Pediatr Nephrol DOI 10.1007/s00467-013-2588-3
ORIGINAL ARTICLE
Circulating endothelial cells in pediatric renal transplant recipients Arife Uslu Gökceoğlu & Sema Akman & Sadi Köksoy & Emel Şahin & Mustafa Koyun & Elif Çomak & Çağla Serpil Doğan & Halide Akbaş & Ayhan Dinçkan
Received: 16 January 2013 / Revised: 15 July 2013 / Accepted: 23 July 2013 # IPNA 2013
Abstract Background An increase in the number of circulating endothelial cells (CEC) indicates endothelial damage and the risk of cardiovascular disease. The aim of our study was to investigate the association of CEC with various clinical parameters in pediatric renal transplant recipients. Methods CEC, defined as CD45−CD146+, were enumerated by flow cytometry from the peripheral blood of 50 pediatric renal transplant recipients and 20 healthy controls. Clinical parameters, including renal function tests, fasting blood glucose, serum cholesterol and triglyceride, cyclosporine A (CsA) (trough and 2nd-hour) and tacrolimus (tac) trough blood levels and their association with CEC numbers were analyzed. Results CEC numbers of patients were higher than those of controls (respectively, 128±89 cells/ml (42–468 cells/ml), Electronic supplementary material The online version of this article (doi:10.1007/s00467-013-2588-3) contains supplementary material, which is available to authorized users. A. Uslu Gökceoğlu (*) : S. Akman : M. Koyun : E. Çomak : Ç. S. Doğan Department of Pediatric Nephrology, Akdeniz University Medical Faculty, Antalya, Turkey e-mail:
[email protected] S. Köksoy Department of Medical Microbiology, Akdeniz University Medical Faculty, Antalya, Turkey S. Köksoy : E. Şahin Laboratory of Organ Transplantation, Akdeniz University Medical Faculty, Antalya, Turkey H. Akbaş Department of Biochemistry, Akdeniz University Medical Faculty, Antalya, Turkey A. Dinçkan Institute of Organ Transplantation, Akdeniz University Medical Faculty, Antalya, Turkey
82±33 cells/ml (32–137 cells/ml), p =0.024). There was a statistically significant negative correlation between CEC numbers and glomerular filtration rate (GFR) (r =−0.300, p =0.012). There was also a statistically positive association between CEC numbers and transplant duration as well as cyclosporine trough level (respectively, r =0.397, p =0.004, r =0.714, p =0.004). CEC numbers in patients on tac and CsA were similar (p =0.716). Conclusions Our results demonstrate that renal transplant recipients with high CsA trough blood level, longer transplant duration, and lower GFR, are at greater risk of developing endothelial damage. Keywords CD146 . Chronic kidney disease (CKD) . Cardiovascular disease (CVD) . Pediatric kidney transplantation . Endothelial damage . Cyclosporine
Introduction Life expectancy of renal transplant recipients is shorter than the general population due to cardiovascular diseases (CVD) [1]. The risk factors for CVD in patients with chronic kidney disease (CKD) are also responsible for the CVD in renal transplant recipients [2, 3]. Immunosuppressive treatment, rejection episodes, and post-transplant diabetes mellitus (DM) also increase the risk of CVD in renal transplant recipients [2, 4]. In particular, calcineurin inhibitors (CNI) remain the mainstay of immunosuppressive therapy, and assessment of their potential contribution to cardiovascular risk is of great importance [5]. Endothelial damage and loss of physiological function of the endothelium are key factors that have a role in the development and progression of CVD [6, 7]. Endothelial cells are the barrier between the immune cells of recipients and the donor tissue. During vascular rejection, endothelial cells of recipients play the key role at the beginning of the immune
Pediatr Nephrol
reaction. Endothelial damage is the most frequent reason of graft loss [8]. Circulating endothelial cells (CEC) are mature endothelial cells that detach from the endothelium [9–11]. These cells are heterogeneous in size (10–50 μm), carry the markers of endothelial cells (WV, CD31, CD144, and CD146), and do not have leukocyte markers (CD45 and CD 33) [12]. CEC are present at very low levels in healthy children and the increase in CEC number demonstrates endothelial damage and thus might have prognostic importance in CVD [13]. The increase in CEC number has also been reported in hemodialysis patients, ANCA associated vasculitis, adult renal transplant recipients, and allograft rejection episodes [14–18]. In a recent study, Metsuyanim et al. [19] evaluated the antigenic potential in pediatric patients undergoing hemodialysis and after renal transplantation by quantifying mRNA expression of endothelial progenitor cell (EPC)-specific molecules (Flk1, Tie-2, CD133, VE-C, CD146, and CD31/PECAM-1) and of growth factors promoting EPC function (VEGF, EPO, and SDF-1) in peripheral blood, and compared this with healthy controls. Their major result was the observation that the number of CECs was decreased in end stage renal disease (ESRD) patients, suggesting a lower antigenic potential. However, their method was neither easy nor convenient to perform in our hospital. We, therefore, aimed to investigate CEC numbers through an easier and more quantitative approach (flow cytometric analysis), to determine their association with various clinical parameters in pediatric renal transplant recipients. We hereby report the first flow cytometric CEC evaluation in pediatric renal transplant recipients.
