Lipocalin: An Independent Predictor of Adverse Outcomes in Acute Kidney Injury

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1 American Journal of Nephrology O riginal Re por t : Pat ie nt- O rie nte d, Translat ional Research A m J N e p h r o l 2010 ; 31: DOI: / Received: December 6, 2009 Accepted: March 30, 2010 Published online: May 4, 2010 Urine N e ut rophil G e lat inase -A ssociate d Lipocalin: An Independent Predictor of Adverse Outcomes in Acute Kidney Injury Ha Na Yang Chang Soo Boo Myung-Gyu Kim Sang-Kyung Jo Won Yong Cho Hyoung-Kyu Kim D i v i s i o n o f N e p h r o l o g y, D e p a r t m e n t o f I n t e r n a l M e d i cine, Korea University Anam Hospital, Korea University College of Medicine, Seoul, Korea Key Words Ac u te k i d n e y i nj u r y Pro g n o sis U r i n e n e u t r o p h i l gelatinase-associated lipocalin Abstract Background: S eve r a l r e ce nt s t u d i e s h ave s h ow n t h at n e u - trophil gelatinase-associated lipocalin (NGAL) may be a promising biomarker for the early detection of acute kidney injury (AKI), but the role of NGAL in predicting adverse clinical outcomes has not been well addressed. The purpose of this study was to evaluate the usefulness of urine NGAL as outcome predictor in patients with AKI. Methods: This was a prospective cohort study enrolling hospitalized AKI patients. Patients were divided into four groups according to initial urine NGAL excretion quartiles. The primary clinical outcome variables were in-hospital mortality and renal function at 4 weeks. Results: Initial urine NGAL was identified as an independent predictor of in-hospital mortality and persistent loss of renal function. In the analysis of predictive performance of urine NGAL, the AUC was and a cutoff v a l u e o f n g /m l p r e d i c te d l o ss o f r e n a l f u n c t i o n w i t h 88.2% sensitivity and 81.0% specificity. Conclusion: This study could suggest that urine NGAL, a new early biomarker might also be served as a reliable clinical outcome predictor in AKI patients. C o p y r i g h t S. K a r g e r AG, B a s e l Introduction Acute kidney injury (AKI) is a significant medical problem with high morbidity and mortality [1]. In clinical practice, AKI is diagnosed based upon changes in serum creatinine or urine output over a short period of time. However, creatinine is not a reliable indicator nor does creatinine represent the degree of kidney damage in acute stage. Lack of an accurate definition and grading system for AKI has made it difficult to conduct largescale multicenter clinical trials, interpret the results of clinical trials and develop prognostic scores. In 2004, the Acute Dialysis Quality Initiative (ADQI) Group proposed a new definition/classification system for AKI called the RIFLE criteria [2], and several recent studies have demonstrated the usefulness of this new definition/ classification system in predicting adverse outcomes from AKI, especially in critically ill patients [3 5]. Rec ent ly, se ver a l prom i si ng u r i na r y biom a rk e r s, including neutrophil gelatinase-associated lipocalin (NGAL) [6], kidney injury molecules-1 [7] and interleukin-18 (IL-18) [8], for the early detection of AKI have been Dr. H.N. Yang and Dr. C.S. Boo contributed equally to this study. Fax karger@karger.ch S. Karger AG, Basel /10/ $26.00/0 Accessible online at: Sang-Kyung Jo, MD, PhD, Assoc. Prof. Department of Internal Medicine Korea University Anam Hospital 126-1, 5-Ka, Anam-Dong, Seongbuk-Ku, Seoul (Korea) Tel , Fax , korea.ac.kr

