- Email: [email protected]
The Impact of ICAM1 and VCAM1 Gene Polymorphisms on Chronic Allograft Nephropathy and Transplanted Kidney Function
The Impact of ICAM1 and VCAM1 Gene Polymorphisms on Chronic Allograft Nephropathy and Transplanted Kidney Function ski, A. Pawlik, M. Wi K. Kłoda, L. Doman sniewska, K. Safranow, and K. Ciechanowski ABSTRACT ICAM-1 and VCAM-1 adhesion molecules play important roles in the immune response and emergence of chronic allograft nephropathy (CAN). The several polymorphisms of ICAM1 and VCAM1 genes are associated with changes in molecular expression therefore affecting allograft function and immune responses after kidney transplantation. The aim of this study was to examine the impact of polymorphisms in ICAM1 and VCAM1 genes on biopsy-proven CAN and renal allograft function. The 270 Caucasian renal transplant recipients (166 men and 104 women) were genotyped for the rs5498 ICAM1 and rs1041163 and rs3170794 VCAM1 gene polymorphisms using real-time polymerase chain reaction. There was no correlation between polymorphisms and CAN. Creatinine concentrations in the first month after transplantation differed between the rs5498 ICAM1 genotypes (P ¼ .095), being higher for GG carriers (AA þ AG vs GG, P ¼.07) albeit not with statistical significance. Creatinine concentrations at 12, 24, and 36 months after transplantation differed significantly among rs5498 ICAM1 genotypes (P ¼ .0046, P ¼.016, and P ¼ .02) and were higher among GG carriers (AA þ AG vs GG, P ¼ .001, P ¼ .004, and P ¼ .006). Rs5498 ICAM1 GG genotype and receipient male gender were independent factors associated with higher creatinine concentrations. These results suggest that the rs5498 ICAM1 GG genotype may be associated with long-term allograft function.
T
HE SUCCESS OF KIDNEY TRANSPLANTATION depends on many immunologic and genetic factors.1 All of these conditions affect transplanted kidney function, as well as determine the possibility of acute and chronic rejection. Clinical manifestations of the ischemia and reperfusion injury are delayed graft function, defined as the need for hemodialysis during the first 7 days after transplantation.2 Apart from the basic parameter of diuresis, the condition of the allograft can be evaluated by a number of laboratory and imaging tests.3 Decreasing transplanted kidney function is assessed by an allograft biopsy. Moreover, an allograft biopsy can be performed to monitor the status of a functioning kidney.4,5 ICAM-1 which is encoded by a gene located on 19th chromosome, locus 19p13.3-p13.2 shows constant expression on the surface of endothelial cells. Through interactions with LFA-1 molecules it initiates immune responses. Leukocytes activated upon adhesion transmigrate to surrounding tissues, liberating proteolytic enzymes and reactive oxygen species that are destructive to the endothelium of the transplanted kidney.6 VCAM-1 (which is encoded on the first chromosome, locus 1p3132) is expressed on endothelial and antigen-presenting cells.