Materials and methods Patients aged between 6 and 18 years who received kidney grafts at Akdeniz University Medical Faculty between August 2000 and February 2012, and were followed periodically at the Pediatric Nephrology Department, were included in the study. The study was approved by the Institutional Review Board of Akdeniz University and informed consent was obtained from every patient and control. Exclusion criteria from the study were: post-transplantation period shorter than 3 months, active infection at the study time, CNI exchange, plasmapheresis treatment, and erythrocyte transfusion within the last 3 months before the study. Age, sex, donor type, renal transplantation time, immunosuppressive treatment, height, weight, and systolic and diastolic blood pressures were recorded. Body mass index (BMI) was calculated with the formula weight/height (m)2. BMI was evaluated according to age and sex of the patients [20]. Medications, history of acute rejection, and hypertension were recorded.
The following laboratory parameters were measured: blood urea nitrogen (BUN), serum creatinine, cystatin C, fasting blood glucose, calcium, phosphorus, uric acid, total cholesterol, triglyceride, C-reactive protein (CRP), erythrocyte sedimentation rate, blood CNI (tacrolimus (tac) or cyclosporine A (CsA)) levels. Estimated glomerular filtration rate (eGFR) was calculated with the Schwartz formula [21]. Patients were categorized according to GFR levels [22]. Twenty healthy volunteers age- and gender-matched to the patient group were chosen from among patients admitted to the Pediatric Nephrology Department for enuresis nocturna. They had no renal disease, hypertension, or history of cardiac disease and were not under any medication at the time of study. Various parameters, including weight, height, blood pressure measurements, BUN, serum creatinine, and cystatin levels of the controls were measured and recorded. Their GFR was also calculated as described above.
Laboratory measurements The evaluation of CEC numbers was performed with flow cytometry using FACSCantoII (BD Biosciences, Franklin Lakes, NJ, USA) and FACSDiva software (BD Biosciences). Peripheral blood samples were incubated with selected antibodies (PE Mouse Anti-human CD146 (BD Pharmingen); FITC Mouse Anti-human CD45 (BD Pharmingen)) at the concentrations suggested by the manufacturer. CECs were defined as CD146 (+) CD45 (−) cells in peripheric blood, and data were expressed as cell count per 1 ml of blood. Respective isotypic controls were also included in the analysis. BUN, serum creatinine, serum calcium, phosphorus, triglyceride, total cholesterol, fasting blood glucose, CRP, and erythrocyte sedimentation rate were studied with routine laboratory techniques (COBAS-8000). Cystatin C was measured using the nephelometric method (BNII). Tac level was studied by the EMIT method (COBAS-integra, Roche) and CsA level by the EMIT method (Twinn, Siemens).
Statistical analysis All analyses were performed using SPSS software (SPSS 16, SPSS Inc., Chicago, IL, USA). Continuous variables are expressed as mean ± SD. Categorical variables are presented as frequencies and percentages. The Mann–Whitney U test was used to compare groups. The Spearman correlation test was used for correlation analysis. Multiple stepwise linear regression analysis was used to show the influences of variables on number of CEC. All tests were performed with p