2 discovered and their usefulness in diagnosing AKI before the rise in serum creatinine has been reported. In addition to facilitating the early diagnosis of AKI, there is also an urgent need for the identification of ideal biomarkers to predict adverse clinical outcomes of AKI. Relatively few studies have examined the usefulness of various urine biomarkers as outcome predictors [9, 10]. The aim of this study was to examine whether urinary excretion of NGAL at the time of diagnosis of AKI could independently predict adverse clinical outcomes in hospitalized patients with AKI. Subjects and Methods Study Population This study was a prospective cohort study of hospitalized patients with AKI conducted between June 2007 and July 2008 at the Korea University Anam Hospital, a tertiary teaching hospital in Seoul, Korea. The patients who were admitted or referred to the Nephrology Department because of a clinical diagnosis of AKI with varying degrees of renal dysfunction and with different types of injuries were eligible for enrollment. The investigation was approved by institutional review board of this center and written informed consent was obtained from each patient or legal guardian before enrollment. Patients under 18 years of age on chronic dialysis, or with a history of kidney transplantation were excluded. Definitions AKI was diagnosed according to the RIFLE criteria. We used the rise in serum creatinine concentration or a decline in glomerular filtration rate (GFR) for patients to be assigned into categories according to the RIFLE classification. The estimated GFR (egfr) was calculated using the MDRD equation. For patients with a history of chronic kidney disease, we used the lowest known serum creatinine value during the preceding 3 months as the baseline from our hospital database. For other patients, we tried to obtain their baseline serum creatinine value from recent records from other hospitals or from their routine laboratory examination annually provided by the National Health Service of Korean Medical Insurance System which is adopting service aimed at the whole population. In some patients, the lowest creatinine during hospitalization was used as the baseline value. The primary clinical outcome variables were in-hospital mortality and renal function 4 weeks after enrollment, classified into recovery or persistent loss. Recovery was defined by the return of the creatinine or egfr to baseline and persistent loss was defined by failure of the creatinine or egfr to return to baseline, or a requirement for maintenance renal replacement therapy (RRT). The secondary clinical outcome variable was the requirement for RRT during the course of treatment. Data Collection The patient demographic data, etiology of AKI, comorbid conditions, requirement for RRT, in-hospital mortality and renal outcome at 1 month of enrollment were prospectively collected. All of the laboratory data were obtained at the time of admission to the hospital or at the time of consultation to the nephrology department. Urine and serum samples were centrifuged at 2,500 rpm at 4 C for 5 min and the supernatants were stored at 80 C until the NGAL assay was performed. Measurement of NGAL Serum and urine NGAL were measured using a NGAL ELISA kit (BioPorto, Gentofte, Denmark), according to the manufacturer s instructions. The detection limit for the assay was between 0.5 and 4.0 pg/ml. The amount of urine NGAL excretion was expressed as ng/ml and for comparison, urine NGAL normalized to mg creatinine (ng/mg creatinine) was also obtained. Statistics All statistical analysis was performed by SPSS version 12.0 (SPSS Inc.). Data were expressed as the mean 8 s t a nd a rd d e v i at ion for normally-distributed data or median (interquartile range) for non-normally distributed data. Comparison between groups was analyzed by an ANOVA test or Kruskal-Wallis test in case of numerical data, and 2 test or Fisher s exact test in case of categorical data. Multiple comparisons were controlled by using the Bonferroni correction. We conducted univariate and multivariate logistic regression analyses to assess the impact of different baseline characteristics at admission on the outcomes. We could select some significant factors in multivariate