Transplantation of renal allografts significantly improves the survival of individuals with end-stage renal disease (ESRD). However, the duration of allograft survival is not uniform across recipient ethnic groups. In the United States, the five-year allograft survival of white recipients is 66.2% compared with a more abbreviated duration of functioning allografts for black recipients of 54.4%1. Several nonimmunologic reasons have been proposed to explain the foreshortened allograft survival of black recipients2. Studies that have explored allelic variants of genes that might determine the differential rate of allograft function associated with recipient ethnicity have been limited to antigens associated with the human leukocyte antigen (HLA) system3 and chemokine receptors such as CCR2 and CCR54,5.
Chemokines are cellular attractants that have defined roles in directing movements of neutrophils and cells necessary for the initiation of the T-cell response. Chemokines attract monocytes, effector T cells, and immature dendritic cells to sites of inflammation6, facilitating the interaction of T cells and antigen-presenting cells, promoting allorecognition that can lead to rejection6,7. Further, chemokines attract neutrophils to sites of ischemia and contribute to alloantigen-independent injury to transplanted organs8. The potential roles of chemokines in allograft function are illustrated by antibody blockade or targeted deletion of particular chemokines in rodents, resulting in reduced inflammatory, vascular, and interstitial changes in allografts6,7,8.
The normal functioning of chemokines requires binding to their respective receptors. However, binding of CC chemokines [regulated on activation normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1)] and CXC chemokines [interleukin-8, melanoma growth-stimulatory factor (MGSA)] to the Duffy antigen receptor complex (DARC) does not result in signal transduction9,10. The absence of DARC expression in mouse knock-out models leads to more intense inflammatory infiltrates in organs after exposure to lipopolysaccharide11,12. These and other findings support the hypothesis that DARC binds to chemokines, limiting systemic inflammation by reducing circulating levels of chemokines.
The DARC antigens are a clinically significant blood group system expressed on erythrocyte membranes, capable of producing hemolytic disease of the newborn and hemolytic transfusion reactions. The DARC antigens, Fy(a) and Fy(b), are codominantly expressed, and the alleles, FyA and FyB, are encoded on chromosome 1. FyA and FyB differ by a single base substitution at nucleotide position 1205, which results in the substitution of aspartic acid for glycine at residue position 4413. The Fy(a- b-) phenotype is the result of a silent FyB allele that arises through the substitution of thymine to cytosine at nucleotide 535. This single nucleotide polymorphism (SNP) disrupts transcription of the gene14. There is extensive linkage disequilibrium between the SNPs at nucleotide positions 535 and 1205, such that the FyB allele resulting from thymine substitution at nucleotide 1205 is always present with the SNP at nucleotide 535 that produces the FyB Null allele as determined by restriction fragment length polymorphisms13. The distribution of DARC antigens varies in different ethnic groups because the null phenotype, Fy (a- b-), is more common in African populations than Caucasians15. Duffy serves as a receptor for Plasmodium vivax, and its absence is associated with increased resistance to infection and may confer a survival advantage where malaria is endemic.
We hypothesize that absence of DARC expression contributes, in part, to the heightened immune response to renal allografts and foreshortened allograft survival observed among African American recipients. In addition, the absence of DARC expression might increase the response of the innate immune system that does not require recognition of foreign antigens to damage allografts, resulting in delayed allograft function (DGF). The objective of this investigation was to examine the potential association of allelic variants that alter the expression of DARC on erythrocytes with the susceptibility of African American recipients to DGF and acute rejection.
METHODS
Patients
A prospective cohort study enrolled recipients of cadaveric renal allografts transplanted during 1997 to 2002 from eight transplant centers in eastern Pennsylvania within the Gift of Life Donor Program (Hospital of the University of Pennsylvania, Thomas Jefferson University Hospital, Hahneman University Hospital, Albert Einstein Medical Center, Lankenau Hospital, Hershey Medical Center, Geisinger Medical Center, and Lehigh Valley Hospital). The unique distribution of DARC alleles associated with ethnicity creates population stratification, which may produce spurious associations of DARC and renal allograft function16,17. As a strategy to minimize ethnic admixture (i.e., confounding by ethnicity), as well as to increase the proportion of subjects with the FyB Null allele to improve the precision of the estimates of associations, this present investigation was limited to African American recipients. Patients were consented for participation at the time of transplantation or at a subsequent outpatient visit at the local transplant clinic. This protocol was approved by the Institutional Review Boards at each of the participating centers.