The interactions of VCAM-1 and its receptor VLA-4 which is found on lymphocytes, macrophages and granulocytes initiate, T-cell responses to alloantigen in the early development of acute and chronic rejection.7 Anti-VCAM-1 and anti-VLA-4 antibodies synergistically block rejection of transplanted organs.8,9 The several polymorphisms among ICAM1 and VCAM1 genes are associated with altered expression of these molecules, which may affect allograft function and immune responses. The aim of this study was to examine the impact of polymorphisms in ICAM1 and VCAM1 genes on biopsy-proven chronic allograft nephropathy (CAN) and long-term renal allograft function. From the Clinical Department of Nephrology, Transplantology and Internal Medicine (K.K., L.D., M.W., K.C.), Department of Pharmacology (A.P.), and Department of Biochemistry and Medical Chemistry (K.S.), Pomeranian Medical University in Szczecin, Szczecin, Poland. ski, Clinical Address reprint requests to Leszek Doman Department of Nephrology, Transplantology and Internal Medicine, Pomeranian Medical University in Szczecin, Szczecin, Poland. E-mail: [email protected]
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.03.043
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2244
Transplantation Proceedings, 45, 2244e2247 (2013)
ICAM1 AND VCAM1 GENE POLYMORPHISMS
METHODS The study enrolled 270 Caucasian renal recipients including 166 men and 104 women of overall mean age 47.63 12.96 years who underwent transplantations between 2000 and 2006. The most frequent causes of renal failure were chronic glomerulonephritis (58%), hypertension (9%), and diabetes (9%). All donors were cadaveric sources. The mean number of matched HLA-A and HLA-B alleles was 1.5 0.8, and the mean number of matched HLA-DR alleles was 1.2 0.5. Blood samples were collected from all patients for genetic analysis and creatinine concentrations evaluation at 1, 3, 6, 12, 24, and 36 months after kidney transplantation. Creatinine concentrations were evaluated with the colorimetric method. CAN was diagnosed in 62 patients by biopsies performed with the PAJUNK DeltaCut biopsy system and scored by a renal pathologist using the Banff working classification. All patients received a calcineurin inhibitor (cyclosporine [75%] or tacrolimus [24%]), azathioprine (55%) or mycophenolate mofetil (37%), and steroids (91%). Our local ethics committee approved the study protocol, which was conducted in accordance with the Helsinki Declaration of 1975.
Genotyping Genomic DNA was extracted from leukocytes contained in 450 mL whole blood samples with ethylenediaminetetraacetic acid as an anticoagulant, using a nonorganic and nonenzymatic extraction method.10 DNA precipitated with 99.5% ethanol was dissolved in distilled water and standardized to 20 ng/mL using a Nanodrop ND1000 spectrophotometer. This material was the matrix for amplification in real-time polymerase chain reactions (PCR). We analyzed 3 single nucleotide polymorhisms (SNPs): rs5498:A > G in exon 6 of the ICAM1 gene, as well as rs3l70794:T > C and rs1041163:T > C in the VCAM1 gene promotor using TaqMan probes in a 7500 Fast Real-Time PCR System machine (Applied Biosystems, USA). The substrates for the reaction were: TaqMan Genotyping Master Mix (Applied Biosystems), 2 starters (900 mmol/L) and 2 TaqMan probes with 50 reporter pigment and 30 nonfluorescent quencher (200 mmol/L). Probes specific for the more frequent allele were marked with 6-FAM reporter pigment and for the less frequent allele, with VIC pigment. For the rs5498:A > G and rs3l70794:T > C SNPs analysis, 2 ready-made assays were used: No. C__8726337_40 and C__30900705_10. For the rs1041163 T > C SNP analysis, we performed a new assay with starter sequences: 50 -GAC CTC TGG GTT ACT TGT TTA TAA GCT-30 , 50 -GAGATGCTGTTTCTAGGGTGTGG-30 and probes: FAMdTAG GGA TCA GAA AAA TTG A, VICdTAG GGA TCA GAG AAA TTG A. The reaction were performed at 95 C for 10 minutes for AmpliTaq Gold polymerase activation, before 40 cycles of 15 seconds in 92 C and 60 seconds in 60 C. Patient genotypes were determined through comparison of 6-FAM and VIC pigment fluorescence. The results were verified by review of the amplification plots.
Statistical Analysis The distributions of genotypes and alleles among patients with CAN compared with patients free of this complication were evaluated statistically with the chi-square test using the Yates correction or Fisher exact test. The concentrations of creatinine and histopathologic changes were estimated with the Kruskal-Wallis or the Mann-Whitney test. The influences of genotypes and alleles on creatinine concentrations were evaluated with a general linear model (GLM). P values <.05 were considered to be statically significant. The statistical power for comparison of allele
2245 frequencies between patients with (n ¼ 62) vs without CAN (n ¼ 208) was sufficient to detect with an 80% probability a true effect size measured as an odds ratio equal to 1.81 or 0.53 for rs5498, 2.1 or 0.31 for rs1041163, and 5.72 for rs3170794.
