The predictive performance of plasma neutrophil gelatinase-associated lipocalin (NGAL) increases with grade of acute kidney injury

The predictive performance of plasma NGAL increases with grade of AKI 3349 15. Bröchner-Mortensen J. A simple method for the determination of glomerular filtration rate. Scand J Clin Lab Invest 1972;30:271 274 16. Christensen PK, Hommel E, Clausen P et al. Impaired autoregulation of the glomerular filtration rate in patients with nondiabetic nephropathies. Kidney Int 1999; 56: 1517 1523 17. Schjoedt KJ, Rossing K, Juhl TR et al. Beneficial impact of spironolactone on nephrotic range albuminuria in diabetic nephropathy. Kidney Int 2006; 70: 536 542 18. Derkx FH, Tan-Tjiong L, Wenting GJ et al. Asynchronous changes in prorenin and renin secretion after captopril in patients with renal artery stenosis. Hypertension 1983; 5: 244 256 19. Altman DG. Clinical trials. Chapter 15 In: Altman DG (ed). Practical Statistics for Medical Research, 1st edn. London: Chapman & Hall, 1991, 440 474 20. Bianchi S, Bigazzi R, Campese VM. Long-term effects of spironolactone on proteinuria and kidney function in patients with chronic kidney disease. Kidney Int 2006; 70: 2116 2123 21. Pratt JH, McAteer JA. Beta-adrenergic enhancement of angiotensin II-stimulated aldosterone secretion. Life Sci 1989; 44: 2089 2095 22. Ehrhart-Bornstein M, Bornstein SR, Gonzalez-Hernandez J et al. Sympathoadrenal regulation of adrenocortical steroidogenesis. Endocr Res 1995; 21: 13 24 23. Gupta P, Franco-Saenz R, Mulrow PJ. Regulation of the adrenal renin angiotensin system in cultured bovine zona glomerulosa cells: effect of catecholamines. Endocrinology 1992; 130: 2129 2134 24. Kim YS, Immink RV, Stok WJ et al. Dynamic cerebral autoregulatory capacity is affected early in Type 2 diabetes. Clin Sci (Lond) 2008; 115: 255 262 25. Christensen PK, Lund S, Parving H-H. The impact of glycaemic control on autoregulation of glomerular filtration rate in patients with non-insulin dependent diabetes. Scand J Clin Lab Invest 2001; 61: 43 50 26. 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Loutzenhiser R, Griffin K, Williamson G et al. Renal autoregulation: new perspectives regarding the protective and regulatory roles of the underlying mechanisms. Am J Physiol Regul Integr Comp Physiol 2006; 290: R1153 R1167 33. Loon NR, Wilcox XS, Unwin RJ. Mechanism of impaired natriuretic response to furosemide during prolonged therapy. Kidney Int 1989; 36: 682 689 34. Onesti G, Bock KD, Heimsoth V et al. Clonidine: a new antihypertensive agent. Am J Cardiol 1971; 28: 74 83 35. Benowitz NL. Antihypertensive agents. Chapter 11, In Katzung BG (ed) Basic and Clinical Pharmacology, 6th edn. CA: Appleton & Lange, 1995, 147 170 36. Price DA, Porter LE, Gordon M et al. The paradox of the low-renin state in diabetic nephropathy. J Am Soc Nephrol 1999; 10: 2382 2391 Received for publication: 3.3.09; Accepted in revised form: 3.6.09 Nephrol Dial Transplant (2009) 24: 3349 3354 doi: 10.1093/ndt/gfp234 Advance Access publication 27 May 2009 The predictive performance of plasma neutrophil gelatinase-associated lipocalin (NGAL) increases with grade of acute kidney injury Anja Haase-Fielitz 1,2, Rinaldo Bellomo 1, Prasad Devarajan 3, Michael Bennett 3,DavidStory 4, George Matalanis 5, Ulrich Frei 2, Duska Dragun 2 and Michael Haase 1,2 1 Department of Intensive Care, Austin Health, Melbourne, Australia, 2 Department of Nephrology and Intensive Care Medicine, Charité University Medicine, Berlin, Germany, 3 Department of Pediatrics and Developmental Biology, Cincinnati Children s Hospital Medical Center, Cincinnati, OH, USA, 4 Department of Anaesthesiology and 5 Department of Cardiac Surgery, Austin Health, Melbourne, Australia Correspondence and offprint requests to: Rinaldo Bellomo; E-mail: rinaldo.bellomo@austin.org.au Abstract Background. In