCR5-59029A promoter allele (OR 18, 95% CI 1.63-464.12; P = 0.005).
Figure 1: Effect of allelic association of chemokine gene polymorphisms on acute rejection in G-1 patients.
CCR2-64I /CCR5-59029A (*) was statistically significant.

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   Discussion Top

By regulating the recruiting of lymphocytes and monocytes from the bloodstream into the sites of inflammation, chemokines and chemo­kine receptors seem to be one of the reasons causing acute allograft rejections in renal trans­plantation. Several functional polymorphisms of these molecules have been described and their association with the susceptibility to acute kidney rejection was reported. [5],[7],[10]

A 32 bp deletion in the CCR5 gene (CCR5­∆32) results in a non-functional surface receptor molecule that is unable to bind its chemokine ligands Rantes, MIP-1α and MIPβ. Fischereder et al [6] reported that homozygous patients for CCR5-∆32 had significantly longer renal trans­plant survival times than CCR5 heterozygous and CCR5 +/+ patients. We cannot address this finding because we did not observe this genotype in our study. Moreover, the CCR5-∆32 heterozygous genotype, which causes less cell surface expression of CCR5, [7] had no sig­nificant association with acute rejection in this study.

No significant differences were observed when patients with or without acute rejection epi­sodes were compared for the distribution of CCR5-59029 genotypes. Our data contrast with the study of Yigit et al who found a signi­ficant difference between the groups with and without acute renal rejection in the A and G allele distribution in both CCR2-V64I and CR5-59029 gene variants. [11]

In contrast to previous results of Kruger et al [12] who found that recipients of renal trans­plants homozygous for the -2518 G mutation of the MCP-1 gene are at risk for premature kidney graft failure, the present study supports the lack of involvement of polymorphism at position-2518 (A/G) of the MCP-1 gene on the susceptibility to acute rejection among renal recipients.

Because of the low number of patients homo­zygous for CCR2-64I, only the effects of its heterozygous variants were addressed in this study. In G-1 patients, the CCR2-64I allele fre­quency was significantly higher in recipients with acute rejection (0.428) compared with those without this complication (0.153) (P = 0.035).

Moreover, recipients with the CCR2-64I allele in combination with the CCR5-59029 A allele had a significantly higher risk of acute re­jection (P = 0.005). A linkage disequilibrium between the two alleles suggested by Hizawa et al [13] may be responsible for this significant association.

Our data revealed an association between acute rejection and CCR2-64I/MCP-1G. In ac­cordance with the results of Kang et al, [14] we hypothesize that the CCR2 dimorphism con­sisting of valine/isoleucine amino acid substitution at position 64 of this chemokine recep­tor, seems to result in significant conforma­tional changes in the structure of the protein. Therefore, the complex of chemokine (MCP­1G) and its receptor (CCR2-64I) may promote the migration of monocytes into the trans­planted kidney.

In conclusion, Tunisian recipients of renal transplantation with the CCR2-64I allele are at increased risk for acute rejection. This variant may be added to the spectrum of immunoge­netic factors known to be involved in allograft renal loss.

   References Top

1.Akalin E, Murphy B. Gene polymorphisme and transplantation. Curr Opin Immunol 2001; 13:572-6.  Back to cited text no. 1
2.Dickinson AM, Middleton PG. Beyond the HLA typing age: genetic polymorphisms pre­dicting transplant outcome. Blood Rev 2005; 19:333-40.  Back to cited text no. 2
3.Lalor PF, Adams DH. Lymphocyte homing to allografts. Transplantation 2000;70:1131-9.  Back to cited text no. 3
4.Nelson PJ, Krensky AM. Chemokines, chemo­kine receptors, and allograft rejection. Immunity 2001;14:377-86.  Back to cited text no. 4
5.Segerer S, Cui Y, Eitner F, et al. Expression of chemokines and chemokine receptors during human renal transplant rejection. Am J Kidney Dis 2001;37:518-31.  Back to cited text no. 5
6.Fischereder M, Luckow B, Hocher B, et al. CC chemokine receptor 5 and renal-transplant survival. Lancet 2001;357:1758-61.  Back to cited text no. 6
7.Abdi R, Huong TT, Sahagun-Ruiz A, et al. Chemokine receptor polymorphism and risk of acute rejection in human renal transplantation. J Am Soc Nephrol 2002;13:754-8.  Back to cited text no. 7
8.Steinmetz OM, Panzer U, Harendza S, et al. No association of the -2518 MCP-1 A/G pro­moter polymorphism with incidence and cli­nical course of IgA nephropathy. Nephrol Dial Transplant 2004;19:596-601.  Back to cited text no. 8
9.Petrek M, Drabek J, Kolek V, et al. CC che­mokine receptor gene polymorphisms in Czech patients with pulmonary sarcoidosis. Am J Respir Crit Care Med 2000;162:1000-3.  Back to cited text no. 9
10.Hancock WW, Gao W, Faia KL, Csizmadia V. Chemokines and their receptors in allagraft rejection. Curr Opin Immunol 2000;12:511-6.  Back to cited text no. 10
11.Yigit B, Bozkurt N, Berber I, Titiz I, Isbir T. Analysis of CC chemokine receptor 5 and 2 polymorphisms and renal transplant survival. Cell Biochem Funct 2007;25:423-6.  Back to cited text no. 11
12.Kruger B, Schroppel B, Ashkan R, et al. A monocyte chemoattractant protein-1 (MCP-1) polymorphism and outcome after renal trans­plantation. J Am Soc Nephrol 2002;13:2585-9.  Back to cited text no. 12
13.Hizawa N, Yamaguchi E, Furuya K, Jinushi E, Ito A, Kawakari Y. The role of the C-C che­mokine receptor 2 gene polymorphism V64I (CCR2-V64I) in sarcoidosis in a Japanese population. Am J Respir 1999;159:2021-3.  Back to cited text no. 13
14.Kang SW, Park SJ, Kim YH, et al. Association of MCP-1 and CCR2 polymorphisms with the risk of late acute rejection after renal trans­plantation in Korean patients. Int J Immuno­genet 2007;35:25-31.  Back to cited text no. 14

Correspondence Address:
Y Gorgi
Immunology Lab, Charles Nicolle Hospital, Bd 9 Avril, 1006 Tunis
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PMID: 21196609

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  [Figure 1]

  [Table 1], [Table 2]

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