RESULTS
The distributions of genotypes among the 3 studied SNPs were concordant with Hardy-Weinberg equilibrium (P > .3). Patients with vs without CAN did not differ as regards age (47.6 14.2 vs 47.6 12.6 years respectively, P ¼ .92) or gender distribution (30.7% vs 40.9% female; P ¼ .18). The distributions of genotypes and alleles of the rs5498 ICAM1 and rs1041163 and rs3170794 VCAM1 gene polymorphisms in patients with vs without CAN are presented in Table 1. None of them showed a significant difference. Creatinine concentrations in the first month after transplantation differed between the rs5498 ICAM1 genotypes (P ¼ .095), being higher albeit not significantly among GG carriers (AA þ AG vs GG, P ¼ .07). There were no significant differences between creatinine concentrations among rs5498 ICAM1 genotypes at 3 and 6 months after transplantation. Creatinine concentrations at 12, 24, and 36 months after transplantation differed significantly between rs5498 ICAM1 genotypes (P ¼ .0046 and P ¼ .016; P ¼ .02) being greater among GG carriers (AA þ AG vs GG, P ¼ .001, and P ¼ .004; P ¼ .006). The percentage of patients with serum creatinine >2 mg/dL at 12 months after transplantation was also significantly higher in GG carriers (GG vs AA þ AG: 47.5% vs 21.4%; P ¼ .0012). When the rs5498 GG genotype was analyzed as a marker to predict a serum creatinine >2 mg/dL at 12 months after transplantation, its sensitivity was 28.4% but specificity 89.3%, with a positive predictive value of 47.5% and a negative predictive value of 78.6%. There were no significant differences regarding creatinine concentrations between the rs1041163 VCAM1 and rs3170794 VCAM1 genotypes. To examine whether the rs5498 ICAM1 GG genotype was an independent risk factor associated with increased creatinine concentrations, a GLM was constructed including the studied polymorphism, recipient gender, and age. In this analysis, rs5498 ICAM1 GG genotype and recipient male gender were independent factors associated with higher creatinine concentrations at l2, 24, and 36 months after transplantation (Table 2). DISCUSSION
In recent years, the frequency of acute rejection episodes after kidney transplantation has decreased, but CAN remains the major cause of kidney loss. The long-term changes affecting transplanted kidney function depend on many factors.11 HLA compatibility, age and sex of the donor, panel-reactive antibodies, delayed graft function, immunosuppressive treatment, acute rejection episodes, and infections.12e14 Kidney function in the first months after transplantation predict long-term outcome. Evaluation of creatinine concentrations is a basic way to monitor allograft
KŁODA, DOMANSKI, PAWLIK ET AL
2246
Table 1. Distributions of the Genotypes and Alleles of the rs5498 ICAM1 and rs1041163 and rs3170794 VCAM1 Gene Polymorphisms in Patients With and Without CAN Without CAN (n ¼ 208)
CAN (n ¼ 62) Genotypes and alleles
rs5498 AA AG GG A G rs1041163 TT TC CC T C rs3170794 TT TC T C
n
%
n
%
Comparison
OR (95% CI) for CAN
P
17 32 13 66 58
18.5 23.4 31.7 20.6 26.5
75 105 28 255 161
81.5 76.6 68.3 79.4 73.5
GG þ AG vs AA GG vs AG þ AA GG vs AA G vs A
1.49 1.71 2.05 1.39
(0.80e2.79) (0.82e3.54) (0.88e4.76) (0.93e2.09)
NS NS NS NS
43 17 2 103 21
21.3 27.9 28.6 22.2 28.0
159 44 5 362 54
78.7 72.1 71.4 77.8 72.0
CC þ TC vs TT CC vs TC þ TT CC vs TT C vs T
1.43 1.35 1.48 1.37
(0.77e2.69) (0.26e7.15) (0.28e7.89) (0.79e2.37)
NS NS NS NS
59 3 121 3
22.4 42.9 22.7 42.9
204 4 412 4
77.6 57.1 77.3 57.1
TC vs TT
2.59 (0.56e11.91)
NS
C vs T
2.55 (0.56e11.57)
NS
P values calculated with Fisher exact test. CAN, chronic allograft nephropathy; OR, odds ratio; NS, not significant.