Data
Whole blood that remained from routine clinical testing was used as the source of recipient DNA. Data related to donor and recipient characteristics at the time of transplantation (e.g., ethnicity, gender, age) were collected in a prospective manner from patient interviews. Post-transplantation characteristics were prospectively abstracted from inpatient and outpatient medical records at six-month intervals. The first primary outcome for this analysis was a presumed episode of acute rejection within the first year post-transplant, defined by the initial use of antibody therapy (OKT3, thymoglobulin, antithymocyte globulin, or antilymphocyte globulin) or two or more doses of intravenous methylprednisolone after the first 72 hours post-transplant. The second primary outcome, delayed allograft function (DGF), was the requirement for dialytic therapy of any duration within the first week post-transplant.
Genotyping
DNA was extracted from whole blood specimens or from buffy coats using Qiagen blood DNA extraction kits (Valencia, CA, USA). Alleles of DARC were determined by allele-specific polymerase chain reaction (ASPCR) Four separate genotyping reactions were performed on each genomic DNA sample using the primers described by Hessner et al18. Two microliters (
L) of sample DNA (50 ng/L) were added to a mixture of 2 L of 10
(AmpliTaq; Roche Applied Science, Indianapolis, IN, USA), 0.2 L of Tag gold (5 U/L), 0.4 L of forward and reverse primers (20 mol/L), 0.2 L of 20 micromolar (mol/L) forward and reverse human growth hormone primers, 0.4 L of 10 mmol/L dNTPs, in 10 mmol/L Tris-HCL, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, in a total reaction volume of 20 L. Samples were amplified on a Peltier type thermal cycler using the following program: for FyA primers-94°C for 10 minutes, 35 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute; for FyB primers-94°C for 10 minutes, 35 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 2 minutes. All amplified samples were subjected to a final extension step at 72°C for 10 minutes, and then stored at 4°C until electrophoresis.
Statistical analysis
Descriptive analysis
The analyses of this investigation focused on the SNP at position 535 because the functionality of the DARC gene related to chemokine binding is associated with the allele at this locus and the high level of linkage disequilibrium17 between this locus and the locus at nucleotide 1235. The distributions of the alleles and genotypes among the entire cohort were examined for the presence of Hardy-Weinberg equilibrium (HWE)19. Recipients were divided into two groups based on the genotype: individuals who were homozygous for the FyB Null allele, and individuals with other genotypes. Continuous variables were compared by nonparametric testing. Frequencies of categorical variables were compared by chi-square testing.
Quantitative analysis
Cox proportional hazards models were fit to examine the independent association of the number of FyB Null alleles, or the FyB Null genotype of DARC with the time to the first episode of acute rejection within the first year post-transplant. Multivariable models were fit with the factors that had unadjusted associations (P < 0.10) with DARC genotypes. Logistic regression analysis was utilized to examine the associations of the FyB Null genotype and the number of FyB Null alleles with the odds of the development of DGF. The same algorithmic strategy as implemented for acute rejection was used to develop a multivariable model. All analyses were performed using SAS 8.01 (Cary, NC, USA). P values were two-sided.
RESULTS
DARC alleles and genotype frequencies
A total of 222 African Americans received a cadaveric renal allograft during the study. The frequencies of alleles and genotypes observed in the study population did not differ significantly from what was expected under Hardy-Weinberg equilibrium (P value = 0.28) Table 1.
The examination of the association of the DARC gene with the time to acute rejection, as well as the development of DGF, entailed the exclusion of six recipients because of primary nonfunction (allograft nephrectomy) of the allograft, and of 35 recipients who had no available follow-up data. Among this final group of 181 subjects, the most common genotype was homozygosity for the FyB Null allele (69.6%), and FyB Null allele was the most frequent (76.7%), consistent with previous studies that have reported a predominance of the FyB Null allele among non-Caucasians11. The data for the remaining 181 subjects remained consistent with HWE for this locus (P = 0.20).
DARC and baseline characteristics
The baseline characteristics of the cohort according to the FyB Null genotype of the recipient are presented in Table 2. The causes of end-stage renal disease (ESRD) are not mutually exclusive, and hypertension is most frequent in the cohort as anticipated from the ethnicity of the recipients. There are no factors that have a statistically significant association with the genotypes. There is an indication of a possible association between FyB Null genotype and levels of panel reactive antibodies (PRA) (P = 0.09).
DARC genotype and episodes of acute rejection
At least one episode of acute rejection occurred among 16.0% of the cohort during the first year post-transplant. The Kaplan-Meier curves in Figures 1a and b present the rejection-free survival according to the number of FyB Null alleles and to the FyB Null genotype, respectively. There were no detectable unadjusted associations of acute rejection with the number of alleles or these genotypes (P = 0.66 and P = 0.83 by log rank tests, respectively) Figure 1a and b. As well, there were no detectable unadjusted associations in Cox models between the number of FyB Null alleles (P = 0.83) or with the FyB Null genotype (P = 0.59) and the rate of acute rejection Table 3. The absence of detectable associations between FyB Null genotype or the number of FyB Null alleles with acute rejection persisted after adjustment for most recent PRA Table 4. Adjusting for the levels of HLA mismatching, type of calcineurin inhibitor at the time of transplantation, and the use of antibody induction therapy, previously described factors that have associations with acute rejection, by forcing these variables into the model did not impact the observations.
