KIDNEY TRANSPLANTATION Association of Serum Fetuin-A Levels with Allograft Outcome in Renal Transplant Recipients Abdolrasoul Mehrsai,1 Gholamreza Pourmand,1 Hamed Azhdari Tehrani,2 Hossein Keyhan,2 Mohamad Reza Rahmati,1 Ayat Ahmadi,3 Sanaz Dehghani,1 Rahil Mashhadi1* Purpose: To determine serum fetuin-A pattern after renal transplantation and its association with graft outcome. Materials and Methods: In 41 renal transplant recipients, serum pretransplant fetuin-A levels and serum fetuin-A concentrations on days 7 and 30 after transplantation were measured using the enzyme-linked immunosorbent as- say (ELISA) method. Also, the association between serum fetuin-A levels with clinical and laboratory parameters was evaluated. Results: A significant decrease in serum fetuin-A levels was noted in the first week after transplantation (P < .001). Subsequently, it started to increase and surpass pretransplant values during the first month (P < .001). Pretransplant fetuin-A levels did not differ among patients with different diethylenetriamine pentaacetic acid (DTPA) results. In addition, serum fetuin-A levels did not significantly correlate with metabolic parameters. Conclusion: In this prospective study there was no increase in serum fetuin-A levels during the first month and pretransplant fetuin-A levels are not predictive for allograft outcome in renal transplant recipients. Keywords: kidney transplantation; postoperative complications; kidney failure; kidney function tests; prospective studies. INTRODUCTION Kidney transplantation is an important and effective treatment for most patients with end-stage renal disease, which confers a survival benefit compared to hemodialysis.(1) Improved technical approaches, new advances in immunosuppression, antibody definition and improvements in the overall care of the transplant recipient during the past decades, have significantly in- creased the short-term kidney allograft survival rate.(2,3) In spite of these advances, long-term allograft survival after the first year has not improved, and the length of kidney allograft survival is still shorter than that of the recipient’s even with the use of immunosuppressive drugs in sufficient doses to prevent acute rejection.(2,3) Reasons for allograft failure are not well understood. (2) Renal allograft failure occurs by both immune and nonimmune mechanisms; for example, graft rejection, graft thrombosis, interstitial fibrosis and tubular atrophy (IF/TA), infection, chronic inflammation, nephrotoxici- ty from calcineurin inhibitors and calcification of the al- lografts.(2) Therefore, access to innovative risk-identify- ing strategies in order to increase the long-term allograft survival can be considered as the best target for studies.(4) In patients with end-stage renal disease (ESRD) and chronic kidney disease (CKD), the risk of subsequent cardiovascular events is higher than the age- and sex- matched populations with normal kidney function.(5) Recent observations have shown rapid progression of cardiovascular calcification in patients with CKD and ESRD.(5) Cardiovascular calcification is a tightly regulated process affected by multiple mechanisms and also several serum proteins.(6) Although calcium and phosphate precipita- tion occur in several tissues due to multiple risk factors such as age or inflammation reactions, but some serum precipitation inhibitory proteins would not allow this process to proceed. A number of calcification inhibitors such as fetuin-A, have been identified in recent years.(5,6) Fetuin-A is a vertebrate plasma protein, belongs to the cystatin superfamily. Cystatins can inhibit cysteine peptidases and play key roles in inflammation.(7) Fetu- in-A (molecular weight of about 60 kDa), also known as α-2 Heremans-Schmid glycoprotein (AHSG), is synthesized by hepatocytes and ubiquitously pres- ent in the serum.(8) Fetuin-A serum concentrations are relatively high (0.5 and 1.0 g/L) in the healthy population.(9) Fetuin-A has been recognized as a ma- jor serum-based inhibitor of vascular and soft-tissue calcification, limiting hydroxyapatite crystal forma- tion and stabilizing calcium–phosphate in a complex, which enables its clearing by the phagocytic system. (9-11) In animal studies, fetuin A-deficient mice devel- oped calcifications in soft-tissue and vessels, acceler- ated by a mineral-rich diet, as well as renal failure.