logistic regression by the backward selection based on likelihood ratio (LR). To further examine the predictive performance of urinary NGAL in short-term renal outcome from AKI, we plotted receiver operating characteristics (ROC) curve by using sensitivity and specificity of urinary NGAL. Results Patient Characteristics One hundred patients with a clinical diagnosis of AKI were enrolled. The mean age was 60.2 years, 58% were males and the etiologies of AKI were diverse (ischemic, toxic, sepsis and multifactorial). Because patients with varying degrees of severity were enrolled, we did not obtain any acute illness score that is usually used in patients in ICU. The severity of illness of the patient cohort in this study was as follows: 30% of patients needed ICU admission during the course of admission and 13% needed mechanical ventilator support. RRT was provided to 32% of the patients and the overall in-hospital mortality was 12%. Failure of renal recovery assessed 1 month after enrollment occurred in 35% of the patients. For further characterization of the patient cohorts in our study, we separately analyzed patients who needed RRT in comparison with patients who did not require RRT during the course of hospitalization. As expected, patients who were dependent on RRT had a significantly increased prevalence of diabetes and increased initial serum creatinine 502 Am J Nephrol 2010;31: Yang/Boo/Kim/Jo/Cho/Kim

3 Table 1. Patient characteristics according to urine NGAL excretion quartiles (n = 100) ng/ml: 1st quartile (n = 24) ( ) 2nd quartile (n = 25) ( ) 3rd quartile (n = 24) ( ) 4th quartile (n = 24) ( ,788.3) p Gender 3, male 19 (79.2) 15 (60.0) 12 (52.2) 11 (45.8) Age 1, years DM 3 3 (12.5) 6 (24) 9 (39.1) 11 (45.8) Hypertension 3 14 (58.3) 12 (48) 14 (60.9) 15 (62.5) Proteinuria 3 3 (12.5) 2 (8) 10 (41.7) 14 (58.3) <0.001 Cause of AKI Multifactorial 13 (54.2) 15 (60) 14 (58.3) 6 (25.0) Ischemic 6 (25) 9 (36) 3 (12.5) 4 (16.7) Sepsis 1 (4.2) 1 (4) 3 (12.5) 11 (45.8) Nephrotoxic 4 (16.7) 0 2 (8.3) 1 (4.2) Baseline Cr 2, mg/dl 1.0 ( ) 1.1 ( ) 1.8 ( ) 1.72 ( ) Initial Cr 2, mg/dl 2.45 ( ) 2.9 ( ) 4.1 ( ) 5.15 ( ) a Initial egfr 2, ml/min/1.73 m ( ) 24.0 ( ) 14.3 ( ) 10.1 ( ) Initial BUN 2, mg/dl 38.0 ( ) 43.0 ( ) 57.0 ( ) 60.0 ( ) RIFLE criteria Risk 9 (37.5) 5 (20.0) 7 (30.4) 1 (4.2) Injury 6 (25.0) 8 (32.0) 3 (13.0) 6 (25.0) Failure 9 (37.5) 12 (48) 13 (56.5) 17 (70.8) Mortality (4.0) 5 (20.8) 6 (25.0) RRT 3 3 (12.5) 3 (12.0) 8 (34.8) 16 (66.7) a, b Recovery (1 month) 3 23 (95.8) 23 (92) 11 (45.8) a, b 6 (25.0) a, b <0.001 p values were obtained by ANOVA or Kruskal-Wallis test in case of numerical data and 2 or Fisher s exact test in case of categorical data. a p < 0.05 (by Bonferroni correction) vs. 1st quartile; b p < 0.05 (by Bonferroni correction) vs. 2nd quartile. Cr = Creatinine; BUN = blood urea nitrogen. 1 Means 8 SD. 2 Median (IQR). 3 Numbers of patients (%). level, and 87.5% of patients with RRT fell into the failure category of RIFLE. Sepsis was the predominant cause of AKI in patients who required RRT (40.6%), while only 6% of patients who did not require RRT had sepsis as the cause of AKI (p! 0.001). The rate of ICU admission was significantly higher in patients who underwent RRT (78.1 vs. 28.4%, p! 0.001). Mortality rate and failure of recovery of renal function was also significantly higher in patients who needed RRT (mortality: 34.4 vs. 1.5%, p! 0.001; recovery of kidney function: 28.1 vs. 83.6%, p! 0.001). Patient Characteristics according to Initial Urinary NGAL Excretion Initial urinary NGAL excretion varied between 3.1 and 1,788.3 ng/ml. We divided patients into quartiles according to the urine NGAL excretion and the baseline characteristics and clinical outcomes were compared ( table 1 ). In comparison with the baseline characteristics, age, gender and the presence of comorbid conditions were not significantly