Urinary Calprotectin and Posttransplant Renal Allograft Injury

Objective Current methods do not predict the acute renal allograft injury immediately after kidney transplantation. We evaluated the diagnostic performance of urinary calprotectin for predicting immediate posttransplant allograft injury. Methods In a multicenter, prospective-cohort study of 144 incipient renal transplant recipients, we postoperatively measured urinary calprotectin using an enzyme-linked immunosorbent assay and estimated glomerular filtration rate (eGFR) after 4 weeks, 6 months, and 12 months. Results We observed a significant inverse association of urinary calprotectin concentrations and eGFR 4 weeks after transplantation (Spearman r = −0.33; P<0.001). Compared to the lowest quartile, patients in the highest quartile of urinary calprotectin had an increased risk for an eGFR less than 30 mL/min/1.73 m2 four weeks after transplantation (relative risk, 4.3; P<0.001; sensitivity, 0.92; 95% CI, 0.77 to 0.98; specificity, 0.48; 95% CI, 0.31 to 0.66). Higher urinary calprotectin concentrations predicted impaired kidney function 4 weeks after transplantation, as well as 6 months and 12 months after transplantation. When data were analyzed using the urinary calprotectin/creatinine-ratio similar results were obtained. Urinary calprotectin was superior to current use of absolute change of plasma creatinine to predict allograft function 12 months after transplantation. Urinary calprotectin predicted an increased risk both in transplants from living and deceased donors. Multivariate linear regression showed that higher urinary calprotectin concentrations and older donor age predicted lower eGFR four weeks, 6 months, and 12 months after transplantation. Conclusions Urinary calprotectin is an early, noninvasive predictor of immediate renal allograft injury after kidney transplantation.


Introduction
The immediate function of a renal allograft within the first weeks after kidney transplantation may vary considerably. The renal allograft may immediately start urine production. In contrast, the renal allograft may be damaged. An impaired renal allograft function within the first weeks or within the first year is detrimental to the longevity of the allograft and accounts for a 40% decrease in long-term graft survival [1][2][3][4][5]. A major role of ischemia-reperfusion injury for impaired renal allograft function has been confirmed in the literature [6].
Current methods do not predict the acute renal allograft injury immediately after kidney transplantation [7]. Recently, urinary calprotectin concentrations have been identified as a marker of acute kidney injury in the non-transplant population [8,9]. Calprotectin is a calcium-binding complex of two proteins of the so-called S100 group. It serves as a mediator protein of the innate immune system. Calprotectin is derived predominantly from neutrophils and mononuclear cells and activates Toll-like receptor 4 thereby amplifying inflammatory activity [10]. The innate immune system plays a crucial role in the pathophysiology of ischemia-reperfusion injury in the kidney. Neutrophils and macrophages migrate into the transplant within 6 h of reperfusion and release proinflammatory cytokines and other soluble inflammatory mediators [11]. After ischemia-reperfusion injury of the tubular epithelial cells, Toll-like receptor 4 maintains and amplifies the inflammatory response [12]. In the present study we tested the hypothesis that urinary calprotectin at the first postoperative days predicts acute renal allograft injury and allograft function, i.e. estimated glomerular filtration rate (eGFR) 4 weeks, 6 months, and 12 months after transplantation.

Approval
The study protocol was in accordance with the ethical standards of the Declarations of Helsinki and Istanbul. The study was approved by the local ethics committees (Den Videnskabsetiske Komite for Region Syddanmark, Projekt-ID:  and Ethik-Komitee Charité Universitä tsmedizin Berlin).