adult cardiac surgery, the predictive value for AKI of neutrophil gelatinase-associated lipocalin (NGAL) appears to have wide variability. The choice of definition of acute kidney injury (AKI) might, at least in part, account for such variability. Methods. In a prospective study of 100 adult cardiac surgery patients, we assessed the value of postoperative plasma NGAL in predicting AKI according to the degree of severity used for its definition. Results. The predictive value of plasma NGAL varied according to the AKI definition used and was higher for more C The Author 2009. Published by Oxford University Press [on behalf of ERA-EDTA]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

3350 A. Haase-Fielitz et al. severe AKI (increase in creatinine >50%: mean AUC ROC 0.79 ± 0.01) compared to less severe AKI (>25%: mean AUC ROC 0.65 ± 0.02); P = 0.001. The discriminatory ability of NGAL for AKI also increased with increasing RI- FLE classes (AUC ROC R: 0.72, I: 0.79, F: 0.80) or AKIN stages (AUC ROC 1: 0.75, 2: 0.78, 3: 0.81); P = 0.015. It was highest for the prediction of renal replacement therapy (AUC ROC: 0.83). Conclusions. In adult cardiac surgery patients, the predictive value of NGAL increases with grade of AKI. This observation needs to be taken into account when interpreting any future studies of this biomarker. Keywords: acute kidney injury; cardiac surgery; neutrophil gelatinase-associated lipocalin (NGAL); RIFLE; grade In experimental and clinical studies, neutrophil gelatinaseassociated lipocalin (NGAL) appears to be one of the most frequently investigated and most promising biomarkers for the early diagnosis of acute kidney injury (AKI) [1 5]. NGAL was found to be an excellent biomarker for the early diagnosis of AKI in children undergoing cardiac surgery [1,4,5], in adults admitted to hospital via the emergency department [3] and in children and adults undergoing renal transplantation [2], with areas under the curve for the receiver operating characteristic (AUC ROC) >0.9. However, with accumulating evidence, a wide range of NGAL s predictive values for AKI after cardiac surgery have been reported in adult patients with an AUC ROC ranging from 0.53 to 0.80 [6 9]. These observations raise concern about the robustness of NGAL as a biomarker. Unfortunately, different definitions of AKI after cardiac surgery have been used to assess the predictive value of NGAL in different adult studies creating a definitionrelated confounder in our understanding of NGAL s usefulness as a biomarker. Most AKI definitions have been based on serum creatinine changes, including >25% or >50% creatinine increases from the baseline during varying periods of time and creatinine-based criteria of RIFLE (R-renal risk, I-injury, F-failure, L-loss of renal function, E-end stage renal disease) [10] or AKI Network (AKIN) [11] classification [1,4 9]. Such diversity of AKI definitions might have influenced the predictive value of NGAL with less stringent definitions of AKI potentially decreasing the performance of NGAL as a predictive biomarker [12]. However, the impact of the grade and/or choice of the definition of AKI on the performance of NGAL have not yet been studied. In this investigation, we assessed the impact of the definition of AKI on the performance of plasma NGAL as renal biomarker in adult patients after cardiac surgery. Subjects and methods Introduction Patients In the present study, we took advantage of a previous prospective cohort study [9], where we enrolled 100 adult patients who underwent cardiac surgery necessitating the use of cardiopulmonary bypass at a tertiary hospital. We excluded patients undergoing emergency operation (time between hospital admission to operation <24 h) or off-pump surgery, patients presenting with advanced chronic kidney disease (serum creatinine >300 µmol/l), kidney