Noninvasive Diagnostic And Predictive Value In Renal Transplant Recipients By Measurement Of Urine STim-3, Fractalkine And BCA-1

Kidney transplantation has become the best replacement therapy for patients with chronic renal failure, one-year survival rate after transplantation has improved markedly. Graft failure from immunologic factors and death from opportunistic infection in the first year after transplantation are no longer common clinical outcomes other than 30 years ago. However, the long-term outcome of the graft is still poor because of allograft dysfunction after 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. How to detect rejection and distinguish it from other causes of renal dysfunctionn is still an challenging topic in the transplantation field.With the application of new immunosuppressive agents, more and more patients presented with atypical clinical manifestation of acute rejection, 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. Some patients with stable renal function were detected with abnormal pathological presentation in protocol biopsies. Current Banff criteria is not sufficient for clinical practice in some atypical cases. To target individualized immunosuppressive regemin, it is important to evaluate the immune state of this special population.In recent years, researchers have been dedicated to explore more sensitive and timely indicators for the diagnosis of early renal allograft dysfunction. Many noninvasive test supplementing the biopsy has been used for monitoring acute rejection. For example, noninvasive tests in kidney transplants by measure of Mig, IP 10, Tim-3, Fractalkine in urine.The aim of this study is to investigate whether level of sTim-3, Fractalkine and BCA-1 in urine is a useful noninvasive tool for the assessment of acute rejection in renal transplant recipients. Our topic is expected to provide new noninvasive dignostic marks for acute renal rejection and to provide some clinical basis for future research.Part I Noninvasive Diagnostic and Predictive Value in Renal Transplant Recipients by Measurement of urine sTim-3Objective:To investigate the relationship between early-stage renal acute rejection and the level of sTim-3 in urine, explore the diagnostic value and noninvasive monitoring in early stage after transplantation by measurement of urine sTim-3.MethodsWe examined urine samples from renal transplant patients between January 2006 and October 2009.155 patients were enrolled, including 49 with biopsy-proved acute rejection,58 patients with stable renal function and no abnormal histological findings, 10 patients with subclinical rejection in protocol biopsy,9 patients with biopsy-proven acute tubular necrosis and 29 patients with biopsy-proven chronic allograft nephropathy. Additionally, urinary samples were also collected from 40 healthy controls. sTim-3 was measured in urinary samples using a commercial human sTim-3 enzyme-linked immunosorbent assay (ELISA) kit (GBD Systems). All samples were tested in duplicate by a standardized ELISA technique. Statistical analysis was performed by using SPSS software package. (Version 16.0) Because of nonparametric distribution, comparisons of sTim-3 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 sTim-3 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. sTim-3 levels were expressed per millimole of urinary creatinine to correct for difference in urinary concentration. Immunohistochemistry for Tim-3 expression was performed on biopsies from renal transplant patients with acute rejection and No-AR.ResultsPatient with acute rejection excreted urinary sTim-3 at a significantly higher level (4257±439.4,95%CI:3373-5142 ng/mmol creatine) than levels of patients with No-AR and healthy controls (P<0.001). Patients with acute tubular necrosis excreted urinary sTim-3 at a significantly lower level (2180±217,95%CI:1679-2680 ng/mmol creatine) than levels of patients with acute rejection. ROC curve was constructed to determine the discriminatory power of sTim-3 levels for diagnosis of acute rejection. The area under ROC curve was 0.874 (95%CI:0.8-0.948), which showed that sTim-3 was a suitable marker for the diagnosis of acute rejection. At a cut point of 1836 ng/mmol creatinine, the sensitivity was 89.4% and the specificity was 82.5%(P<0.001). Patients with steroid-resistant acute rejection had significantly greater urinary sTim-3 concentration than patients with steroid-sensitive acute rejection (5548±613.5,95%CI: 4287-6809 ng/mmol creatinine vs 2653±391.7,95%CI:1830-3476 ng/mmol creatinine, P=0.0002).The dynamic level of urinary sTim-3 in acute renal rejection patients within 3 weeks after transplantation is about 6000 ng/mmol creatine, which is remarkably higher than patients with stable renal function (2000 ng/mmol creatinine). We also find that sporadic expression of Tim-3 was present on biopsies from patients with acute rejection and none in patients with No-AR.ConclusionsThe monitoring of sTim-3 in urine may be a new and noninvasive approach for detection acute rejection as well as useful to predict response to antirejection therapy. It has good sensitivity and specificity. Besides, measurement of sTim-3 in urine is a simple, inexpensive method for the routine clinical monitoring after kidney transplantation.PartⅡNoninvasive Diagnostic and Predictive Value in Renal Transplant Recipients by Measurement of urine FractalkineObjectiveTo investigate the relationship between early-stage renal acute rejection and the level of Fractalkine in urine, explore the diagnostic value and noninvasive monitoring in early stage after transplantation by measurement of urine Fractalkine.MethodsWe examined urine samples from renal transplant patients between January 2006 and October 2009.155 patients were enrolled, including 49 with biopsy-proved acute rejection,58 patients with stable renal function and no abnormal