Study population
We performed a multicenter prospective-cohort study of 144 patients receiving kidney transplants. Patients who were at least 18 years old and who were scheduled to receive living donor kidney transplants or deceased donor kidney transplants were recruited. Written informed consent was obtained from all patients before entry into the study. Baseline characteristics of donors and recipients and information on organ procurement were prospectively obtained from medical records. Induction therapy, immunosuppressive therapy, concomitant medications, and transplant biopsy were all made by the clinicians at each institution according to their local protocols. Delayed graft function was defined by at least one dialysis session within 7 days of transplantation [13]. Hemodialysis within 1 week after transplantation was performed because of uremic symptoms (29% of cases), hypervolemia causing dyspnoe (50% of cases), hyperkalemia (21% of cases). The treating physicians were unaware of the calprotectin concentrations. During follow up, one recipient died during the first year posttransplant due to a cardiovascular event. Six recipients lost graft function during the first year posttransplant. eGFR was available after 12 months in 123 patients.
Enzyme-linked immunosorbent assay for urinary calprotectin and urinary kidney injury molecule-1 We collected one urine sample postoperatively. We aliquoted urine supernatants into cryovials, labeled each with a random barcode, and stored samples at 220uC. The enzyme-linked immunosorbent assay (ELISA) for urinary calprotectin was performed as previously published by our group using the PhiCal-Calprotectin assay kit (Immundiagnostik AG, Bensheim, Germany) according to the manufacturer's protocol [8,9]. Calprotectin concentrations were measured by personnel blinded to patient information. The coefficient of variation was less than 6% Urinary kidney injury molecule-1 (KIM-1) concentrations was measured using ELISA kits according to the manufacturer's protocol (dianova, Hamburg, Germany). The coefficient of variation was less than 10%.

Assessment of allograft function
Plasma creatinine was routinely measured. The absolute change in plasma creatinine was calculated as plasma creatinine preoperatively minus the first postoperative day. Four weeks, 6 months, and 12 months after transplantation we determined estimated glomerular filtration rate (eGFR) in kidney recipients according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation [14]. eGFR = 1416min(Cr/k,1) a 6max(Cr/k,1) 21 where Cr is plasma creatinine in mg/dL, k is 0.7 for females and 0.9 for males, a is 20.329 for females and 2 0.411 for males, min indicates the minimum of Cr/k or 1, and max indicates the maximum of Cr/k or 1.
CKD-EPI equation is recommended in transplanted patients because it more accurately gives true glomerular filtration rate measured by plasma clearance of (99 m)Tc-diethylenetriamine pentaacetic acid [15].

Histological findings
In 98 patients a baseline biopsy was performed at transplantation as benchmark for comparison of post-transplantation histology in case a post-transplant indication biopsy was necessary. Median number of glomeruli in the baseline biopsy was 7 (IQR, 3 to 11). The severity of lesions (arteriolar hyalinosis, arteriolosclerosis, interstitial fibrosis) was scored semiquantitatively as published [16,17].

