A Study Of The Association Between Acute Rejection And Fractalkine & Vascular Endothelial Growth Factor

IntroductionAcute allograft rejection is one of the most frequent complications in renal transplantation. Intensity of acute rejection and effect of treatment have direct impact on the long-term outcome of the graft. It is crucial that a definite diagnosis should be as early as possible. Histopathologic examination of renal biopsy is regarded as the gold standard of acute rejection. But biopsy is limited by the invasive nature of the procedure and cost. Morever, because of the inadequate sample size and sampling error, there have been conflicting results of the histological findings. Noninvasive test that could be used for monitoring acute rejection would be of considerable value in supplementing the biopsy. The clinical diagnosis of acute rejection is difficult toobtain because typical clinical manifestations of acute rejection have changed with modern immunosuppression. However, blood and urinary measurement may provide a simple tool for diagnosis of acute rejection. The development of noninvasive techniques for detection of acute rejection would be desirable in the transplantation community. The aim of this study was to investigate whether level of fractalkine in urine and blood was a useful noninvasive tool for the assessment of acute rejection in renal transplant recipients.Patients and Methods Source of blood and urinary samplesWe examined urine and blood samples from renal transplant patients between June 2001 and Dec 2005. There were 67 patients with biopsy-proven acute rejection within six months after transplant, 11 patients with biopsy-proven acute tubular necrosis and 14 patients biopsy-proven with chronic allograft nephropathy. There were 119 patients with stable renal function and no abnormal histological findings (No-AR) as well as 13 patients with subclinical rejection in protocol biopsy. Fresh first-morning urine samples from patients were collected every two weeks during two months after transplant. On the day of biopsy, blood and urinary samples were collected before biopsy. Additionally, blood and urinary samples were also collected from 80 healthy controls. ELISA: Determination of fractalkine in urine and bloodFractalkine was measured in urinary and blood samples using a commercial human fractalkine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). All samples were tested in duplicate by a standardized ELISA technique. ImmunohistochemistryImmunohistochemistry for fractalkine expression was performed on biopsies from renal transplant patients with acute rejection and No-AR. We also examinedfractalkine expression on biopsie from donors. Statistical AnalysisStatistical analysis was performed by using SPSS software package. (Version 14.0; SPSS Inc, Chicago, IL) Because of nonparametric distribution, comparisons of fractalkine levels among different groups were performed by Kruskal-Wallis test or Mann-Whitney U test and Wilcoxon Signed Ranks Test. A conventional receiver operating characteristic (ROC) curve was applied to determine the sensitivities and specificities for fractalkine measurements for patients with acute rejection, subclinical rejection and steroid-resistant acute rejection. Area under the curve was also calculated. Results were expressed in the text as mean ± SEM unless otherwise stated. P<0.05 was considered statistically significant. Fractalkine levels were expressed per millimole of urinary creatinine to correct for difference in urinary concentration.ResultsLevels of urinary fractalkine in patients with No-AR during 8 weeks after transplantLevels of urinary fractalkine in patients with No-AR were similar during the first 8 weeks after transplant. Urinary fractalkine was a marker of acute renal allograft rejectionPatient with acute rejection excreted urinary fractalkine at a significantly higher level (407.52±57.02, 95%CI: 293.68-521.35 ng/mmol creatinine) than levels of patients with No-AR and healthy controls (P<0.001). Patients with acute tubular necrosis and chronic allograft nephropathy excreted urinary fractalkine at a significantly lower level (103.55±36.24, 95%CI: 22.80-184.29 ng/mmol creatinine, P=0.004 and 96.02±32.91, 95%CI: 24.92-167.12 ng/mmol creatinine, P=0.001, respectively) than levels of patients with acute rejection. A ROC curve was constructed to determine the discriminatory power of fractalkine levels for diagnosisof acute rejection. The area under ROC curve was 0.834 (95%CI: 0.770-0.899), which showed that fractalkine was a suitable marker for the diagnosis of acute rejection. At a cut point of 102.88 ng/mmol creatinine, the sensitivity was 82.1% and the specificity was 76.5% (P<0.001). Level of urinary fractalkine in cellular and humoral rejectionPatients with cellular rejection excreted urinary fractalkine (395.43±72.00 ng/mmol creatinine, 95%CI: 250.13-540.73 ng/mmol creatinine) and patients with humoral rejection excreted urinary fractalkine (429.17±95.14 ng/mmol creatinine, 95%CI: 232.37-625.98 ng/mmol creatinine). However, no statistical difference was observed between the two groups (P=0.824). High levels of urinary