transplant patients and patients <18 years. Plasma NGAL and serum creatinine For the measurement of plasma NGAL, we obtained blood samples at 6 h after commencement of cardiopulmonary bypass closely corresponding to2haftertheendofcardiopulmonary bypass measured by TriageMeter (Biosite, San Diego, CA, USA). We centrifuged the samples at 2000 g for 5 min and stored the supernatant in equal volumes at 80 C until measurement. Serum creatinine was measured preoperatively and daily from first to seventh day postoperatively utilizing the modified Jaffé method standardized by isotope dilution mass spectroscopy. The inter- and intra-assay coefficients of variation of plasma NGAL and creatinine measurement were <5%. Endpoints For analysis of diagnostic value of plasma NGAL, we used sustained (>2 days) increases in serum creatinine of (1) >25% and (2) >50% from the baseline to the postoperative peak value within (1) 48 h, (2) 72 h, (3) 120 h and (4) 168 h of surgery. We also assessed the value of plasma NGAL in relation to the full definition of the RIFLE [10] classes R, I and F within 7 days postoperatively and the AKIN [11] stages 1, 2 and 3 within 48 h postoperatively (Table 1). For the grading of patients according to the RIFLE classification, we considered the serum creatinine concentration, Table 1. Classification systems for acute kidney injury RIFLE criteria a (within 7 days postoperatively) AKIN criteria b (within 48 h postoperatively) Class GFR Urine output Stage Creatinine Urine output R-Risk Crea increase 1.5 or GFR loss <0.5 ml/kg/h >6 h 1 Crea increase 1.5 or Crea <0.5 ml/kg/h >6h >25% increase >0.3 mg/dl I-Injury Crea increase 2 or GFR loss >50% <0.5 ml/kg/h >12 h 2 Crea increase 2or <0.5 ml/kg/h >12 h F-Failure Crea increase 3 or GFR loss <0.3 ml/kg/h >24 h 3 c Crea increase 3orCrea <0.3 ml/kg/h >24 h >75% or Crea increase >4 mg/dl (acute rise >0.5 mg/dl) or anuria >12 h increase >4 mg/dl (acute rise >0.5 mg/dl) L-Loss Persistent loss of kidney function >4 E-End-stage renal disease weeks End-stage renal disease >3 months a Bellomo et al. [10]. b Mehta et al. [11]. c Patients on renal replacement therapy were classified as stage 3.

The predictive performance of plasma NGAL increases with grade of AKI 3351 Table 2. Patients characteristics (N = 100) procedures were valve surgery and coronary artery bypass grafting surgery. Age (years) 69.5 ± 8.7 Seven studies reported on the predictive value of NGAL Sex, n (female) 39 Arterial hypertension, n 84 for AKI after cardiac surgery in children and adults with a Diabetes mellitus a, n 28 wide predictive range of AUC ROC (Table 3). Five studies Preoperative chronic renal impairment, n 27 defined AKI as an increase in serum creatinine of >50% Preoperative serum creatinine (µmol/l) 90.8 ± 25.7 and two used selected criteria from the AKIN classification. Also, the timing of the creatinine increase considered Preoperative egfr (ml/min/1.73 m 2 ) b 73.9 ± 19.7 Congestive heart failure c, n 25 Chronic obstructive pulmonary disease, n 14 for AKI definition varied between studies (Table 3). While Peripheral vascular disease, n 6 the timing of NGAL measurement in relation to the commencement of surgery was similar, the predictive value of EuroScore, points d 5.3 ± 2.2 Coronary revascularization, n 52 NGAL ranged from 0.53 to 0.80 in adults (Table 3). Valve surgery, n 25 Simultaneous coronary revascularization 17 The frequencies of serum creatinine increases, RIFLE and valve surgery, n 17 classes and AKIN stages in the study population are shown Duration of cardiopulmonary bypass (min) 138 ± 39 in Tables 4 6. Length of stay in hospital (days) 10.8 ± 6.5 Table 4 displays the performance characteristics of Hospital deaths, n 2 plasma NGAL according to grade of AKI. The predictive value of plasma NGAL varied (AUC ROC 0.64 0.83) a Patients on oral antidiabetic medication or on insulin. b egfr, estimated glomerular filtration rate according to the MDRD (Modification of Diet in Renal Disease) study equation [13]. vere AKI. Considering a creatinine increase of >50% at c New York Heart Association class III/IV or impaired left ventricular postoperative Days 2, 3, 5 and 7, the average predictive according to definition used and was higher for more se- function (defined as left ventricular ejection fraction <35%). value of plasma NGAL was higher for AKI defined as an d [15]. Linear values denote mean ± standard deviation. increase in serum creatinine of >50% (AUC ROC 0.79 ± 0.01) compared to >25% (AUC ROC 0.65 ± 0.02); P = the estimated glomerular filtration rate (egfr) by the Modified Diet in 0.001. When plasma NGAL was evaluated for its ability to Renal Disease formula re-expressed for use with the serum creatinine predict the need for renal replacement therapy, its AUC values standardized to isotope dilution mass spectroscopy [13] and the urine output criteria. For AKIN, we considered the criteria referring to ROC further increased to 0.83. Tables 5 and 6 present the creatinine increases, use of renal replacement therapy and urine output performance characteristics of plasma NGAL according to decreases. Urine output was documented hourly. At our centre, renal replacement therapy was initiated if the patient fulfilled at least one of the of AKI. After pooling corresponding RIFLE classes with the RIFLE classification and the AKI network definition following clinical criteria: oliguria (urine output <100 ml/6 h) that has AKIN stages (R with 1, I with 2, F with 3, I+F with 2+3), been unresponsive to fluid resuscitation measures, hyperkalaemia ([K+] >6.5 mmol/l), severe acidaemia (ph <7.2) or clinically significant organ the predictive value of plasma NGAL increased with grade oedema (e.g. lung) in the setting of renal failure. of AKI; P = 0.015. Plasma NGAL performance when using RIFLE classification was similar to that of AKIN definition (Table Statistics We searched Pubmed, EMBASE and Medline (as of 2 February 2009) and 5,6). The cut-off value of plasma NGAL for best sensitivity considered all clinical studies of paediatric and adult patients where the and specificity in predicting a subsequent creatinine increase, RIFLE classes or AKIN stages increased from less diagnostic value of NGAL to predict AKI after cardiac surgery was reported as the area under the curve for the receiver-operating characteristic severe to more severe AKI (Tables 4 6) and ranged between (AUC ROC). We performed a sensitivity analysis on the value of early >145 ng/ml and >340 ng/ml. plasma NGAL in predicting AKI after cardiac surgery according to AKI definitions used in previous publications in this setting [1 9] by calculation of the AUC ROC. For AUC ROC analysis, the control group was not develop subsequent AKI (as identified by a creatinine A considerable proportion of patients (45.5%) who did no AKI and grade of AKI was the event group. The AUC ROC value increase > 50%) showed mild postoperative increases in of plasma NGAL for a creatinine increase >25% was compared with that plasma NGAL with concentrations ranging from >100 ng/ for a creatinine increase >50% by the paired student t-test. In addition, we assessed the performance of plasma NGAL in this cohort using the ml [ = above normal range (normal range: 40 100 ng/ml RIFLE and AKIN classifications. The relationship between increasing [16])] to <150 ng/ml (just below our cut-off for best sensitivity and specificity to predict AKI). The predictive value RIFLE classes and AKIN stages with the AUC ROC value of plasma NGAL was tested by analysis of variance. Comparison of an AUC ROC of plasma NGAL was not related to timing of the creatinine was performed as previously described [14]. For each parameter, we calculated the best threshold (defined as the renal biomarker concentration increase (P = 0.98). that is closest to the point on the receiver-operating characteristic curve where sensitivity = 1-specificity = 1), sensitivity and specificity. We used SPSS Version 16.0 (SPSS Inc, Chicago, IL, USA) and MedCalc Version 9.3.9.0 (MedCalc Software, Mariakerke, Belgium). Discussion Results Demographic data are shown in Table 2. Significant comorbidities included arterial hypertension, diabetes mellitus and preoperative renal impairment with egfr <60 ml/min/1.73 m 2 [13]. The most common surgical In a cohort of 100 adult patients, we performed a sensitivity analysis to determine the influence of AKI definition on the predictive value of plasma NGAL for AKI after cardiac surgery. We found that the predictive value of plasma NGAL increased with grade of AKI from an AUC ROC of 0.64 for a >25% increase in serum creatinine to an AUC ROC value of 0.83 for the prediction of the need for

3352 A. Haase-Fielitz et al. Table 3. Definition of acute kidney injury after cardiac surgery and the predictive value of early measured NGAL AKI definition No. of Creatinine Timing of postoperative Timing of NGAL measurement AUC ROC to predict Reference patients Setting increase creatinine increase (after end of CPB) AKI (plasma/urine) Mishra et al. [1] 71 Paediatric >50% Within 5 days At 2 h 0.91/0.99 Dent et al. [4] 120 Paediatric >50% Within 5 days At 2 h 0.96/ Bennett et al. [5] 196 Paediatric >50% Within 5 days At 2 h /0.95 Wagener et al. [6] 81 Adult >50% Within 5 days At 3 h /0.74 Wagener et al. [7] 426 Adult >50% or Within 2 days At 3 h /0.60 >0.3 mg/dl Koyner et al. [8] 72 Adult >25% or need Within 3 days At 2ho 0.53/0.70 for RRT Haase-Fielitz et al. [9] 100 Adult >50% Within 5 days At 2h 0.80/ RRT, renal replacement therapy. Six hours after the start of cardiopulmonary bypass (CPB) or the intensive care unit arrival value. Table 4. Performance characteristics of plasma NGAL at 6 h after start of CPB according to AKI severity AKI definition (no. of patients) AUC ROC (95% CI) Sensitivity (%) Specificity (%) Cut-off (ng/ml) >25% within 48 h, (N = 36) 0.66 (0.48 0.84) 63.2 72.7 >145 >25% within 72 h, (N = 38) 0.64 (0.46 0.83) 61.1 69.6 >145 >25% within 120 h, (N = 39) 0.67 (0.48 0.85) 68.4 63.6 >145 >25% within 168 h, (N = 40) 0.64 (0.45 0.83) 61.1 69.6 >145 >0.3 mg/dl or >50% within 48 h, (N = 32) 0.66 (0.48 0.84) 63.2 72.7 >145 >25% or RRT within 72 h, (N = 42) 0.68 (0.51 0.85) 65.0 76.2 >145 >50% within 48 h, (N = 20) 0.78 (0.61 0.96) 75.0 75.9 >155 >50% within 72 h, (N = 21) 0.79 (0.62 0.95) 75.5 76.2 >155 >50% within 120 h, (N = 23) 0.80 (0.63 0.96) 79.0 78.0 >150 >50% within 168 h, (N = 23) 0.80 (0.63 0.96) 79.0 78.0 >150 RRT, (N = 4) 0.83 (0.60 0.98) 75.0 100.0 >340 The increase in serum creatinine defined as an increase from a preoperative to a postoperative peak value. NGAL, neutrophil gelatinase-associated lipocalin; CPB, cardiopulmonary bypass; AKI, acute kidney injury; RRT, renal replacement therapy. Table 5. Performance characteristics of plasma NGAL at 6 h after start of cardiopulmonary bypass to predict AKI according to RIFLE classes. AKI definition (no. of patients) AUC ROC (95% CI) Sensitivity (%) Specificity (%) Cut-off (ng/ml) RIFLE R(N = 31) 0.72 (0.54 0.91) 70.0 71.4 >150 I(N = 13) 0.79 (0.56 0.99) 85.7 75.0 >150 F(N = 6) 0.80 (0.53 1.00) 66.7 100.0 >240 I + F(N = 19) 0.79 (0.59 0.99) 80.0 75.0 >150 AKI, acute kidney injury. RIFLE [10]. Table 6. Performance characteristics of plasma NGAL at 6 h after the start of cardiopulmonary bypass to predict AKI according to AKIN stages AKI definition (no. of patients) AUC ROC (95% CI) Sensitivity (%) Specificity (%) Cut-off (ng/ml) AKIN 1(N = 29) 0.75 (0.59 0.92) 71.4 73.7 >150 2(N = 11) 0.78 (0.59 0.99) 80.0 80.0 >150 3(N = 6) 0.81 (0.58 1.00) 66.7 100.0 >240 2 + 3(N = 17) 0.79 (0.57 0.99) 75.0 78.9 >150 AKI, acute kidney injury. AKIN [11]. renal replacement therapy. We found no association with the timing of the creatinine increase. NGAL has been reported to represent an early and highly specific biomarker for AKI in several studies in paediatric cardiac surgery with an AUC ROC >0.9 [1,4,5]. However, more recent prospective clinical studies found a surprisingly low predictive value of early NGAL for AKI after adult cardiac surgery, with an AUC ROC 0.7 [6 8] Different AKI definitions used in these studies might have contributed to these discrepancies. Most studies have

The predictive performance of plasma NGAL increases with grade of AKI 3353 analysed NGAL concentrations to predict AKI defined as an increase in serum creatinine >50% from the baseline to the postoperative peak value. However, two studies [6,8] have reported the predictive value of NGAL using selected criteria from the AKIN classification which excluded its urine output criteria. In these studies, even patients who did not subsequently develop AKI had a substantial early rise in NGAL concentration. This finding may reflect not only the choice of AKI grade but also the relatively short time frame during which AKI was assessed for (48 h). Such a short evaluation time may miss the diagnosis of AKI in patients who develop a substantial and clinically relevant creatinine increase 60 or 72 h after cardiopulmonary bypass [17]. In the present study, according to the AKI definition used, we found large variations in the plasma NGAL s predictive value with an AUC ROC ranging from 0.64 (for a >25% creatinine increase) to 0.83 (for renal replacement therapy). In terms of a common classification of the value of biomarkers in general, this corresponds to a shift from poor to good predictive performance or from no useful to a useful risk predictor (as defined by AUC ROC >0.7 [18]), respectively. The results of our study provide a possible explanation for the large range of AUC ROC for NGAL s predictive value for AKI. Importantly, early but mild NGAL increases might also indicate equally mild AKI or renal stress that will not be detected by a subsequent creatinine increase. Such increases may also represent a response to systemic inflammation and may not be related to AKI itself. As a consequence, a patient with increased NGAL levels (100 150 ng/ml) and a creatinine increase of <50% will be classified no AKI although tubular injury or stress might well be present. On the other hand, prerenal azotaemia, which can meet the proposed creatinine- or urine output-based definitions of AKI without any actual tubular injury, degrades the diagnostic ability and tests performance of the marker. NGAL probably does not increase in prerenal azotaemia as previously shownbynickolaset al.[3]. Our study has limitations. This study was a post hoc analysis, yet one of the largest in adults in this field. Other factors beyond AKI definition might be of importance for the predictive value of NGAL such as the measurement of NGAL in plasma or urine. In this regard, there is speculation that due to its origin from the renal tubules, urine NGAL might be of a higher predictive value compared to plasma NGAL originating mostly from neutrophils, liver and lung cells [19]. However, to date, there is no conclusive evidence for urine NGAL being superior to plasma NGAL. As we did not measure urine NGAL, we cannot provide comparison with plasma NGAL. Also, the degree of inflammation or diseases of liver and lungs may contribute to different findings in the literature on the predictive performance of NGAL. To address such potential confounders, we assessed NGAL in the same patient cohort using different AKI definitions using each patient as his/her own control. Furthermore, we did not only assess the most commonly used definitions of AKI in this study but we also reused those definitions previously used in the setting of the predictive performance of NGAL enabling comparability. Also, other potential confounders of the value of NGAL such as setting of AKI, comorbidities, storage conditions of samples or assay used should be investigated in separate studies and are beyond the scope of this study. Finally, as most AKI definitions are based on changes in serum creatinine and urine output markers with many known limitations they may not at all reflect the true value of NGAL in detecting acute kidney injury or tubular injury or tubular stress but rather the limitations of current surrogates for the presence or absence of AKI. A biomarker can only be as good as the surrogate endpoint it predicts in the case of NGAL, serum creatinine- and urine output-based AKI, insensitive or unspecific markers of loss of GFR, respectively. In future studies, more sensitive AKI definitions using markers other than serum creatinine or urine output should be evaluated. Once confirmed in large multicentre trials as reliable, robust and independent predictors of patient outcomes emerging novel renal biomarkers such as NGAL or fatty acid-binding protein [20] may be used to define the presence or absence or degree of AKI independent of changes in urine output or serum creatinine. In conclusion, the results of this study support the view that, in adult cardiac surgery, the predictive value of NGAL increases with grade of AKI. This observation needs to taken into account when interpreting future studies of this biomarker. Acknowledgements. Dr Haase holds a postdoctoral Feodor-Lynen research fellowship from the Alexander von Humboldt-Foundation, Germany. This study was partly funded by a grant from the Australian and New Zealand College of Anaesthetists and by the Austin Hospital Anesthesia and Intensive Care Trust Fund. R.B. and M.H. conceived the study. M.H., A.H.F., P.D. and R.B. participated in the design of the study, performed the statistical analysis and drafted the manuscript. D.S., D.D., M.B., G.M. and U.F. participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Conflict of interest statement. Drs Bellomo and Devarajan have acted as paid consultant to Abbott Diagnostics and Biosite. Dr Devarajan has received limited research grant support from Biosite and Abbott Diagnostics. Dr Haase has received lecture fees and travel expenses from Abbott Diagnostics and Biosite, both of which are involved in the development of NGAL assays to be applied in clinical practice. All other authors have nothing to declare. (See related article by S. S. Waikar et al. Creatinine as the gold standard for kidney injury biomarker studies? Nephrol Dial Transplant 2009; 24: 3263 3265.) References 1. Mishra J, Dent C, Tarabishi R et al. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 2005; 365: 1231 1238 2. Parikh CR, Jani A, Mishra J et al. Urine NGAL and IL-18 are predictive biomarkers for delayed graft function following kidney transplantation. Am J Transplant 2006; 6: 1639 1645 3. Nickolas TL, O Rourke MJ, Yang J et al. Sensitivity and specificity of a single emergency department measurement of urinary neutrophil gelatinase-associated lipocalin for diagnosing acute kidney injury. Ann Intern Med 2008; 148: 810 819 4. Dent CL, Ma Q, Dastrala S et al. Plasma neutrophil gelatinaseassociated lipocalin predicts acute kidney injury, morbidity and mortality after pediatric cardiac surgery: a prospective uncontrolled cohort study. Crit Care 2007; 11: R127

3354 M. P. Schneider et al. 5. Bennett M, Dent CL, Ma Q et al. Urine NGAL predicts severity of acute kidney injury after cardiac surgery: a prospective study. Clin J Am Soc Nephrol 2008; 3: 665 673 6. Wagener G, Jan M, Kim M et al. Association between increases in urinary neutrophil gelatinase-associated lipocalin and acute kidney injury after adult cardiac surgery. Anesthesiology 2006; 105: 485 491 7. Wagener G, Gubitosa G, Wang S et al. Urinary neutrophil gelatinaseassociated lipocalin and acute kidney injury after cardiac surgery. Am J Kidney Dis 2008; 52: 425 433 8. Koyner JL, Bennett MR, Worcester EM et al. Urinary cystatin C as an early biomarker of acute kidney injury following adult cardiothoracic surgery. Kidney Int 2008; 74: 1059 1069 9. Haase-Fielitz A, Bellomo R, Devarajan P et al. Novel and conventional serum biomarkers predicting acute kidney injury in adult cardiac surgery a prospective cohort study. Crit Care Med 2009; 37: 553 560 10. 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European system for cardiac operative risk evaluation (EuroSCORE). Eur J Cardiothorac Surg 1999; 16: 9 13 16. Axelsson L, Bergenfeldt M, Ohlsson K. Studies of the release and turnover of a human neutrophil lipocalin. Scand J Clin Lab Invest 1995; 55: 577 588 17. Haase M, Haase-Fielitz A, Bagshaw SM et al. A phase II randomized controlled trial of high-dose N-acetylcysteine in high-risk cardiac surgery patients. Crit Care Med 2007; 35: 1324 1331 18. Swets JA. Measuring the accuracy of diagnostic systems. Science 1988; 240: 1285 1293 19. Schmidt-Ott KM, Mori K, Li JY et al. Dual action of neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 2007;18:407 413 20. Portilla D, Dent C, Sugaya T et al. Liver fatty acid-binding protein as a biomarker of acute kidney injury after cardiac surgery. Kidney Int 2008; 73: 465 472 Received for publication: 12.3.09; Accepted in revised form: 28.4.09 Nephrol Dial Transplant (2009) 24: 3354 3359 doi: 10.1093/ndt/gfp318 Advance Access publication 30 June 2009 Gender is related to alterations of renal endothelial function in type 2 diabetes Markus P. Schneider, Martin Ritt, Ulrike Raff, Christian Ott and Roland E. Schmieder Department of Nephrology and Hypertension, University of Erlangen-Nuremberg, Germany Correspondence and offprint requests to: Roland E. Schmieder; E-mail: roland.schmieder@rzmail.uni-erlangen.de Abstract Background. Gender has been shown to affect endothelial function of the forearm circulation in patients with type 2 diabetes, but data on the renal circulation are lacking. We hypothesized that renal vascular nitric oxide (NO) availability is higher, and oxidative stress lower, in female compared to male patients with type 2 diabetes. Methods. In 41 male and 39 female patients with type 2 diabetes, renal plasma flow (RPF) was determined by constant infusion input clearance at baseline and following infusion of the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA, 4.25 mg/kg) to assess basal renal vascular NO availability. After a subsequent infusion of L-arginine (100 mg/kg) to restore baseline conditions, vitamin C (45 mg/kg) was co-infused to determine levels of oxidative stress in the renal circulation. Results. Baseline renal haemodynamics were similar between genders. L-NMMA-induced renal vasoconstriction was more pronounced in females compared to males ( 89 ± 69 versus 60 ± 52 ml/min/1.73 m 2, P = 0.03). After administration of L-arginine to restore baseline perfusion, the co-infusion of vitamin C led to a lesser increase of RPF in females than in males (+37 ± 86 versus +60 ± 52 ml/min/1.73 m 2, P = 0.05). Conclusions. Our data demonstrate that NO availability in the renal circulation is greater in female than in male patients with type 2 diabetes that is associated with reduced levels of oxidative stress in females. The role of this genderrelated difference in renal endothelial function for the initiation and progression of diabetic nephropathy should be addressed in future studies. Keywords: endothelium; gender; kidney; nitric oxide; oxidative stress C The Author 2009. Published by Oxford University Press [on behalf of ERA-EDTA]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org