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American Journal of Transplantation 2006; 6: 1639–1645  2006 The Authors Journal compilation C  2006 The American Society of Transplantation and the American Society of Transplant Surgeons Urine NGAL and IL-18 are Predictive Biomarkers for
Delayed Graft Function Following Kidney
Transplantation
C. R. Parikha, A. Janib, J. Mishrac, Q. Mac,
C. Kellyc, J. Baraschd, C. L. Edelsteinb
and P. Devarajanc,∗
Acute renal failure (ARF) represents a common and poten-tially devastating problem in clinical medicine, with a persis- aNephrology, Yale University, New Haven, Connecticut, tently high mortality and morbidity (1–3). Renal ischemia- reperfusion injury is the leading cause of ARF in the na- bNephrology, University of Colorado, Denver, Colorado, tive as well as the transplanted kidney. Although advances in basic research have identified successful interventions cNephrology, Cincinnati Children's Hospital Medical in animal models, the lack of early markers for human Center, University of Cincinnati, Cincinnati, Ohio, USA ARF has precluded initiation of therapy in a timely manner dNephrology, Columbia University, New York, USA (1–4). In current clinical practice, ARF is typically diagnosed Corresponding author: P. Devarajan, by measuring serum creatinine. Unfortunately, creatinine is an unreliable indicator during acute kidney injury, and doesnot reflect the degree of damage until a steady state has Delayed graft function (DGF) due to tubule cell
been reached, which may require several days (5).
injury frequently complicates deceased donor kid-
ney transplants. We tested whether urinary neu-
Acute kidney injury due to ischemia-reperfusion occurs trophil gelatinase-associated lipocalin (NGAL) and
to some extent almost invariably in deceased donor re- interleukin-18 (IL-18) represent early biomarkers for
nal allografts, and even in some live donor transplants, of- DGF (defined as dialysis requirement within the first
week after transplantation). Urine samples collected
ten resulting in varying degrees of early renal dysfunction on day 0 from recipients of living donor kidneys (n =
(6). ARF due to delayed graft function (DGF) complicates 23), deceased donor kidneys with prompt graft func-
4–10% of live donor and 5–50% of deceased donor kidney tion (n = 20) and deceased donor kidneys with DGF
transplants (7–9). In addition to the well-known complica- (n = 10) were analyzed in a double blind fashion by
tions of ARF and dialysis, DGF predisposes the graft to both ELISA for NGAL and IL-18. In patients with DGF, peak
acute and chronic rejection (10–12), is an independent risk postoperative serum creatinine requiring dialysis typi-
factor for suboptimal graft function at one year post trans- cally occurred 2–4 days after transplant. Urine NGAL
plant, and increases the risk of chronic allograft nephropa- and IL-18 values were significantly different in the
thy and graft loss (13–20). A variety of clinical algorithms three groups on day 0, with maximally elevated levels
have been proposed for prediction of DGF based on pre- noted in the DGF group (p < 0.0001). The receiver–
operating characteristic curve for prediction of DGF
operative risk factors (21,22), but no objective and validated based on urine NGAL or IL-18 at day 0 showed an area
tools are currently available for the early diagnosis of lesser under the curve of 0.9 for both biomarkers. By mul-
degrees of acute kidney injury following kidney transplan- tivariate analysis, both urine NGAL and IL-18 on day
tation. Several clinical definitions of DGF that employ urine 0 predicted the trend in serum creatinine in the post-
output, creatinine reduction ratios, or dialysis requirement transplant period after adjusting for effects of age, gen-
have been reported in the literature (7–22). However, these der, race, urine output and cold ischemia time (p <
clinical variables typically identify DGF only several days af- 0.01). Our results indicate that urine NGAL and IL-18
ter kidney transplantation. Consequently, early therapeutic represent early, predictive biomarkers of DGF.
interventions that have ameliorated DGF in animal modelshave been ineffective in human studies (6), at least in part Key words: Acute kidney injury, biomarkers, delayed
due to the paucity of early biomarkers for kidney injury.
graft function, interleukin-18
Received 4 November 2005, revised 16 February 2006
We recently utilized a genome-wide interrogation strategy and accepted for publication 6 March 2006
to identify kidney genes that are induced very early afterischemia in animal models, whose protein products mightserve as novel biomarkers for the initiation phase of ARF.