Statistics
Continuous data are presented as median and interquartile range (IQR). Non-parametric Mann-Whitney test or Kruskal-Wallis test with Dunn's Multiple Comparison Test were used as appropriate to detect differences between the groups. Frequency counts were calculated for categorical data. Differences in these categorical variables between the groups were analyzed by Chisquare test or Fisher's exact text as appropriate. Associations between variables were determined using non-parametric Spearman correlation. We performed receiver operating characteristic (ROC) curve analysis to detect the accuracy of urinary calprotectin, urinary KIM-1, and absolute change in plasma creatinine for predicting eGFR after transplantation.
We performed linear regression for associations between urinary calprotectin and eGFR four weeks after kidney transplantation while adjusting for donor age, donor gender, living-donor vs. deceased-donor status, recipient age, recipient gender, delayed graft function, recipient months on dialysis before transplantation, and use of prednisolone. For regression analyses urinary calprotectin concentrations were log-transformed to obtain normal distributed data to obtain normal distributed data as tested by Kolmogoroff-Smirnov-test. Multivariate models were constructed with backward variable selection, using P,0.05 for variable retention.
Data were analyzed using GraphPad prism software (version 5.0, GraphPad Software, San Diego, CA, USA) and SPSS for windows (version 15.0; SPSS, Chicago, IL, USA). All statistical tests were two-sided. Two-sided p-values less than 0.05 were considered to indicate statistical significance.
We observed that urinary calprotectin was superior to current use of absolute change of plasma creatinine to predict allograft function. Urinary calprotectin resulted in an AUC of 0.76 (95% CI, 0.65 to 0.88; P,0.001) to predict eGFR less than 30 mL/ min/1.73 m 2 12 months after transplantation, whereas the   Tables 2 and 3. Multivariate linear regression showed that higher urinary calprotectin concentrations (P,0.001), older donor age (P,0.001), and delayed graft function (P = 0.039) predicted lower eGFR four weeks after transplantation, but not donor gender, living-donor vs. deceased-donor status, recipient age, recipient gender, recipient months on dialysis before transplantation, or prednisolone. As shown in Table S1 using mixed model analyses for estimated glomerular filtration rate 4 weeks after kidney transplantation confirmed these results. We also found that that higher urinary calprotectin concentrations and older donor age predicted lower eGFR 6 months as well as 12 months after transplantation.
We also analyzed urinary calprotectin levels in a subgroup of 38 patients who were anuric before renal transplantation. We would like to emphasize that that subgroup of 38 patients were anuric before renal transplantation, but not after transplantation. 5 out of 10 previously anuric patients (50.0%) with urinary calprotectin concentrations in the highest quartile showed a creatinine clearance in the lowest quartile. On the other hand, none out of 10 previously anuric patients (0.0%) with urinary calprotectin concentrations in the lowest quartile showed a creatinine clearance in the lowest quartile (P = 0.03).

Discussion
In the present study we showed that postoperative urinary calprotectin predicts kidney allograft function after transplantation. We observed that use of urinary calprotectin was superior to current use of absolute change of plasma creatinine to predict allograft function after transplantation. It is noteworthy to acknowledge the biologically plausible role of calprotectin in the mechanisms of ischemic reperfusion injury after kidney transplantation. The tubular damage leads to a secondary activation of the innate immune system with immigration of granulocytes and monocytes. Moreover it leads to an amplification and activation of toll-like receptors [18]. Calprotectin is a ligand of toll-like receptors, which is constitutively expressed in both proximal and distal tubules [19]. Hall et al. [20] reported that neutrophil gelatinase-associated lipocalin predicted dialysis within one week after kidney transplantation. They reported that 34 out of 91 patients (37%) had delayed graft function, defined by at least one dialysis session within the first postoperative week [20]. However, it is well-known that the clinical decision to perform postoperative hemodialysis may be influenced by several factors beyond renal allograft function, for example fluid overload and clinical uremic status of the patient.
Looking at the results for urinary calprotectin and urinary KIM-1 it may be speculated that an increased neutrophil and macrophage response to ischemia-reperfusion injury may cause increased calprotectin levels. This may be supported by the finding that urinary calprotectin but not urinary KIM-1 was significantly associated with renal allograft function.
A study by Szeto et al. in 63 kidney transplant recipients investigated urinary mRNA of neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, interleukin-18, surfactant protein-C, and S100 calcium-binding proteins A8 and A9 according to histologic groups observed in biopsies from biopsy because of progressive worsening of allograft function. They showed that urinary mRNA of KIM-1 may provide prognostic information about renal function decline, irrespective of the kidney pathology [21]. No data are available for urinary calprotectin protein and pathology because of progressive worsening of allograft function.
The present study confirms previous reports that donor age has a large impact on allograft function. However, there is no treatment to adjust for the fact of using allografts from older subjects. It should be noted that urinary calprotectin predicted allograft function even after adjustment for donor age. Hence, measurements of urinary calprotectin may add to the diagnostic procedures.
Beside the use of urinary calprotectin for prediction of immediate renal allograft injury, its determination immediately after kidney transplantation may be helpful and a necessary step for the implementation of future strategies to improve outcome after kidney transplantation. In summary, urinary calprotectin is an early, noninvasive predictor of acute renal allograft injury after kidney transplantation.