histological findings, 10 patients with subclinical rejection in protocol biopsy,9 patients with biopsy-proven acute tubular necrosis and 29 patients with biopsy-proven chronic allograft nephropathy. Additionally, urinary samples were also collected from 40 healthy controls. Fractalkine was measured in urinary 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. Statistical analysis was performed by using SPSS software package. (Version 16.0) 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. Immunohistochemistry for Fractalkine expression was performed on biopsies from renal transplant patients with acute rejection and No-AR.ResultsPatient with acute rejection excreted urinary Fractalkine at a significantly higher level (429.1±56.1,95%CI:316.2-541.9 ng/mmol creatine) than levels of patients with No-AR and healthy controls (P<0.001). Patients with acute tubular necrosis excreted urinary Fractalkine at a significantly lower level (133±9.8,95%CI:110.3-155.7 ng/mmol creatine) than levels of patients with acute rejection. ROC curve was constructed to determine the discriminatory power of Fractalkine levels for diagnosis of acute rejection. The area under ROC curve was 0.909 (95%CI:0.856-0.962), which showed that Fractalkine was a suitable marker for the diagnosis of acute rejection. At a cut point of 144.6 ng/mmol creatinine, the sensitivity was84% and the specificity was 81%(P<0.001). Patients with steroid-resistant acute rejection had significantly greater urinary Fractalkine concentration than patients with steroid-sensitive acute rejection (526.8±85.5,95%CI:351-702.5 ng/mmol creatinine vs 309.6±81.6,95%CI: 135.7-483.4 ng/mmol creatinine, P=0.01).The dynamic level of urinary Fractalkine in acute renal rejection patients within 3 weeks after transplantation fluctuated during 400-600 ng/mmol creatine, which is remarkably higher than patients with stable renal function (100-150 ng/mmol creatinine). We also find that widespread expression of Fractalkine was present on biopsies from almost patients with acute rejection and few patients with No-AR.ConclusionsAs a noninvasive monitoring, Fractalkine in urine may be a new approach for detection acute rejection as well as useful to predict response to antirejection therapy. It has good sensitivity and specificity. Besides, measurement of Fractalkine in urine is a simple, inexpensive method for the routine clinical monitoring after kidney transplantation.Part III Noninvasive Diagnostic and Predictive Value in Renal Transplant Recipients by Measurement of urine BCA-1ObjectiveTo investigate the relationship between early-stage renal acute rejection and the level of BCA-1 in urine, explore the diagnostic value and noninvasive monitoring in early stage after transplantation by measurement of urine BCA-1.MethodsWe examined urine samples from renal transplant patients between January 2006 and October 2009.155 patients were enrolled, including 49 with biopsy-proved acute rejection,58 patients with stable renal function and no abnormal histological findings,9 patients with biopsy-proven acute tubular necrosis and 29 patients with biopsy-proven chronic allograft nephropathy. Additionally, urinary samples were also collected from 40 healthy controls. BCA-1 was measured in urinary samples using a commercial human BCA-1 enzyme-linked immunosorbent assay (ELISA) kit ((R&D Systems). All samples were tested in duplicate by a standardized ELISA technique. Statistical analysis was performed by using SPSS software package. (Version 16.0) Because of nonparametric distribution, comparisons of BCA-1 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 BCA-1 measurements for patients with acute rejection, acute humoral 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. BCA-1 levels were expressed per micromole of urinary creatinine to correct for difference in urinary concentration.ResultsPatient with acute rejection excreted urinary BCA-1 at a significantly higher level (8.1±2.1,95%CI:3.9-12.4 pg/μmol creatine) than levels of patients with No-AR and healthy controls (P<0.001). Patients with acute tubular necrosis excreted urinary BCA-1 at a significantly lower level (2.52±0.57,95%CI:1.21-3.84pg/μmol creatinine) than levels of patients with acute rejection. ROC curve was constructed to determine the discriminatory power of BCA-1 levels for diagnosis of acute rejection. The area under ROC curve was 0.881 (95%CI:0.811-0.951), which showed that BCA-1 was a suitable marker for the diagnosis of acute rejection. At a cut point of 0.23 pg/μmol creatine, the sensitivity was 81.6% and the specificity was 87.9%(P<0.001). Patients with steroid-resistant acute rejection had significantly greater urinary BCA-1 concentration than patients with steroid-sensitive acute rejection (11±2.82,95%CI:5.22-16.8 pg/μmol creatinine vs 5.34±4.28,95%CI:3.8-14.5 pg/μmol creatinine, P=0.0032). Patients with acute humoral rejection had significantly higher urinary BCA-1 concentration than patients with acute celluar rejection (24.2±6.68,95%CI:9.5-38.9 pg/μmol creatinine vs 2.91±0.65,95%CI:1.6-4.22 pg/μmol creatinine, P=0.0002). ROC curve was constructed to determine the discriminatory power of BCA-1 levels for diagnosis of acute humoral rejection. The area under ROC curve was 0.867 (95%CI:0.725-1.005), which showed that BCA-1 was a suitable marker for the diagnosis of acute humoral rejection. At a cut point of 8.3 pg/μmol creatine, the sensitivity was 75% and the specificity was 91.9% The dynamic level of urinary BCA-1 in acute renal rejection patients within 3 weeks after transplantation is above 2 pg/μmol creatine, however, the level of patients with stable renal function is below 2 pg/μmol creatine.ConclusionsThe monitoring of BCA-1 in urine may be a new and noninvasive approach for detection acute rejection as well as useful to discriminate the type of rejection. It has good sensitivity and specificity.