We identified neutrophil gelatinase-associated lipocalin Parikh et al.
(NGAL) as one of the most dramatically up-regulated genes ELISA for IL-18 quantitation
in the kidney after ischemia (23–25). NGAL protein was The urine IL-18 ELISA was performed as previously described (27), using a also markedly induced in kidney tubule cells early after is- human IL-18 ELISA kit (Medical and Biological Laboratories, Nagoya, Japan) chemia in mouse models (24). Importantly, NGAL protein that specifically detects the mature form of IL-18. All measurements were was easily detected in the urine very early after ischemic made in a blinded fashion. The inter- and intra-assay coefficient variations injury in animals, and was found to be a highly predictive were 5–10%, corresponding to that reported by the kit manufacturer. Theresults were expressed in ng/mg creatinine to standardize for changes in biomarker of acute kidney injury in patients undergoing car- diac surgery (26). We therefore tested the hypothesis thaturine NGAL represents a novel early biomarker of renal in-jury in another representative human population, namelypatients undergoing kidney transplantation. Since our pre- vious studies have shown that urine interleukin-18 (IL-18) The results are expressed as means ± SD or medians with ranges. TheSAS 8.2 statistical software (SAS Institute, Cary, NC) was utilized for the levels are also increased in patients with established DGF analysis. NGAL and IL-18 values were noted to have a skewed distribution, (27), a secondary objective of this study was to compare and nonparametric tests were therefore employed. The relationships be- the utility of NGAL and IL-18 measurements for the pre- tween urine NGAL or IL-18 at day 0 and clinical variables were assessed diction of DGF.
by chi-square test (for categorical variables) and analysis of variance (forcontinuous variables). The Kruskal-Wallis test was used to compare livingrelated, deceased donor with prompt graft function, and deceased donor Materials and Methods
DGF. A conventional receiver operating characteristic (ROC) curve was gen-erated for urine NGAL and IL-18 at day 0 post transplant in deceased donor patients with DGF. The area under the curve was calculated to determine This investigation was approved by the Institutional Review Boards of par- the quality of NGAL and IL-18 as predictive biomarkers of DGF. An area ticipating centers. Written informed consent was obtained from each pa- of 0.5 is no better than expected by chance, whereas a value of 1.0 sig- tient or legal guardian before enrollment. Consecutive patients undergo- nifies a perfect biomarker. Univariate and multivariate stepwise multiple ing living related or deceased donor kidney transplantation at our centers logistic regression analyses were performed to assess predictors of DGF.
were prospectively enrolled. The immunosuppressive regimen was similar For some logistic regression models, there was a complete separation of in all patients, consisting of tacrolimus with prednisone and mycopheno- points and the maximum likelihood estimates did not exist. Ad hoc ad- late mofetil. Spot urine samples were collected within the first 24 hours justments were therefore used to fit the final logistic models. Potential (day 0) following transplantation. In the majority of patients, the urine sam- independent predictor variables included age, gender, race, donor source, ple was obtained soon after admission to the hospital unit, prior to admin- urine NGAL on day 0 post transplant, urine IL-18 on day 0 post transplant, istration of tacrolimus, and no patient received more than a single dose of creatinine reduction ratio (defined as a percentage decrease in serum cre- tacrolimus prior to urine collection. Urine samples were centrifuged at 2000 atinine in the first 2 days post transplant) and cold ischemia time. Rather g for 5 min, and the supernatants stored in aliquots at −80◦C. Serum creati- than analyzing as continuous variables, we categorized urine output (as ≥ nine was measured at baseline, just before the kidney transplantation, and or < 1 L/day) and creatinine reduction ratio (as ≥ or < 30%), as previ- routinely monitored at least daily in the postoperative period. The primary ously described in studies of DGF 28. Univariate and multivariate analysis outcome variable was the development of DGF, defined as the need for for predicting trends in serum creatinine were calculated using general- dialysis within the first week after transplantation. The decision to initiate ized linear regression with repeated measures analysis, with Greenhouse- dialysis originated from the primary transplant nephrologists and transplant Geisser correlation for p-values. A p-value of <0.05 was considered surgeons, without any involvement from the study investigators. Other vari- ables collected included age, gender, race, original kidney disease, cold is-chemic time, urine output, serial serum creatinines and urine creatinine.