different. However, the cause of AKI was different among the four groups. Sepsis was the main cause of AKI in patients who belonged to the 4th quartile of urine NGAL. The initial serum creatinine was significantly higher in patients who belonged to the 4th quartiles of urine NGAL excretion (p = 0.002, Bonferroni s correction). Although the proportion of failure in the RIFLE classification was high (70.8%) in the 4th quartile, it was not statistically significant. In univariate analyses of clinical outcomes, the requirement for RRT or in-hospital mortality was higher in the 4th quartiles of the initial urine NGAL excretion. In addition, renal recovery at 1 month was also significantly lower in the 3rd or 4th quartiles of urine NGAL excretion (p! 0.001). We also examined the presence of dipstick 1+ or more proteinuria and observed that significantly higher number of patients in the 4th quartile of urine NGAL had proteinuria ( table 1 ). Additionally, be- Urine NGAL Predicts Outcomes in AKI Am J Nephrol 2010;31:

4 Table 2. Patient characteristics according to normalized urine NGAL excretion quartiles (n = 65) ng/mg Cr: First (n = 16) ( ) Second (n = 16) ( ) Third (n = 18) ( ,752.95) Fourth (n = 15) (1, ,004.74) p Gender 3, male 15 (93.8) 9 (56.3) 9 (50.0) 8 (53.3) Age 1, years DM 3 3 (18.8) 6 (37.5) 6 (33.3) 8 (53.3) Hypertension 3 7 (43.8) 9 (56.3) 10 (55.6) 13 (86.7) Proteinuria 3 1 (6.3) 2 (12.5) 10 (55.6) 8 (53.3) Cause of AKI Multifactorial 10 (62.5) 11 (68.8) 11 (68.8) 5 (33.3) Ischemic 2 (12.5) 3 (18.8) 3 (18.8) 2 (13.3) Sepsis 0 2 (12.5) 2 (12.5) 7 (46.7) Nephrotoxic 4 (25.0) (6.7) Baseline Cr 2, mg/dl 1.2 ( ) 1.3 ( ) 1.2 ( ) 1.8 ( ) Initial Cr 2, mg/dl 2.5 ( ) 3.3 ( ) 3.7 ( ) 5.2 ( ) a 0.01 Initial BUN 2, mg/dl 39.0 ( ) 44.0 ( ) 57.1 ( ) 60.5 ( ) RIFLE criteria Risk 6 (37.5) 4 (25.0) 4 (22.2) 0 Injury 5 (31.3) 3 (18.8) 1 (5.6) 4 (26.7) Failure 5 (31.3) 9 (56.3) 13 (72.2) 11 (73.3) Mortality (4.0) 5 (20.8) 6 (25.0) RRT 3 3 (12.5) 3 (18.8) 9 (50.0) 12 (80.0) a, b <0.001 Recovery (1 month) 3 15 (93.8) 12 (75.0) 7 (38.9) a, b 2 (13.3) a, b <0.001 p values were obtained by ANOVA or Kruskal-Wallis test in case of numerical data and 2 or Fisher s exact test in case of categorical data. a p < 0.05 (by Bonferroni correction) vs. 1st quartile; b p < 0.05 (by Bonferroni correction) vs. 2nd quartile. Cr = Creatinine; BUN = blood urea nitrogen. 1 Means 8 SD. 2 Median (IQR). 3 Numbers of patients (%). cause urine NGAL that is normalized to urine creatinine might be more accurate, we performed all analyses with a normalized NGAL value in 65 patients and observed similar findings ( table 2 ). Urinary NGAL Excretion for Prediction of Adverse Clinical Outcomes in AKI We performed a multivariate logistic regression analysis to identify factors that determine in-hospital mortality or renal recovery. In the final model by backward (LR) variable selection, we found that failure in RIFLE criteria (OR 15.71) and the 3rd and 4th quartiles of urine NGAL excretion appeared to be independent predictors of inhospital mortality (OR and 14.18, respectively; table 3 ). Urinary NGAL excretion, in addition to prediction of in-hospital mortality, was also found to be an independent predictor of loss of renal function (OR and for the 3rd and 4th quartiles, respectively; table 4 ). Serum NGAL was also found to be an independent risk factor in predicting the failure of renal recovery (OR for the 4th quartile of serum NGAL; table 4 ). However, because patients in the risk or injury group were expected to have a relatively lower risk of death, we performed an additional analysis to see whether urinary NGAL level could predict clinical outcomes only in patients in the failure category. We observed that initial urine NGAL excretion in patients who belonged to the failure group independently predicted in-hospital mortality and loss of renal function. The odds ratio for