function.15 The aim of this study was to examine the impact of polymorphisms in ICAM1 and VCAM1 genes on CAN and creatinine concentrations. There were no significant associations between the studied polymorphisms and the CAN development. Nevertheless, the creatinine concentrations at 12, 24, and, 36 months after transplantation were higher among rs5498 ICAM1 genotype GG carriers. Cottone et al reported that adhesion molecules play a significant role in transplanted kidney damage. That long-term function of the renal graft as estimated through the glomerular filtration rate values was associated with serum concentrations of both ICAM-1 and VCAM-1 adhesion molecules.16 The up-regulated adhesion molecules in kidney tissue undergoing rejection seems to be a step in the cascade of inflammatory tissue rejection. The pathogenic importance of adhesion molecule expression, however, is not clearly understood. Adhesion molecules may not only mediate the infiltration process, but may also serve as costimulatory signals for T-cell activation by antigen-presenting cells. Previous studies have examined the expressions of adhesion molecules in inflammatory compared with noninflammatory renal diseases, revealing
up-regulated adhesion molecules in biopsies showing chronic histologic damage. Increased ICAM-1 and VCAM-1 expression was observed in various renal diseases to be correlated with structural tubular damage and interstitial fibrosis.17 McLaren et al evaluated factors that affect the gradual deterioration of transplanted kidney function. They observed that episodes of acute rejection, proteinuria, and serum triglyceride concentrations were associated with chronic allograft failure.18 In another study, the same group of researchers analyzed correlations between E-selectin, L-selectin, and ICAM1 gene polymorphisms and clinical/biopsy-proven decreased kidney function. They observed that the rs5498 ICAM1 GG genotype was associated with fast onset and more severe chronic allograft failure, suggesting that ICAM1 gene polymorphisms can play a role in the pathogenesis of gradual deterioration of transplanted kidney.19 We demonstrated here that male gender and rs5498 ICAM1 GG genotype were associated with higher creatinine concentrations at 12, 24, and 36 months after transplantation, suggesting it is associated with long-term allograft function.
Table 2. Analysis of the Risk Factors Associated With Creatinine concentration 12, 24, and 36 Months After Kidney Transplantation
REFERENCES
Risk factor
Creatinine concentration after 12 mo P
Creatinine concentration after 24 mo P
Creatinine concentration after 36 mo P
Male gender Age of recipient rs5498GG
.000003 .42 .0004
.000003 .81 .02
.0003 1.00 .01
P values calculated with general linear model.
1. Cecka JM. Kidney transplantation in the United States. Clin Transpl. 2008:1e18. 2. Carter JT, Chan S, Roberts JP, Feng S. Expanded criteria donor kidney allocation: marked decrease in cold ischemia and delayed graft function at a single center. Am J Transplant. 2005;5: 2745e2753. 3. Doma nski L, Safranow K, Dołe˛gowska B, et al. Hypoxanthine as a graft ischemia marker stimulates catalase activity in the renal vein during reperfusion in humans. Transplant Proc. 2006;38:35e38.