ELISA for NGAL quantitation
The urine NGAL ELISA was performed as previously described (26). Briefly,microtiter plates pre-coated with a mouse monoclonal antibody raised against human NGAL (HYB211–05, AntibodyShop, Gentofte, Denmark) A total of 53 patients were included in the study, whose were blocked with buffer containing 1% BSA, coated with 100 lL of sam- demographic characteristics, diagnoses and outcome vari- ples (urine or serum) or standards (NGAL concentrations ranging from 1–1000 ng/mL), and incubated with a biotinylated monoclonal antibody against ables are shown in Tables 1–3. Urine samples obtained human NGAL (HYB211–01B, AntibodyShop) followed by avidin-conjugated within the first 24 hours after transplantation were avail- HRP (Dako, Carpinteria, CA, USA). TMB substrate (BD Biosciences, San able from 23 patients with living related donor (LRD, Jose, CA, USA) was added for color development, which was read after 30 Table 1), 20 subjects with deceased donor and prompt graft min at 450 nm with a microplate reader (Benchmark Plus, BioRad, Hercules, function (CAD, Table 2) and 10 individuals with deceased CA, USA). All measurements were made in triplicate. Pre-coated plates can donor and delayed graft function (CAD DGF, Table 3). There be refrigerated and used for several days, and the entire ELISA procedure were no differences between these three groups in gen- is typically completed in 4 h. The inter- and intra-assay coefficient varia- der, race, original kidney disease or serum creatinine on tions were 5–10% for batched samples analyzed on the same day. The day 0. The LRD group tended to be slightly younger in age, laboratory investigators were blinded to the sample sources and clinical but the ages of the CAD group with prompt graft function outcomes until the end of the study. Urine creatinine was measured usinga quantitative colorimetric assay kit (Sigma, St. Louis, MO, USA), and urine and DGF were similar. In patients with DGF, peak postoper- NGAL was expressed in ng/mg creatinine to standardize for changes in urine ative serum creatinine requiring dialysis typically occurred 2–4 days after transplant.
American Journal of Transplantation 2006; 6: 1639–1645 NGAL and IL-18 in DGF
Table 1: Clinical characteristics of patients with living related donor (LRD) kidney transplantation and prompt graft function
Pt ID = patient identification; Orig dis = original disease; FSGS = focal segmental glomerulosclerosis; GN = glomerulonephritis; HTN =hypertension; AA = African-American.
Table 2: Clinical characteristics of patients with deceased donor (CAD) kidney transplantation and prompt graft function
Pt ID = patient identification; Orig dis = original disease; FSGS = focal segmental glomerulosclerosis; GN = glomerulonephritis; DM =diabetes mellitus; HTN = hypertension; AA = African-American.
Urine NGAL and IL-18 measurements—ELISA
0.05 ng/mg creatinine, n = 71). Median urinary NGAL val- Urine NGAL levels were consistently low in healthy volun- ues were significantly different in the three groups on day teers (0.2 ± 0.05 ng/mg creatinine, n = 10) and in hospital- 0 (p < 0.0001, Figure 1). Similar results were noted for uri- ized control subjects with normal kidney function (0.16 ± nary IL-18 values, as previously published (27). Both urine American Journal of Transplantation 2006; 6: 1639–1645 Parikh et al.
Table 3: Clinical characteristics of patients with deceased donor (CAD) kidney transplantation and delayed graft function (DGF)
Pt ID = patient identification; Orig dis = original disease; FSGS = focal segmental glomerulosclerosis; GN = glomerulonephritis; DM =diabetes mellitus; HTN = hypertension; ADPKD = autosomal dominant polycystic kidney disease; AA = African-American.