inhospital mortality and failure to recovery was 1.1 and 1.4, respectively, per every 100 ng/ml increase in urine NGAL level ( table 5 ). Prognostic Performance of Urine NGAL in Predicting Adverse Clinical Outcomes The predictive performance of urinary NGAL excretion for predicting in-hospital mortality or loss of renal function was analyzed. Figure 1 a and b shows the area under the curve (AUC) for the ROC curve of urine NGAL for adverse clinical outcomes. Urine NGAL had a good diagnostic performance for predicting in-hospital mortality (AUC 0.803) or renal recovery (AUC 0.882). From 504 Am J Nephrol 2010;31: Yang/Boo/Kim/Jo/Cho/Kim

5 Table 3. U nivariate and multivariate analyses for predicting in-hospital mortality Univariate analysis M ultivariate analysis OR (95% CI) p OR ( 95% CI) p Sex 0.98 ( ) Age 1.03 ( ) ( ) Hypertension Absent 1 1 Present 0.48 ( ) ( ) Diabetes mellitus Absent 1 Present 1.11 ( ) Proteinuria Absent 1 Present 3.77 ( ) Baseline creatinine 0.94 ( ) Initial creatinine 1.03 ( ) Initial BUN 1.01 ( ) RIFLE criteria (vs. risk and injury) Failure ( ) ( ) Serum NGAL (vs. 1st and 2nd quartile group) 3rd quartile group 0.48 ( ) th quartile group 4.74 ( ) Urine NGAL (vs. 1st and 2nd quartile group) 3rd quartile group ( ) ( ) th quartile group ( ) ( ) In the multivariate analysis model, a backward (LR) selection approach was adopted. the ROC curve, we derived the optimum cutoff values with the highest AUC. At a cutoff value of ng/ml, the sensitivity and specificity for the prediction of in-hospital mortality were 91.7% and 67.1%, respectively. At a cutoff value of ng/ml, the sensitivity and specificity for the prediction of renal recovery were 88.2 and 81.0%, respectively, suggesting that the urine NGAL level might be a useful biomarker for predicting clinical outcomes ( fig. 1 ). Discussion In this prospective cohort study enrolling hospitalized AKI patients, we demonstrated that initial urine NGAL excretion was an independent predictor of both in-hospital mortality and short-term renal recovery. AKI is a complex disorder that has a wide variety of etiologies with a persistently high mortality [1]. D e s pite tremendous progress in understanding the pathophysiology of AKI, translation of this knowledge from bench-tobedside has been hampered by a lack of standard definitions and grading systems, and also a lack of clinically meaningful biomarkers. The recently proposed new definition/grading system for AKI (RIFLE) has been demonstrated to reliably predict adverse clinical outcomes, such as dialysis requirements or hospital mortality, in several studies [3 5]. T he s e s t u d ie s e n rol le d s e verel y i l l patients and relatively homogenous patient populations, such as those who were admitted to the ICU [11], burn units [5] a nd t hose u ndergoi ng c a rd iac su rger y [12] or liver transplantation [13]. Our study, however, enrolled a very heterogenous patient population. We did not only include critically ill AKI patients in the ICU setting, but also included nephrotoxicant- or ischemia-induced AKI patients who had been admitted to general medical or surgical wards. Relatively low in-hospital mortality (12%) might reflect this patient cohort characteristic. In our study, we observed that initial serum creatinine at admission was significantly higher in patients who belonged to the 3rd or 4th quartile of urine NGAL excretion. Although relatively Urine NGAL Predicts Outcomes in AKI Am J Nephrol 2010;31:

6 Table 4. U nivariate and multivariate analyses for predicting loss of renal function following AKI Univariate analysis M ultivariate analysis OR (95% CI) p OR (95% CI) p Sex 0.59 ( ) Age 1.03 ( ) ( ) Hypertension Absent 1 Present 1.91 ( ) Diabetes mellitus Absent 1 Present 2.96 ( ) Proteinuria Absent ( ) Present ( ) <0.001 Baseline creatinine 5.22 ( ) <0.001 Initial creatinine 1.21 ( ) Initial BUN 1.02 ( ) RIFLE criteria (vs. risk and injury) Failure 2.29 ( ) Serum NGAL (vs. 1st and 2nd quartile group) 3rd quartile group 2.95 ( ) th quartile group ( ) < ( ) Urine NGAL (vs. 1st and 2nd quartile group) 3rd quartile group ( ) < ( ) th quartile group ( ) < ( ) In multivariate analysis model, a backward (LR) selection approach was adopted. Table 5. OR of urinary NGAL for predicting in-hospital mortality and loss of renal function following AKI in the failure group Mortality (in-hospital death) N o recovery at 1 month after admission OR 95% CI p OR 95% CI p Urine NGAL In multivariate analysis model, a backward (LR) selection approach was adopted. Other parameters except urine NGAL were not displayed. later presentation in their course of illness might be associated with worse outcome, there is also the possibility that the initial insult was more severe in these groups of patients, leading to increased urinary NGAL excretion ultimately a with worse outcome. Currently, there is huge heterogeneity in clinical presentation of AKI and there is almost no way to identify the precise timing and severity of renal injury in patients with different degrees of susceptibility. Despite varying severities of illness, the RIFLE criteria were also found to be an independent predictor of in-hospital mortality in AKI patients in our study, confirming the results from previous studies. Contrary to in-hospital mortality, there have been few studies that aimed to examine the usefulness of the RIFLE criteria in predicting renal outcomes. This might be related in part to the inclusion of severely 506 Am J Nephrol 2010;31: Yang/Boo/Kim/Jo/Cho/Kim

7 1.00 ROC 1.00 ROC Sensitivity 0.50 Sensitivity specificity 1 specificity Fig. 1. Performance of urinary NGAL excretion for prediction of in-hospital mortality ( a ) and failure of recovery ( b ). a AUC: (p = 0.001) Cutoff: ng/ml sensitivity 91.7% specificity 67.1% b AUC: (p < 0.001) Cutoff: ng/ml sensitivity 88.2% specificity 81.0% ill patient cohorts with high mortality in most studies. In our study, we examined the possible usefulness of the RIFLE criteria in predicting renal outcome in surviving patients. However, we found that the RIFLE criteria was not a good prognostic factor in predicting renal outcome among survivors. The recent discovery of new, promising biomarkers has been mostly focused on their ability to detect AKI as early as possible. However, in addition to early detection, discovery of biomarkers that can be used as surrogate endpoints in assessing the response to certain therapeutic interventions or that can predict clinical outcomes is also urgently needed. Until recently, relatively few studies that focused on the ability of new biomarkers to predict adverse clinical outcomes have been performed [8 10]. NGAL is one of the most promising biomarkers for the early detection of AKI. Despite several controversies, urinary NGAL excretion has been consistently shown to be increased well before the increase in serum creatinine or the decrease in urine output [6, 14]. However, its ability to predict clinical outcomes in patients with AKI has rarely been assessed. Parikh et al. [8] reported that urinary NGAL and IL-18 on the day of transplantation were predictive biomarkers for delayed graft function. Although that study only included 53 kidney transplant recipients, they demonstrated that every 100 ng/ml increase in urine NGAL was associated with increased odds of delayed graft function (DGF) by 20%. In our study, we enrolled 100 AKI patients and observed that urinary NGAL excretion was an independent predictor of not only in-hospital mortality, but also short-term renal recovery. Patients in the 3rd or 4th quartiles of urine NGAL excretion had tremendously increased odds for in-hospital mortality and also failure of recovery. Because patients in the risk and injury categories were expected to have a better prognosis, we