ICAM1 AND VCAM1 GENE POLYMORPHISMS 4. Solez K, Axelsen RA, Benediktsson H, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int. 1993;44:411e422. 5. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant. 2008;8:753e760. 6. Kim I, Moon SO, Kim SH, et al. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells. J Biol Chem. 2001;276:7614e7620. 7. Jeong HJ, Lee HH, Kim YS, Kim SI, Moon JI, Park K. Expression of ICAM-1 and VCAM-1 in renal allograft rejection. Transplant Proc. 1998;30:2953e2954. 8. Crews GM, Erickson L, Pan F, et al. Downregulation of TGFbeta and VCAM-1 is associated with successful treatment of chronic rejection in rats. Transplant Proc. 2005;37:1926e1928. 9. Burkhart C, Heusser C, Morris RE, et al. Pharmacodynamics in the development of new immunosuppressive drugs. Ther Drug Monit. 2004;26:588e592. 10. Lahiri DK, Bye S, Nurnberger JI Jr. A non-organic and nonenzymatic extraction method gives higher yields of genomic DNA from whole-blood samples that do nine other methods tested. J Biochem Biophys Methods. 1992;25:193e205. 11. Chapman J. Addressing the challenges for improving longterm outcomes in renal transplantation. Transplant Proc. 2008;40:2e4.
2247 12. Barocci S, Valente U, Fontana I, et al. Long-term outcome on kidney retransplantation: a review of 100 cases from a single center. Transplant Proc. 2009;41:1156e1158. 13. Heinze G, Oberbauer R, Kainz A, et al. Calcineurin inhibitor-based immunosuppressive therapy, donor age, and longterm outcome after kidney transplantation. Transplantation. 2009;87:1821e1829. 14. Süsal C, Döhler B, Sadeghi M, Ovens J, Opelz G. HLA antibodies and the occurrence of early adverse events in the modern era of transplantation: a collaborative transplant study report. Transplantation. 2009;87:1367e1371. 15. Resende L, Guerra J, Santana A, Mil-Homens C, Abreu F, da Costa AG. First year renal function as a predictor of kidney allograft outcome. Transplant Proc. 2009;41:846e848. 16. Cottone S, Palermo A, Vaccaro F, et al. Inflammation and endothelial activation are linked to renal function in long-term kidney transplantation. Transpl Int. 2007;20:82e87. 17. Mrowka C, Sieberth HG. Detection of circulating adhesion molecules ICAM-1, VCAM-1 and E-selectin in Wegener’s granulomatosis, systemic lupus erythematosus and chronic renal failure. Clin Nephrol. 1995;43:288e296. 18. McLaren AJ, Fuggle SV, Welsh KI, Gray DW, Morris PJ. Chronic allograft failure in human renal transplantation: a multivariate risk factor analysis. Ann Surg. 2000;232:98e103. 19. McLaren AJ, Marshall SE, Haldar NA, et al. Adhesion molecule polymorphisms in chronic renal allograft failure. Kidney Int. 1999;55:1977e1982.
T
HE SUCCESS OF KIDNEY TRANSPLANTATION depends on many immunologic and genetic factors.1 All of these conditions affect transplanted kidney function, as well as determine the possibility of acute and chronic rejection. Clinical manifestations of the ischemia and reperfusion injury are delayed graft function, defined as the need for hemodialysis during the first 7 days after transplantation.2 Apart from the basic parameter of diuresis, the condition of the allograft can be evaluated by a number of laboratory and imaging tests.3 Decreasing transplanted kidney function is assessed by an allograft biopsy. Moreover, an allograft biopsy can be performed to monitor the status of a functioning kidney.4,5 ICAM-1 which is encoded by a gene located on 19th chromosome, locus 19p13.3-p13.2 shows constant expression on the surface of endothelial cells. Through interactions with LFA-1 molecules it initiates immune responses. Leukocytes activated upon adhesion transmigrate to surrounding tissues, liberating proteolytic enzymes and reactive oxygen species that are destructive to the endothelium of the transplanted kidney.6 VCAM-1 (which is encoded on the first chromosome, locus 1p3132) is expressed on endothelial and antigen-presenting cells.