Figure 1.
The rate of acute rejection within the first year post-transplant according to the number of FyB Null alleles (A). The rate of acute rejection within the first year post-transplant according to the FyB Null homozygosity or other genotype (B).
Full figure and legend (24K)DARC genotype and delayed allograft function
The incidence of DGF was 42.5%. In an unadjusted model, as well as after adjustment for most recent PRA, there was no apparent trend in the association between the odds of DGF and FyB Null genotype or the number of FyB Null alleles Tables 5 and 6. Consideration of the duration of cold ischemia time in the adjusted model did not impact the observations.
DISCUSSION
The present study examined the relationship between allelic variants of the DARC gene and acute rejection and DGF among African American recipients or cadaveric renal allografts. Homozygosity for the FyB Null allele was the most prevalent genotype and the FyB Null allele was most frequent, consistent with previous investigations. However, an association of the allelic variants of DARC within this group of recipients with the rate of acute rejection and the odds of DGF was not detected.
DARC binds to various types of chemokines and prevents the binding of chemokines to receptors, impairing an inflammatory response. Selective pressure by nature has created the prevalence of an allele that prevents the expression of DARC on erythrocytes among African Americans that might lead to heightened activity of inflammatory cells and plausibly increase the risk for events post-transplant that are detrimental to allograft survival. Immunocytochemistry studies confirm that DARC expression is not limited to erythrocytes and occurs in the endothelium of glomeruli, peritubular capillaries, vasa recta, and principal cells of collecting ducts in normal kidney tissue20,21. Several recent investigations revealed that the expression of DARC is unlikely to be constitutive within the kidney because the expression is up-regulated in renal allografts during episodes of acute rejection22,23. The present investigation utilized a DNA-based method to genotype for an allelic variant that has well-described functional implications on the transcription of the DARC gene. However, the alteration of the expression of the DARC genes associated with the allelic variant considered in this study is erythroid-specific; persons who are serologically negative for Duffy expression on erythrocytes can still express the protein in other tissues20.
A recent investigation enrolled 163 African American recipients of renal allografts from Emory University who were serologically typed for DARC. The majority (71.8%) of the study population was Duffy negative Fy (a- b-), and this phenotype did not have an association with allograft survival that attained statistical significance (P = 0.169). Further, no descriptive association was observed between episodes of acute rejection within six months post-transplant and the serologic Duffy type24. However, the conclusions from this investigation may have been limited by the accuracy of serologic methods to determine the absence of Duffy expression due to variation in strength of antibody reactions both within and between various anti-FyB reagents25,26, and the proportion of immature erythrocytes that continue to manufacture protein in peripheral blood between subjects27. The DNA-based method used here obviated these concerns and allowed for the opportunity of a more conclusive investigation. This study, in agreement with the previous serologic study, did not reveal an association between DARC genotype and AR and DGF. There are several possible interpretations for our findings. First, the level of erythrocyte expression of DARC may be unimportant in the immune response to an allograft. Second, a relationship could not be excluded between the genotypic expression of donor-related DARC and transplant outcomes. Third, the DARC gene in isolation does not determine the susceptibility to acute rejection or to DGF.
There are several limitations to this study. First, we cannot exclude with certainty that the DARC gene may have an association with acute rejection that is dependent upon products of other genes that have immune-related functions, such as interleukins 4 and 10, which were not considered in this study. Second, potential misclassification of acute rejection (phenotypic heterogeneity) would have led to an underestimation of effects because this observational study did not require all episodes of acute deterioration of renal function to be defined by a biopsy. Third, the severity of DGF represented by the duration of the need for dialysis post-transplant was not considered in this investigation, and we cannot eliminate the possibility that DARC impacts duration of DGF. Fourth, although genotyping error may result in misclassification of individuals and obscure true associations, this was not suggested by the presence of HWE. Finally, the magnitude of associations of the allelic variants of DARC may have not have been detectable with the sample size of this investigation.
CONCLUSION
The susceptibility of African American recipients to acute rejection or to DGF according to recipient-related variants of the DARC gene was not observed. Investigation of variants of this gene that are related to the expression of DARC within renal tissue (i.e., donor-related variants), should be considered in future investigations.
References
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Acknowledgments
We would like to express our appreciation to John Abrams at the Gift of Life Donor Organ Procurement Organization, to the transplant staff of the participating centers, and to Megan Wolfe and Sarah Jahn, for their technical assistance. Funding for this project was provided in part from NIAID to KCM (K23-AI001838) and HIF (R01-AI43295).
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