wanted to confirm these findings in a more severe patient group. In the analysis including the patients in the failure category only, every 100-ng/ml increase in urine NGAL excretion was associated with increased odds of in-hospital mortality by 10% and of failure of renal recovery by 40%, confirming the usefulness of NGAL in predicting clinical outcomes. In quantitating urine NGAL excretion, there has been controversy about the issue that simple urine NGAL or NGAL normalized to urine creatinine is better. However, according to the literature that have both data, a significant difference does not seem to exist. We also performed analyses both with NGAL ( table 1 ) and NGAL/cr ( table 2 ) and found no difference. Recently, McIlroy et al. [15] reported that optimal discriminatory power of urine NGAL in predicting AKI following cardiac surgery is thought to be only in patients with normal baseline renal function (egfr ml/ min). Therefore, we also examined whether there is a difference in the discriminatory power of initial urine Urine NGAL Predicts Outcomes in AKI Am J Nephrol 2010;31:

8 NGAL in predicting outcomes according to baseline egfr, but observed no difference (data not shown). However, because the number of patients in each group was small, further studies to identify the possible differential discriminatory power of urine NGAL according to baseline egfr in predicting outcomes should be performed. This is the first prospective study demonstrating that urinary NGAL can be used as a potential predictor of clinical outcomes in patients with AKI, in addition to its established usefulness as an early biomarker. In addition, we also observed the possibility that serum NGAL might also be used to predict worse renal outcome. Serum NGAL has been demonstrated to be a useful biomarker in predicting AKI in pediatric septic shock patients [16] as well as after cardiac surgery [17]. Whereas many studies of biomarkers have been performed in very homogenous populations, our study enrolled very heterogenous AKI patients at different times of admission, with different etiologies and different severities of illness. This heterogeneity, in fact, is a reality and discovering biomarkers that can be useful, even in a very heterogenous population of AKI patients, is thought to be more important and we identified that urine NGAL might be a useful biomarker for predicting clinical outcomes. We also generated conventional ROC to assess the predictive performance of urinary NGAL. The AUC was in predicting in-hospital mortality and in predicting renal recovery, suggesting a relatively good diagnostic performance for urinary NGAL. De spite prom i si ng re su lt s su g ge s t i n g t he p ot e nt i a l clinical usefulness of urinary NGAL as an outcome predictor, our study had some important limitations. First, this was a single center study enrolling a relatively small number of patients (n = 100) and needs to be confirmed in multicenter studies. Second, the impact of increased urinary NGAL excretion on long-term renal function should also be assessed. Third, because a combination of several biomarkers might improve the diagnostic performance in predicting outcome, simultaneous measurement of several other biomarkers would be more prudent. In conclusion, we have demonstrated that urinary NGAL excretion at the time of diagnosis of AKI can predict adverse clinical outcomes, suggesting the important clinical utility of this new biomarker in patient care. Further studies enrolling more patients from multiple centers is necessary to confirm these findings in an effort to improve patient prognosis. Conflict of Interest This work was supported by Korean Society of Nephrology Grant. Part of this work was presented in a poster session of the annual meeting of American Society of Nephrology Meeting held in Philadelphia in Acknowledgements We thank Ms. Yoon Suk Ko and