(12) Fetuin-A is a multifunctional molecule with several bio- logic functions such as inhibition of calcification-induc- ing effects of transforming growth factor-β, inhibition 1 Urology Research Center, Tehran University of Medical Sciences, Tehran, Iran. 2 Department of Urology, Bouali Hospital, Islamic Azad University, Tehran Medical Branch, Tehran, Iran. 3 Research Development Center, Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran. *Correspondence: Hassan-Abad Sq., Urology Research Center, Sina Hospital, Tehran, Iran. Tel: +98 21 66348560. Fax: +98 21 66348560. E-mail: rh_mashhadi@yahoo.com. Received October 2014 & Accepted February 2015 Kidney Transplantation 2182 of insulin receptor autophosphorylation, tyrosine kinase activity and protease.(8) Serum fetuin-A reacts as a neg- ative acute phase glycoprotein and is down regulated in acute and chronic inflammatory states.(13) Some studies permit consideration of fetuin-A deficiency as a new mortality risk factor in patients with chronic kidney dis- ease (CKD).(4,9) In hemodialysis patients, serum levels of fetuin-A is significantly lower than controls.(14) In ad- dition, lower fetuin-A is associated with increased vas- cular calcification and inflammation and consequently high cardiovascular mortality.(9,14) Few data was availa- ble regarding effect of fetuin-A in renal transplant recip- ients. One study showed that fetuin-A levels increased after renal transplantation.(15) In another study, no asso- ciation was found between fetuin-A levels and allograft rejection.(9) However, due to the reduction of serum fe- tuin-A levels during inflammation(16) and its inhibitory role in calcification and fibrosis,(8,17) fetuin-A serum levels may be considered as a predictor of allograft out- come and rejection episodes.(4) We assumed that serum fetuin-A levels would be related to the kidney function. Therefore, the aim of this study was to determine serum fetuin-A pattern after renal transplantation and to exam- ine whether fetuin-A is associated with graft outcome. MATERIALS AND METHODS Study Subjects In this single-center prospective study, 65 patients (48 males) who had undergone kidney transplantation were included. Of these patients, 41 were included in the final analysis and 24 were excluded due to incomplete data. Transplantations were performed in Sina Hospital, Iran, from October 2012 to May 2013. There were 30 (73%) deceased donor and 11 (27%) living donor. The follow- ing groups of recipients were excluded from the study: recipients of combined organs such as kidney/pancreas, children and patients with hepatitis B or C. The caus- es of renal failure were diabetic nephropathy (n = 7), hypertensive ischemic nephropathy (n = 16), reflux ne- phropathy (n = 10), nephrotic syndrome (n = 3), chronic nephrolithiasis (n = 3) and unknown etiology (n = 2). An informed consent was obtained from all patients prior to their inclusion in the study. This study was approved by the Medical Ethics Committee of the Tehran Universi- ty of Medical Sciences. Patients’ immunosuppressive protocols were based on calcineurin inhibitor (cyclo- sporine A, 6-7 mg/kg/day) along with mycophenolate mofetil hydrochloride (1200 mg/m2). Additionally, all patients received prednisone prior to transplantation (methylprednisolone, 10 mg/kg prior to transplantation rapidly converting into prednisolone, tapering the dose over 12 weeks to the dose of 0.3 mg/kg); also, anti-thy- mocyte globulin (ATG rabbit, 1-1.5 mg/kg) was giv- en to 5 patients with possible renal allograft rejection. Renal function was evaluated by measuring urine vol- ume and serum creatinine before transplantation and on days 7 and 30 after transplantation (creatinine clear- ance was calculated using the Cockcroft/Gault formu- la). In order to evaluate renal perfusion and function, technetium-99 diethylenetriamine pentaacetic acid (99mTc-DTPA) scan was performed along with Dop- pler ultrasonography at the end of the first week after renal transplantation. Routine laboratory parameters were measured by standard clinical chemistry methods. Serum Fetuin-A Five milliliters of venous blood were withdrawn from every patient. Serum was collected after centrifugation at 2500 g for 20 min and stored at −80°C for subsequent analyses. Serum fetuin-A levels were measured using the enzyme-linked immunosorbent assay (ELISA, BioVen- dor Laboratory Medicine, Brno, Czech