Table 4: Summary of baseline and clinical characteristics in de-
ceased donor transplants Serum creatinine at Serum creatinine at Serum creatinine at Type and function of the graft
Fractional decrease in s creat > 30%, Figure 1: Urinary NGAL levels in the first 24 h after kidney
transplantation. Box and whisker plots show the 10th, 25th, 50th
Urine output > 1 L, (median), 75th and 90th percentile values. Median urinary NGAL levels (ng/mg creatinine) were greater in patients who received Cold ischemia time a deceased donor kidney and subsequently developed delayed graft function (deceased-DGF; n = 10) compared with patients 756 (12–2500) 0.0017∗ who received a deceased donor kidney with prompt graft function (deceased-PGF; n = 20) and with patients who received living donor kidneys and showed prompt graft function (living-PGF, n = IL-18, day 0, pg/mg, 217 (33.5–1005) 38.75 (0–773) 23; p < 0.0001).
†Fractional decrease in serum creatinine calculated by serumcreatinine day 0 - serum creatinine day 1/serum creatinine day NGAL and IL-18 values on day 0 were highest in patients 0∗100 (if serum creatinine on day 1 is greater than serum creatinine with deceased donor kidney transplants who subsequently on day 0 then the fractional decrease in serum creatinine will be developed DGF (Table 4).
∗p-value by Wilcoxon rank-sum test.
NGAL and IL-18 for prediction of acute renal injury
Table 4 demonstrates baseline and clinical parameters in
patients with deceased kidney transplantation, with either
urinary NGAL and IL-18 values were significantly different DGF or prompt graft function. Age, gender, race, cold is- in the two groups.
chemia time and urine output (< 1 L/day) were not sig-nificantly different in the two groups. Only 30% (six pa- A univariate analysis of our data revealed that the follow- tients) displayed a fractional decrease in serum creatinine ing outcomes were not predictive of DGF: age, gender, of > 30% in the group with prompt graft function. Median race and serum creatinine on day 0. By multivariate logistic American Journal of Transplantation 2006; 6: 1639–1645 NGAL and IL-18 in DGF
Table 5: Multivariate analysis for predicting DGF after deceased
ROC Curve
donor transplantation Fractional s creat decrease Urine output <1 L Cold ischemia time (h) Urine NGAL, 100 ng/mg regression analysis, urine NGAL on day 0 predicted DGF in the early posttransplant period, after adjusting for ef- fects of fractional decrease in serum creatinine, urine out-put and cold ischemic time (p = 0.01). Every 100 ng/mgincrease in urine NGAL was associated with an increased odds of DGF by 20%, after adjusting for other variables(Table 5).
Since dialysis requirement represents a delayed definition of DGF, we also studied the trends in serum creatinine dur-ing the first three postoperative days. Lower urine NGAL 1 - Specificity
values on day 0 predicted a steeper postoperative declinein serum creatinine on a multivariate generalized linearmodel with repeated measures testing (p < 0.01), after adjusting for age, gender, urine output and cold ischemia time. A similar finding was noted for urine IL-18, as pre-viously reported (27). An ROC curve was constructed to determine the discriminatory power of urine NGAL mea- surements on day 0 for the early prediction of DGF. The area under the curve was 0.9 (CI 0.71–1.0) at day 0 posttransplant and the performance of NGAL with respect to derived sensitivities, specificities and predictive values at different cut-off levels are shown in Figure 2. A cut-off of 1000 ng/mg creatinine yielded the optimal sensitivityand specificity at day 0 post transplant. For urine IL-18, the area under the curve for predicting DGF was also 0.9(CI 0.81–0.98). The sample size was insufficient to con- Figure 2: Performance of urine NGAL on day 0 for prediction
duct a multivariate analysis of a combination of the two of DGF. The area under the receiver operating characteristic curve
was 0.90, indicative of an excellent biomarker. The table belowshows the range of sensitivities and specificities for various cut-offs of NGAL. The sensitivity and specificity of NGAL are optimal at a cut-off of 1000 ng/mg.