The interactions of VCAM-1 and its receptor VLA-4 which is found on lymphocytes, macrophages and granulocytes initiate, T-cell responses to alloantigen in the early development of acute and chronic rejection.7 Anti-VCAM-1 and anti-VLA-4 antibodies synergistically block rejection of transplanted organs.8,9 The several polymorphisms among ICAM1 and VCAM1 genes are associated with altered expression of these molecules, which may affect allograft function and immune responses. The aim of this study was to examine the impact of polymorphisms in ICAM1 and VCAM1 genes on biopsy-proven chronic allograft nephropathy (CAN) and long-term renal allograft function. From the Clinical Department of Nephrology, Transplantology and Internal Medicine (K.K., L.D., M.W., K.C.), Department of Pharmacology (A.P.), and Department of Biochemistry and Medical Chemistry (K.S.), Pomeranian Medical University in Szczecin, Szczecin, Poland. ski, Clinical Address reprint requests to Leszek Doman Department of Nephrology, Transplantology and Internal Medicine, Pomeranian Medical University in Szczecin, Szczecin, Poland. E-mail: [email protected]
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.03.043
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2244
Transplantation Proceedings, 45, 2244e2247 (2013)
ICAM1 AND VCAM1 GENE POLYMORPHISMS
METHODS The study enrolled 270 Caucasian renal recipients including 166 men and 104 women of overall mean age 47.63 12.96 years who underwent transplantations between 2000 and 2006. The most frequent causes of renal failure were chronic glomerulonephritis (58%), hypertension (9%), and diabetes (9%). All donors were cadaveric sources. The mean number of matched HLA-A and HLA-B alleles was 1.5 0.8, and the mean number of matched HLA-DR alleles was 1.2 0.5. Blood samples were collected from all patients for genetic analysis and creatinine concentrations evaluation at 1, 3, 6, 12, 24, and 36 months after kidney transplantation. Creatinine concentrations were evaluated with the colorimetric method. CAN was diagnosed in 62 patients by biopsies performed with the PAJUNK DeltaCut biopsy system and scored by a renal pathologist using the Banff working classification. All patients received a calcineurin inhibitor (cyclosporine [75%] or tacrolimus [24%]), azathioprine (55%) or mycophenolate mofetil (37%), and steroids (91%). Our local ethics committee approved the study protocol, which was conducted in accordance with the Helsinki Declaration of 1975.
Genotyping Genomic DNA was extracted from leukocytes contained in 450 mL whole blood samples with ethylenediaminetetraacetic acid as an anticoagulant, using a nonorganic and nonenzymatic extraction method.10 DNA precipitated with 99.5% ethanol was dissolved in distilled water and standardized to 20 ng/mL using a Nanodrop ND1000 spectrophotometer. This material was the matrix for amplification in real-time polymerase chain reactions (PCR). We analyzed 3 single nucleotide polymorhisms (SNPs): rs5498:A > G in exon 6 of the ICAM1 gene, as well as rs3l70794:T > C and rs1041163:T > C in the VCAM1 gene promotor using TaqMan probes in a 7500 Fast Real-Time PCR System machine (Applied Biosystems, USA). The substrates for the reaction were: TaqMan Genotyping Master Mix (Applied Biosystems), 2 starters (900 mmol/L) and 2 TaqMan probes with 50 reporter pigment and 30 nonfluorescent quencher (200 mmol/L). Probes specific for the more frequent allele were marked with 6-FAM reporter pigment and for the less frequent allele, with VIC pigment. For the rs5498:A > G and rs3l70794:T > C SNPs analysis, 2 ready-made assays were used: No. C__8726337_40 and C__30900705_10. For the rs1041163 T > C SNP analysis, we performed a new assay with starter sequences: 50 -GAC CTC TGG GTT ACT TGT TTA TAA GCT-30 , 50 -GAGATGCTGTTTCTAGGGTGTGG-30 and probes: FAMdTAG GGA TCA GAA AAA TTG A, VICdTAG GGA TCA GAG AAA TTG A. The reaction were performed at 95 C for 10 minutes for AmpliTaq Gold polymerase activation, before 40 cycles of 15 seconds in 92 C and 60 seconds in 60 C. Patient genotypes were determined through comparison of 6-FAM and VIC pigment fluorescence. The results were verified by review of the amplification plots.
Statistical Analysis The distributions of genotypes and alleles among patients with CAN compared with patients free of this complication were evaluated statistically with the chi-square test using the Yates correction or Fisher exact test. The concentrations of creatinine and histopathologic changes were estimated with the Kruskal-Wallis or the Mann-Whitney test. The influences of genotypes and alleles on creatinine concentrations were evaluated with a general linear model (GLM). P values <.05 were considered to be statically significant. The statistical power for comparison of allele
2245 frequencies between patients with (n ¼ 62) vs without CAN (n ¼ 208) was sufficient to detect with an 80% probability a true effect size measured as an odds ratio equal to 1.81 or 0.53 for rs5498, 2.1 or 0.31 for rs1041163, and 5.72 for rs3170794.
RESULTS
The distributions of genotypes among the 3 studied SNPs were concordant with Hardy-Weinberg equilibrium (P > .3). Patients with vs without CAN did not differ as regards age (47.6 14.2 vs 47.6 12.6 years respectively, P ¼ .92) or gender distribution (30.7% vs 40.9% female; P ¼ .18). The distributions of genotypes and alleles of the rs5498 ICAM1 and rs1041163 and rs3170794 VCAM1 gene polymorphisms in patients with vs without CAN are presented in Table 1. None of them showed a significant difference. Creatinine concentrations in the first month after transplantation differed between the rs5498 ICAM1 genotypes (P ¼ .095), being higher albeit not significantly among GG carriers (AA þ AG vs GG, P ¼ .07). There were no significant differences between creatinine concentrations among rs5498 ICAM1 genotypes at 3 and 6 months after transplantation. Creatinine concentrations at 12, 24, and 36 months after transplantation differed significantly between rs5498 ICAM1 genotypes (P ¼ .0046 and P ¼ .016; P ¼ .02) being greater among GG carriers (AA þ AG vs GG, P ¼ .001, and P ¼ .004; P ¼ .006). The percentage of patients with serum creatinine >2 mg/dL at 12 months after transplantation was also significantly higher in GG carriers (GG vs AA þ AG: 47.5% vs 21.4%; P ¼ .0012). When the rs5498 GG genotype was analyzed as a marker to predict a serum creatinine >2 mg/dL at 12 months after transplantation, its sensitivity was 28.4% but specificity 89.3%, with a positive predictive value of 47.5% and a negative predictive value of 78.6%. There were no significant differences regarding creatinine concentrations between the rs1041163 VCAM1 and rs3170794 VCAM1 genotypes. To examine whether the rs5498 ICAM1 GG genotype was an independent risk factor associated with increased creatinine concentrations, a GLM was constructed including the studied polymorphism, recipient gender, and age. In this analysis, rs5498 ICAM1 GG genotype and recipient male gender were independent factors associated with higher creatinine concentrations at l2, 24, and 36 months after transplantation (Table 2). DISCUSSION
In recent years, the frequency of acute rejection episodes after kidney transplantation has decreased, but CAN remains the major cause of kidney loss. The long-term changes affecting transplanted kidney function depend on many factors.11 HLA compatibility, age and sex of the donor, panel-reactive antibodies, delayed graft function, immunosuppressive treatment, acute rejection episodes, and infections.12e14 Kidney function in the first months after transplantation predict long-term outcome. Evaluation of creatinine concentrations is a basic way to monitor allograft
KŁODA, DOMANSKI, PAWLIK ET AL
2246
Table 1. Distributions of the Genotypes and Alleles of the rs5498 ICAM1 and rs1041163 and rs3170794 VCAM1 Gene Polymorphisms in Patients With and Without CAN Without CAN (n ¼ 208)
CAN (n ¼ 62) Genotypes and alleles
rs5498 AA AG GG A G rs1041163 TT TC CC T C rs3170794 TT TC T C
n
%
n
%
Comparison
OR (95% CI) for CAN
P
17 32 13 66 58
18.5 23.4 31.7 20.6 26.5
75 105 28 255 161
81.5 76.6 68.3 79.4 73.5
GG þ AG vs AA GG vs AG þ AA GG vs AA G vs A
1.49 1.71 2.05 1.39
(0.80e2.79) (0.82e3.54) (0.88e4.76) (0.93e2.09)
NS NS NS NS
43 17 2 103 21
21.3 27.9 28.6 22.2 28.0
159 44 5 362 54
78.7 72.1 71.4 77.8 72.0
CC þ TC vs TT CC vs TC þ TT CC vs TT C vs T
1.43 1.35 1.48 1.37
(0.77e2.69) (0.26e7.15) (0.28e7.89) (0.79e2.37)
NS NS NS NS
59 3 121 3
22.4 42.9 22.7 42.9
204 4 412 4
77.6 57.1 77.3 57.1
TC vs TT
2.59 (0.56e11.91)
NS
C vs T
2.55 (0.56e11.57)
NS
P values calculated with Fisher exact test. CAN, chronic allograft nephropathy; OR, odds ratio; NS, not significant.
function.15 The aim of this study was to examine the impact of polymorphisms in ICAM1 and VCAM1 genes on CAN and creatinine concentrations. There were no significant associations between the studied polymorphisms and the CAN development. Nevertheless, the creatinine concentrations at 12, 24, and, 36 months after transplantation were higher among rs5498 ICAM1 genotype GG carriers. Cottone et al reported that adhesion molecules play a significant role in transplanted kidney damage. That long-term function of the renal graft as estimated through the glomerular filtration rate values was associated with serum concentrations of both ICAM-1 and VCAM-1 adhesion molecules.16 The up-regulated adhesion molecules in kidney tissue undergoing rejection seems to be a step in the cascade of inflammatory tissue rejection. The pathogenic importance of adhesion molecule expression, however, is not clearly understood. Adhesion molecules may not only mediate the infiltration process, but may also serve as costimulatory signals for T-cell activation by antigen-presenting cells. Previous studies have examined the expressions of adhesion molecules in inflammatory compared with noninflammatory renal diseases, revealing
up-regulated adhesion molecules in biopsies showing chronic histologic damage. Increased ICAM-1 and VCAM-1 expression was observed in various renal diseases to be correlated with structural tubular damage and interstitial fibrosis.17 McLaren et al evaluated factors that affect the gradual deterioration of transplanted kidney function. They observed that episodes of acute rejection, proteinuria, and serum triglyceride concentrations were associated with chronic allograft failure.18 In another study, the same group of researchers analyzed correlations between E-selectin, L-selectin, and ICAM1 gene polymorphisms and clinical/biopsy-proven decreased kidney function. They observed that the rs5498 ICAM1 GG genotype was associated with fast onset and more severe chronic allograft failure, suggesting that ICAM1 gene polymorphisms can play a role in the pathogenesis of gradual deterioration of transplanted kidney.19 We demonstrated here that male gender and rs5498 ICAM1 GG genotype were associated with higher creatinine concentrations at 12, 24, and 36 months after transplantation, suggesting it is associated with long-term allograft function.
Table 2. Analysis of the Risk Factors Associated With Creatinine concentration 12, 24, and 36 Months After Kidney Transplantation
REFERENCES
Risk factor
Creatinine concentration after 12 mo P
Creatinine concentration after 24 mo P
Creatinine concentration after 36 mo P
Male gender Age of recipient rs5498GG
.000003 .42 .0004
.000003 .81 .02
.0003 1.00 .01
P values calculated with general linear model.
1. Cecka JM. Kidney transplantation in the United States. Clin Transpl. 2008:1e18. 2. Carter JT, Chan S, Roberts JP, Feng S. Expanded criteria donor kidney allocation: marked decrease in cold ischemia and delayed graft function at a single center. Am J Transplant. 2005;5: 2745e2753. 3. Doma nski L, Safranow K, Dołe˛gowska B, et al. Hypoxanthine as a graft ischemia marker stimulates catalase activity in the renal vein during reperfusion in humans. Transplant Proc. 2006;38:35e38.
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