Republic) meth- od according to manufacturer’s instructions. The intra- and interassay coefficients of variation were both < 5.0%. Statistical Analysis Categorical variables are expressed as frequencies and percentages and continuous variables are reported as mean ± standard deviation (SD). Subgroups of DTPA were compared on serum fetuin-A at 7th and 30th days after transplantation using repeated measure analysis Figure 1. Individual course of fetuin-A levels from pretranspl- antation up to day 30 after kidney transplantation. Parameters Values Age, years 41.27 ± 12.6 Sex, male % 73.17 Body Mass Index, kg/m2 27.48 ± 2 Smoking, % 51.2 Serum albumin, g/L 4.16 ± 0.69 Serum calcium, mg/dL 9.12 ± 0.79 Serum potassium, mmol/L 4.47 ± 0.53 Systolic blood pressure, mmHg 127.8 ± 21 Diastolic blood pressure, mmHg 79.76 ± 13.69 Serum fetuin levels, mg/L Before transplantation 1543 ± 1372.09 Day 7 465.99 ± 630.34 Day 30 1878.38 ± 1556.11 Serum creatinine levels, mg/dL Before transplantation 5.49 ± 2.06 Day 7 2.38 ± 1.5 Day 30 1.68 ± 0.8 All continuous data are expressed as mean ± SD. Table. Demographic and clinical characteristics of study subjects. Fetuin-A in Renal Transplant Recipients-Mehrsai et al. Vol 12 No 03 May-June 2015 2183 of variance. To assess relationship between serum fe- tuin-A with age and serum creatinine levels the Pearson correlation coefficient was used at different time points. RESULTS Clinical and demographic characteristics of the patients are shown in Table. Based on DTPA scan results at the end of the first week after transplantation, the patients were divided into three subgroups. The first subgroup in- cluded 25 patients with fairly proper renal perfusion and function (DTPA-1). The second subgroup comprised of 11 patients with proper renal perfusion but decreased renal function and excretion (DTPA-2), and the third subgroup is composed of 5 patients who had decreased renal perfusion, function and excretion (DTPA-3). In the first week after transplantation, there was a sig- nificant decrease in serum fetuin-A levels (P < .001), it then started to increase and surpass pretransplant values on day 30 (P < .001) (Figure 1). Further- more, pretransplant serum fetuin-A concentrations were correlated with serum fetuin-A levels on days 7 (r = 0.638, P < .001) and 30 (r = 0.438, P < .004). We evaluated serum levels of fetuin-A over time by ter- tile of DTPA subgroups (Figure 2). Subsequently, we found that changes in serum fetuin-A levels did not dif- fer in patients with different DTPAs (P = .541). In order to investigate whether pretransplant levels of serum fe- tuin-A can be a predictor of renal transplant outcome, the mean changes of pretransplant levels of serum fetuin-A in DTPA subgroups were statistically analyzed and there was no statistically significant difference (P = .461). Also, we performed a correlation analysis in order to evaluate the correlation between serum fetuin-A levels and creatinine levels. There was no significant correla- tion between serum fetuin-A levels and creatinine. Fi- nally, we assessed the role of age in fetuin-A levels and no relationship was observed (Figure 3). Moreover, no association was found between serum fetuin-A levels and gender (P = .337) and type of donors (P = .987). DISCUSSION Renal allograft failure is the result of cumulative dam- age caused by various stressors and factors(18) such as IF/TA, chronic inflammation and calcification process- es.(2) Fetuin-A is one of the most potent calcification inhibitor.(19) Since, soft tissue calcifications occurs in fetuin-A knockout mice,(12) and serum fetuin-A lev- els are reduced in uremic conditions, fetuin-A can be considered as an inhibitor of vascular calcification in healthy people as well as uremic patients.(20) Mori and colleagues showed that serum fetuin-A levels are re- duced in patients with ESRD.(21) Similarly, it has been shown that low serum fetuin-A levels are independently associated with higher overall cardiovascular mortali- ty in patients with renal failure.(22) One of the reasons indicating an association between low serum fetuin-A levels and increased mortality can be due to down reg- ulation of fetuin-A in inflammation.(23) Previous studies have shown that renal transplantation leads to improved vascular function and increased serum levels of fetu- in-A.(24) Hence, it can be expected that improvement in renal function is associated with increased serum lev- els of fetuin-A after kidney transplantation.(9) There- fore we hypothesized that serum fetuin-A concentra- tions increase by the time after renal transplantation. However, the results of our study did not demonstrate an increase of serum fetuin-A levels within the first post-transplant month. Moreover, in the first week af- ter transplantation, we observed a significant decrease in serum fetuin-A levels, although it started to increase and surpass pretransplant values on day 30. Thus, there is an obvious disagreement between study results and study hypothesis. In line with our results, Urbanova and colleagues in a study on 30 deceased donor kidney recipients showed that serum fetuin-A concentrations decreased 2 weeks after kidney transplantation.(9) Sim- ilarly, Argani and colleagues observed lower serum fe- tuin-A concentrations in renal transplant (RT) patients than hemodialysis (HD) patients.(8) In contrast, Caglar and colleagues reported an increase in serum fetuin-A concentrations on days 30 and 90 after transplantation. (15) Moe and colleagues studied calcification inhibitors and reported an increase in serum fetuin-A levels after kidney transplantation in 11 patients with a well-func- tioning allograft.(25) Since serum fetuin-A is involved in inflammatory processes, and inflammation is asso- Figure 2. Changes in serum fetuin-A levels from pretransplantation up to day 30 after transplantation by tertile of diethylenetriamine pentaacetic acid (DTPA) groups. Figure 3. Relationship between serum fetuin-A levels and age. A) Before renal transplantation; B) one week after renal transplantation and C) one month after renal transplantation. Fetuin-A in Renal Transplant Recipients-Mehrsai et al. Kidney Transplantation 2184 ciated with allograft outcome in renal transplant recipi- ents,(26,27) we expected that serum fetuin-A pretransplant serum levels are associated with subgroups of DTPA. However, we found no significant correlation between pretransplant serum fetuin-A levels and DTPA results. This might be due to the few number of subgroups. One of the most important factors for explaining the differences observed in serum fetuin-A behaviors may be systemic inflammation in the early post-transplant period.(28) Severe systemic inflammation may be caused by infection, surgical trauma and immune response against the graft in the early period after transplantation. (9) Previous studies have shown that the down regulation of fetuin-A in inflammation or trauma.(13,28) In addition, serum fetuin-A reacts as a negative acute phase glyco- protein and has a negative association with concentra- tion of the tumor necrosis factor α, C-reactive protein (CRP) and inflammatory cytokines interleukin (IL)-1 β , IL-6,(4,28) supporting the hypothesis of inflammation-de- pendent down regulation of fetuin-A expression.(4) Hence, one possible explanation for our findings could be the difference in age recipients, which leads to a dif- ference in the intensity of postoperative inflammation and stress. The mean age of patients in our study and in Urbanova and colleagues’ study(9) was higher than Caglar and colleagues’ study(15) (41.3, 50.2 and 25.2, respectively). On the other hand, it has been recently shown that serum fetuin-A levels may decrease after a single hemodialysis session, that supports the hypothe- sis of dialysis-induced inflammation. Therefore, serum fetuin-A levels can be affected by possible dialysis ses- sions.(29) Another reason to explain the observed differ- ences may be due to pharmacological interventions (for example, treatment with calcium-phosphate medica- tion) that affect the fetuin-A levels.(30) Coglar and col- leagues showed that short-term sevelamer (phosphate binder) treatment significantly increases serum fetu- in-A levels.(30) In this regard, we cannot ignore the pos- sible impact of vitamin D preparations and the severity of glomerular dysfunction on the serum fetuin-A levels. CONCLUSION Our study demonstrated that fetuin-A levels decrease early after transplantation (day 7) and then start to in- crease in the first month. Fetuin-A down regulation early after transplantation may be caused by inflam- mation and trauma after surgery. However, further studies are needed to better clarify fetuin-A patterns after renal transplantation. 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