Our major findings are that urinary NGAL and IL-18 mea-sured on Day 0 of deceased donor kidney transplantation 25 mL/min on postoperative day 7 (29). The present study predict DGF earlier than other clinical definitions that are adds urinary NGAL to the short list of promising biomarkers currently in use. There has been an active quest for urinary for allograft dysfunction that can be noninvasively assayed biomarkers that predict DGF. We have previously demon- for during the early posttransplant period.
strated that urinary levels of IL-18 increase within 24 hin patients with deceased donor transplants who subse- The potential clinical utility of our findings lies in the fact quently developed DGF (27). The present study confirms that several modalities of therapy have succeeded in ani- these previous observations in a larger cohort of patients.
mal models of ischemia-reperfusion injury, but have gen- Similarly, elevated urinary IL-6, IL-8 and actin levels have erally been ineffective in human studies of delayed graft been demonstrated on day 0 in nine recipients of a de- function (6). This may be, at least in part, due to the lack ceased donor renal allograft who displayed sustained acute of reliable methodologies to distinguish between initial de- renal failure (defined as a creatinine clearance of less than grees of kidney injury. It is likely that the availability of early American Journal of Transplantation 2006; 6: 1639–1645 Parikh et al.
predictive biomarkers such as urine NGAL will allow for a Translational Research Initiative Grant from Cincinnati Children's Hospital more accurate stratification of acute kidney injury and for Medical Center. The authors would like to thank Heather Thissen-Philbrook the rational selection of patients who might benefit most for her assistance with the statistical analysis.
from a variety of interventions. Based on this report, it is en-visioned that urinary NGAL and IL-18 determination may be relevant to patients receiving deceased donor transplants.
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Dr. Parikh is supported by NIH/NIDDK (K23-DK064689). Dr. Barasch is sup- 18. Prommool S, Jhangri GS, Cockfield SM, Halloran PF. Time de- ported by NIH/NIDDK (RO1-DK55388, RO1 DK-58872) and a Research pendency of factors affecting renal allograft survival. J Am Soc Grant from the March of Dimes Foundation. Dr. Edelstein is supported Nephrol 2000; 11: 565–573.
by NIH/NIDDK (RO1-DK56851, PO1-DK34039). Dr. Devarajan is supported 19. Halloran PF, Hunsicker LG. Delayed graft function: state of the art, by NIH/NIDDK (RO1-DK53289, P50-DK52612, R21-DK070163), a Grant- Nov 10–11, 2000. Summit Meeting, Scottsdale, Arizona, USA. Am in-Aid from the American Heart Association Ohio Valley Affiliate, and a J Transplant 2001; 1: 115–120.
American Journal of Transplantation 2006; 6: 1639–1645 NGAL and IL-18 in DGF
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transplant recipients. J Am Soc Nephrol 2003; 14: 2967–2974.
27. Parikh CR, Jani A, Meinikov VY, Faubel S, Edelstein CL. Urinary 22. Brier ME, Ray PC, Klein JB. Prediction of delayed renal allograft Interleukin-18 is a marker of human acute tubular necrosis. Am J function using an artificial neural network. Nephrol Dial Transplant Kidney Dis 2004; 43: 405–414.
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28. Rodrigo E, Ruiz JC, Pinera C et al. Creatinine reduction ratio on 23. Supavekin S, Zhang W, Kucherlapati R, Kaskel FJ, Moore LC, post-transplant day two as criterion in defining delayed graft func- Devarajan P. Differential gene expression following early renal tion. Am J Transplant 2004; 4: 1163–1169.
ischemia-reperfusion. Kidney Int 2003; 63: 1714–1724.
29. Kwon O, Molitoris BA, Pescovitz M, Kelly KJ. Urinary actin, 24. Mishra J, Ma Q, Prada A et al. Identification of NGAL as a novel interleukin-6, and interleukin-8 may predict sustained ARF after urinary biomarker for ischemic injury. J Am Soc Nephrol 2003; 4: ischemic injury in renal allografts. Am J Kidney Dis 2003; 41: 1074– 25. Devarajan P, Mishra J, Supavekin S, Patterson LT, Potter SS.
30. Mishra J, Mori K, Ma Q et al. Amelioration of ischemic acute renal Gene expression in early ischemic renal injury: Clues towards injury by NGAL. J Am Soc Nephrol 2004; 15: 3073–3082.
American Journal of Transplantation 2006; 6: 1639–1645

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