fractalkine predicted steroid-resistant acute rejectionPatients with steroid-resistant acute rejection had significantly greater urinary fractalkine concentration than patients with steroid-sensitive acute rejection (568.48± 86.29, 95%CI: 393.80-743.17 ng/mmol creatinine vs 183.31±34.93, 95%CI: 111.63-254.99 ng/mmol creatinine, P<0.001). ROC curve showed the sensitivity and specificity of various cut points for levels of fractalkine to determine the diagnosis of steroid-resistant acute rejection. The area under the ROC curve was 0.771 (95%CI: 0.659-0.884). When the cut point was at 233.76 ng/mmol creatinine, the sensitivity was 74.4% and specificity was 75.0%. It seemed that patients with graft loss had greater urinary fractalkine concentration than patients with reversible acute rejection (671.37±266.62, 95%CI: 116.85-1225.89 vs 376.73±57.43, 95%CI: 261.82-491.64 ng/mmol creatinine). But this difference did not reach statistical significance (P=0.065). Levels of urinary fractalkine before and after acute rejectionBefore and after acute rejection, urinary fractalkine was at a significantly lower level than urinary fractalkine during acute rejection.Urinary fractalkine was an indicator of subclinical renal allograft rejectionPatients with subclinical rejection excreted urinary fractalkine (237.70±65.85, 95%CI: 94.21-381.18 ng/mmol creatinine), which significantly differed from fractalkine excreted of No-AR patients and healthy controls. (P<0.001) ROC curve showed the sensitivity and specificity for various cut points for levels of fractalkine to determine the diagnosis of subclinical rejection. The area under the ROC curve was 0.836 (95%CI:0.753-0.920, P<0.001). The cut point that maximized the combined sensitivity and specificity for fractalkine was 79.48ng/mmol creatinine. At this threshold, the sensitivity was 92.3% and specificity was70.6% (P<0.001). Blood fractalkine was a marker of acute renal allograft rejectionLevels of blood fractalkine in patient with acute rejection (2247.95±286.14, 95%CI: 1676.14-2819.76pg/ml) were significantly higher than levels of patients with No-AR and healthy controls (P<0.001). A ROC curve was constructed to determine the discriminatory power of fractalkine levels for diagnosis of acute rejection. The area under ROC curve was 0. 825 (95%CI: 0.765-0.886, P<0.001). At a cut point of 1108.75 pg/ml, the sensitivity was 71.9% and the specificity was 80.0% (P<0.001). Blood fractalkine was an indicator of subclinical renal allograft rejectionFractalkine levels were significantly higher during subclinical rejection (1185.08±154.83, 95%CI: 1065.49-1591.46 pg/ml) than those of patient with No-AR. (P=0.002). A ROC curve was constructed to determine the discriminatory power of blood fractalkine levels for diagnosis of subclinical rejection. The area under ROC curve was 0.770 (95%CI: 0.667-0.873) and at a cut point of 805 pg/ml, the sensitivity was 76.9% and the specificity was 69.4% (P=0.002). ImmunohistochemistryExpression of fractalkine was present on biopsies from almost patients with acute rejection and few patients with No-AR. There was no expression in renal tissues of donor.ConclusionsThe monitoring of fractalkine in urine and blood may be an important, new and noninvasive approach for detection acute rejection and subclinical rejection as well as helpful to predict response to antirejection therapy. Besides, measurement of fractalkine in urine is a simple, inexpensive method for the routine clinical monitoring after kidney transplantation.IntroductionsKidney transplantation is one of the treatments for most patients with end-stage renal disease (ESRD). Despite major advances in transplant immunology and pharmacotherapy, which have led to an improvement of the first-year renal graft survival, late graft loss continues to be a significant problem in renal transplantation. Complications following renal transplantation such as acute rejection may lead to graft loss either immediately or chronic allograft nephropathy. Histological examination of renal allograft tissue is an accepted standard for diagnosing acute rejection. However, because of the inadequate sample size and sampling error, there have been conflicting results of the histological findings. Moreover, biopsy is limited by the invasive nature of the procedure and cost. It would be desirable to use noninvasive techniques to reduce biopsy procedure for the diagnosis of acute rejection. Even after acute rejection has been determined by biopsy it is still difficult to accurately predict therapeutic response. Making the determination of steroid resistant acute rejection as soon as possible would avoid patient exposure to unnecessary high dose steroid. The purpose of this study was to assess whether measurement of blood and urinary vascular endothelial growth factor (VEGF) could be adopted as new noninvasive diagnostic tool for the acute rejection after renal transplant.Patients and Methods Source of blood and urinary samplesWe examined urine and blood samples from renal transplant patients betweenJune 2001 and Dec 2005. There were 67 patients with biopsy-proven acute rejection within six months after transplant, 11 patients with biopsy-proven acute tubular necrosis and 14 patients biopsy-proven with chronic allograft nephropathy. There were 119 patients with stable renal function and no abnormal histological findings (No-AR) as well as 13 patients with subclinical rejection in protocol biopsy. Fresh first-morning urine samples from patients were collected every two weeks during two months after transplant. On the day of biopsy, blood and urinary samples were collected before biopsy. Additionally, blood and urinary samples were also collected from 80 healthy controls. ELISA: Determination of VEGF in urine and bloodVEGF was measured in urinary and blood samples using a commercial human VEGF enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). All samples were tested in duplicate by a standardized ELISA technique. ImmunohistochemistryImmunohistochemistry for VEGF expression was performed on biopsies from renal transplant patients with acute rejection and No-AR. We also examined VEGF expression on biopsie from donors. Statistical AnalysisStatistical analysis was performed by using SPSS software package. (Version 14.0; SPSS Inc, Chicago, IL) Because of nonparametric distribution, comparisons of VEGF levels among different groups were performed by Kruskal-Wallis test or Mann-Whitney U test and Wilcoxon Signed Ranks Test. A conventional receiver operating characteristic (ROC) curve was applied to determine the sensitivities and specificities for VEGF measurements for patients with acute rejection, subclinical rejection and steroid-resistant acute rejection. Area under the curve was also calculated. Results were expressed in the text as mean ± SEM unless otherwise stated. P<0.05 was considered statistically significant. VEGF levels were expressed per millimole of urinary creatinine to correct for difference in urinary concentration.Results Levels of urinary VEGF in patients with No-AR during 8 weeks after transplantLevels of urinary VEGF in patients with No-AR were similar during the first 8 weeks after transplant. Urinary VEGF was an indicator of acute renal allograft rejectionPatient with acute rejection excreted urinary VEGF at a significantly higher level (28.57±6.21, 95%CI: 16.18-40.97 ng/mmol creatinine) than levels of patients with No-AR (3.05±0.45, 95%CI: 2.17-3.93 ng/mmol creatinine,P<0.001), chronic allograft nephropathy (5.96±2.64, 95%CI: 0.25-11.67 ng/mmol creatinine, P=0.001) and healthy controls (2.87±0.35, 95%CI: 2.16-3.57 ng/mmol creatinine, P<0.001). But there was no difference between patients with acute tubular necrosis and acute rejection. A ROC curve was constructed to determine the discriminatory power of VEGF levels for diagnosis of acute rejection. The area under ROC curve was 0.871 (95%CI: 0.820-0.921), which showed that VEGF was a suitable marker for the diagnosis of acute rejection. At a cut point of 3.64 ng/mmol creatinine, the sensitivity was 85.1% and the specificity was 74.8% (P<0.001). Level of urinary VEGF in cellular and humoral rejectionPatients with cellular rejection excreted urinary VEGF (28.75±8.69, 95%CI: 11.21-46.29ng/mmol creatinine ) and patients with humoral rejection excreted urinary VEGF (28.26±7.86 ng/mmol creatinine, 95%CI: 12.0-44.51 ng/mmol creatinine). However, no statistical difference was observed between the two groups (P=0.200). High levels of urinary VEGF predicted steroid-resistant acute rejection and graft loss.Patients with steroid-resistant acute rejection had significantly greater urinary VEGF concentration than patients with steroid-sensitive acute rejection (42.09± 10.00, 95%CI: 21.85-62.34 vs 9.74±2.63, 95%CI: 4.34-15.14ng/mmol creatinine, PO.001). The area under the ROC curve was 0.771 (95%CI: 0.658-0.884). When the cut pointwas at 12.27 ng/nmol creatinine, the sensitivity was 61.5% and specificity was 85.7%; (PO.001). Patients with graft loss had significantly greater urinary VEGF concentration than patients with reversible acute rejection (106.66±38.60, 95%CI: 12.22-201.10 vs 19.46±4.13, 95%CI: 11.21-27.72 ng/mmol creatinine, P=0.001). ROC curve showed the sensitivity and specificity for various cut points for levels of VEGF to predict worse outcomes after acute rejection. The area under the ROC curve was 0.881 (95%CI: 0.760-1.001). When the cut point was at 22.48 ng/mmol creatinine, the sensitivity was85.7% and specificity was78.3% (P=0.001). Levels of urinary VEGF before and after acute rejectionBefore and after acute rejection, urinary VEGF was at a significantly lower level than urinary VEGF during acute rejection. Urinary VEGF was an indicator of subclinical renal allograft rejectionPatients with subclinical rejection excreted urinary VEGF (16.14±4.09, 95%CI: 7.21-25.06 ng/mmol creatinine), which significantly differed from VEGF excreted of No-AR patients and healthy controls (P<0.001). The area under the ROC curve was 0.819 (95%CI: 0.662-0.976). The cut point that maximized the combined sensitivity and specificity for VEGF was 9.90ng/mmol creatinine.. At this threshold, the sensitivity was76.0% and specificity was 90.8% (P<0.001). Blood VEGF was a marker of acute renal allograft rejectionLevels of blood VEGF in patient with acute rejection (126.76±9.76, 95%CI: 107.27-146.26pg/ml) were significantly higher than levels of patients with No-AR (69.10±6.04, 95%CI: 57.09-81.11pg/ml) and healthy controls (55.38±7.87, 95%CI: 39.71-71.06 pg/ml), PO.001. A ROC curve was constructed to determine the discriminatory power of VEGF levels for diagnosis of acute rejection. The area under ROC curve was 0.744(95%CI: 0.664-0.825, P<0.001). At a cut point of 91.71 pg/ml, the sensitivity was62.5% and the specificity was 80.0% (P<0.001).Blood VEGF was an indicator of subclinical renal allograft rejectionVEGF levels were significantly higher during subclinical rejection (126.96± 20.13, 95%CI: 83.10-170.83 pg/ml) than those of patient with No-AR(69.10±6.04, 95%CI: 57.09-81.11 pg/ml, P=0.002). A ROC curve was constructed to determine the discriminatory power of blood VEGF levels for diagnosis of subclinical rejection. The area under ROC curve was 0.773(95%CI: 0.641-0.904) and at a cut point of 105.31 pg/ml, the sensitivity was 69.2% and the specificity was 84.7% (P=0.002). ImmunohistochemistryExpression of VEGF was present on biopsies from almost patients with acute rejection. Expression of VEGF was at lesser extent in renal tissues of donor and patients with No-AR.ConclusionsThe development of noninvasive techniques for detection of acute rejection is a major goal of the renal transplantation community. The monitoring of VEGF in urine and blood may be an important and new noninvasive approach for detection acute rejection and subclinical rejection. Besides, measurement of VEGF in urine is helpful to predict response to antirejection therapy and poor outcome after acute rejection.IntroductionThe use of long term immunosuppressive treatment contributes significantly to its associated increased risks of infection , malignancy and cardiovascular diease. It is important to achieve a balance between the risks and benefits of immunosuppression while avoiding under or over immunosuppressing the patients. It is difficult to individualize the immunosuppressive treatment on the basis of immunological risks. The ability to predict the rejection risk would allow us to tailor the immunosuppressive treatment to the individual need. Recent advances in molecular techniques have resulted in the description of an increasing number of gene polymorphism. Gene polymorphism might result in altered molecular expression, signaling, production, binding and activity. Altered function arising from these polymorphisms provides an excellent explantation for variation in susceptibility to rejection. Recent studies revealed that single nueleotid polymorphism (SNP) of VEGF at site +405(G/C) and -460(T/C) are associated with diabetic retinopathy, possibly through their effect on the inducibility of VEGF synthesis. We tested whether these VEGF SNPs of donor and recipient associated with the risk for acute allograft rejection.Materials and Methods PatientsWe collected blood samples from 224 renal transplant patients between June2001 and June 2006. There were 79 patients with biopsy-proven acute rejection within six months after transplant and 145 patients with stable renal function and no abnormal histological findings (No-AR). Blood samples were also collected from 183 healthy controls. Genomic DNA was extracted from blood samples from those graft recipient and from spleen cells from cadaveric donors. GenotypingRecipients, donors and healthy controls were genotyped for the SNP at positions +405G/C and -460T/C. DNA was isolated from blood mononuclear cells or spleen cells, collected before transplant, using DNA isolation kit. Genotypes were determined in genomic DNA with Taqman allelic discrimination assay. Statistical AnalysisThe Pearson x~2 test was used to assess difference in the distribution of alleles and genotypes amomge groups (healthy controls, acute rejection and No-AR).Results Difference between healthy controls and graft recipientsVEGF +405 genotype and alleles had significant diffence between healthy controls and graft recipients (P<0.001). VEGF -460 genotype had no diffence between healthy controls and graft recipients. Association of VEGF polymorphism with acute rejectionIndividuals with +405 CC demonstrated a 1.89-fold increased risk of acute rejection compared with those with +405 GG genotype. Individuals with C allele demonstrated a 1.799-fold increased risk of acute rejection compared with those with +405 G allele. VEGF -460 genotype and allele had no diffence between patients with acute rejection and No-AR. Association of VEGF polymorphism of donor with acute rejectionVEGF+405 genotype and allele of donor had no effect on acute rejection. Association of different genotype pair of recipients and donors with acute rejectionGenotype pair of recipients and donors with +405 CC and CC demonstrated a 4.074-fold increased risk of acute rejection compared with those with +405 GG and GGConclusionsWe first reported an association between VEGF gene polymorphism at position +405 and acute rejection. Individuals with +405 CC genotype and C allele demonstrated higher risk of acute rejection. Different genotype pair of recipients and donors also had influence on acute rejection.