Hee Young Lee for their excellent technical assistance. References 1 de Mendonca A, Vincent JL, Suter PM, Moreno R, Dearden NM, Antonelli M, Takala J, Sprung C, Cantraine F: Acute renal failure in the ICU: risk factors and outcome evaluated by the SOFA score. Intensive Care Med 2000; 26: Bellomo R, Ronco C, Kellum JA, Mehta RL, Palevsky P: Acute renal failure definition, outcome measures, animal models, fluid therapy and information technology needs: the Second International Consensus Conference of the Acute Dialysis Quality Initiative (ADQI) Group. Crit Care 2004; 8 : R R Ostermann M, Chang RW: Acute kidney injury in the intensive care unit according to RIFLE. Crit Care Med 2007; 35: , quiz Bell M, Liljestam E, Granath F, Fryckstedt J, Ekbom A, Martling CR: Optimal follow-up time after continuous renal replacement therapy in actual renal failure patients stratified with the RIFLE criteria. Nephrol Dial Transplant 2005; 20: Lopes JA, Jorge S, Resina C, Santos C, Pereira A, Neves J, Antunes F, Prata MM: Prognostic utility of RIFLE for acute renal failure in patients with sepsis. Crit Care 2007; 11: Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, Ruff SM, Zahedi K, Shao M, Bean J, Mori K, Barasch J, Devarajan P: Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 2005; 365: Vaidya VS, Ramirez V, Ichimura T, Bobadilla NA, Bonventre JV: Urinary kidney injury molecule-1: a sensitive quantitative biomarker for early detection of kidney tubular injury. Am J Physiol Renal Physiol 2006; 290:F517 F Parikh CR, Abraham E, Ancukiewicz M, Edelstein CL: Urine IL-18 is an early diagnostic marker for acute kidney injury and predicts mortality in the intensive care unit. J Am Soc Nephrol 2005; 16: Liangos O, Perianayagam MC, Vaidya VS, Han WK, Wald R, Tighiouart H, MacKinnon RW, Li L, Balakrishnan VS, Pereira BJ, Bonventre JV, Jaber BL: Urinary N-acetylbeta-(D)-glucosaminidase activity and kidney injury molecule-1 level are associated with adverse outcomes in acute renal failure. J Am Soc Nephrol 2007; 18: Am J Nephrol 2010;31: Yang/Boo/Kim/Jo/Cho/Kim

9 10 van Timmeren MM, Vaidya VS, van Ree RM, Oterdoom LH, de Vries AP, Gans RO, van Goor H, Stegeman CA, Bonventre JV, Bakker SJ: High urinary excretion of kidney injury molecule-1 is an independent predictor of graft loss in renal transplant recipients. Transplantation 2007; 84: Hoste EA, Clermont G, Kersten A, Venkataraman R, Angus DC, De Bacquer D, Kellum JA: RIFLE criteria for acute kidney injury are associated with hospital mortality in critically ill patients: a cohort analysis. Crit Care 2006; 10:R Kuitunen A, Vento A, Suojaranta-Ylinen R, Pettila V: Acute renal failure after cardiac surgery: evaluation of the RIFLE classification. Ann Thorac Surg 2006; 81: O Riordan A, Wong V, McQuillan R, Mc- Cormick PA, Hegarty JE, Watson AJ: Acute renal disease, as defined by the RIFLE criteria, post-liver transplantation. Am J Transplant 2007; 7: Wagener G, Gubitosa G, Wang S, Borregaard N, Kim M, Lee HT: Urinary neutrophil gelatinase-associated lipocalin and acute kidney injury after cardiac surgery. Am J Kidney Dis 2008; 52: McIlroy DR, Wagener G, Lee HT: Neutrophil gelatinase-associated lipocalin and acute kidney injury after cardiac surgery: the effect of baseline renal function on diagnostic performance. Clin J Am Soc Nephrol 2010; 5 : Wheeler DS, Devarajan P, Ma Q, Harmon K, Monaco M, Cvijanovich N, Wong HR: Serum neutrophil gelatinase-associated lipocalin (NGAL) as a marker of acute kidney injury in critically ill children with septic shock. Crit Care Med 2008; 36: Dent CL, Ma Q, Dastrala S, Bennett M, Mitsnefes MM, Barasch J, Devarajan P: Plasma neutrophil gelatinase-associated lipocalin predicts acute kidney injury, morbidity and mortality after pediatric cardiac surgery: a prospective uncontrolled cohort study. Crit Care 2007; 11:R127. Urine NGAL Predicts Outcomes in AKI Am J Nephrol 2010;31: