Coronary Artery Disease. Neutrophil Gelatinase Associated Lipocalin and Contrast-Induced Acute Kidney Injury

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1 Coronary Artery Disease Neutrophil Gelatinase Associated Lipocalin and Contrast-Induced Acute Kidney Injury Cristina Quintavalle, PhD; Chiara Viviani Anselmi, PhD; Francesca De Micco, PhD; Giuseppina Roscigno, PhD; Gabriella Visconti, MD; Bruno Golia, MD; Amelia Focaccio, MD; Bruno Ricciardelli, MD; Enzo Perna, MD; Laura Papa, PhD; Elvira Donnarumma, PhD; Gerolama Condorelli, MD, PhD; Carlo Briguori, MD, PhD Downloaded from by guest on July 22, 2018 Background Neutrophil gelatinase associated lipocalin (NGAL) is an early marker of acute kidney injury (AKI). Methods and Results Urine NGAL and serum NGAL (sngal) were assessed at 2, 6, 24, and 48 hours after contrast media (CM) exposure in 458 high-risk patients (development set). Optimal thresholds in predicting contrast-induced AKI (serum creatinine [scr] increase 0.3 mg/dl at 48 hours after CM administration) were identified. Major adverse events (MAE; death, dialysis, nonfatal myocardial infarction, sustained kidney injury, and myocardial revascularization) at 1 year were assessed. In the development set, optimal thresholds for contrast-induced AKI occurred at 6 hours for both urine NGAL ( 20 ng/ml; 97% negative predictive value and 27% positive predictive value) and sngal ( 179 ng/ml; 93% negative predictive value and 20% positive predictive value). Furthermore, sngal 179 ng/ml at 6 hours was an independent predictor of 1-year MAE. 1-year MAE occurred in 27/198 patients (13.5%) with sngal <179 ng/ml and scr <0.3 mg/dl, in 57/193 (29.5%) patients with only sngal 179 ng/ml, and in 37/67 (55%) patients with scr 0.3 mg/dl. In additional 253 patients (validation set), no patient with urine NGAL <20 ng/ml or sngal <179 ng/ml at 6 hours developed contrast-induced AKI. Furthermore, 6/68 (9%) patients with sngal <179 ng/ml and scr increase <0.3 mg/dl had 1-year MAE versus 17/57 (30%) patients with sngal 179 ng/ml and scr increase <0.3 mg/dl and 8/16 (50%) patients with scr increase 0.3 mg/dl. Conclusions Urine NGAL <20 ng/ml and sngal <179 ng/ml at 6 hours are reliable markers for ruling out contrastinduced AKI. sngal 179 ng/ml at 6 hours predicts 1-year MAE. Clinical Trial Registration URL: Unique identifier: NCT (Circ Cardiovasc Interv. 2015;8:e DOI: /CIRCINTERVENTIONS ) Key Words: contrast media kidney kidney disease NGAL prognostic factor Iodinated contrast media (CM) administration during coronary and peripheral procedures may cause acute kidney injury (AKI). 1 The current recommendations suggest (1) serum creatinine (scr) as the gold standard and (2) an extended observation ( 24 hours) for patients undergoing coronary procedures with significant renal dysfunction even in case of otherwise uncomplicated clinical course. 2 Several biomarkers of kidney injury have shown diagnostic and prognostic value. 3 Neutrophil gelatinase associated lipocalin (NGAL), a ubiquitous 25-kDa protein covalently bound to gelatinase from human neutrophils, is a marker of kidney tubular injury. 4,5 Serum NGAL (sngal) and urine NGAL (ungal) predict AKI in different clinical settings, 6 9 See Editorial by Dauerman including contrast-induced AKI (CI-AKI). 10,11 Overall, NGAL is an useful tool for early diagnosis (within a few hours) and prognosis (prediction of renal replacement therapy and inhospital mortality) of AKI. 9,12,13 However, there is only limited data on (1) the optimal timing and site (urine/serum) for assessment, (2) the cut-off value, (3) comparison with cystatin C, 14 and (4) the clinical and prognostic significance in the setting of CI-AKI. Objectives of the present study are (1) the assessment of the diagnostic usefulness of NGAL in the setting of CI-AKI in patients with moderate-to-severe chronic kidney disease Received April 8, 2015; accepted July 31, From the Department of Molecular Medicine and Medical Biotechnology, Federico II University, Naples, Italy (C.Q., E.P., G.C.); Institute of Genetics and Biomedical Research, Milan Unit, Milan, Italy (C.V.A., L.P.); Humanitas Research Hospital, Rozzano, Italy (C.V.A., L.P.); Laboratory of Interventional Cardiology and Department of Cardiology, Clinica Mediterranea, Naples, Italy (F.D.M., G.V., B.G., A.F., B.R., C.B.); Institute for Endocrinology and Experimental Oncology, National Research Council, Naples, Italy (G.R., G.C.); and IRCCS SDN, Naples, Italy (E.D.). The Data Supplement is available at Correspondence to Carlo Briguori, MD, PhD, Interventional Cardiology, Clinica Mediterranea, Via Orazio, 2, I-80121, Naples, Italy, carlobriguori@clinicamediterranea.it or Gerolama Condorelli, MD, PhD, Department of Molecular Medicine and Medical Biotechnology, Federico II University, Naples, Via Pansini, 5, Naples, Italy, gecondor@unina.it 2015 American Heart Association, Inc. Circ Cardiovasc Interv is available at DOI: /CIRCINTERVENTIONS

2 2 Quintavalle et al NGAL and Contrast-Induced Acute Kidney Injury WHAT IS KNOWN Serum creatinine is the gold standard for the diagnosis of contrast-induced acute kidney injury. However, serum creatinine requires 24 to 48 hours to increase, despite immediate impairment in glomerular filtration rate by contrast media induced injury. Neutrophil gelatinase associated lipocalin has emerged as an early biomarker of contrast-induced acute kidney injury. WHAT THE STUDY ADDS Neutrophil gelatinase associated lipocalin at 6 hours after contrast media exposure is a reliable marker for ruling out contrast-induced acute kidney injury because of its excellent negative predictive value. High neutrophil gelatinase associated lipocalin at 6 hour after contrast media exposure is associated with a significant serum creatinine increase in only 20% of cases, but is an independent predictor of 1-year major adverse events. undergoing coronary or peripheral angiography or angioplasty and (2) the prognostic significance of high NGAL values, used as an index of kidney damage, irrespective to the presence or absence of changes in kidney function, defined on the basis of scr changes. Methods Patient Population Development Set From January 2009 to October 2012, 458 patients scheduled for coronary or peripheral angiography or angioplasty with an estimated glomerular filtration rate (egfr) 30 ml/min per 1.73 m 2 or Mehran risk score 11 were included in this study. Part of these patients (n=280) had been enrolled in the Renal Insufficiency Following Contrast Media Administration Trial (REMEDIAL) II (registration on ID: NCT ). 15 CI-AKI prophylaxis strategies for the 280 patients enrolled in the REMEDIAL II were (1) hydration with sodium bicarbonate solution (154 meq/l) 16 plus high dose of N-acetylcysteine or (2) hydration with normal saline plus N- acetylcysteine controlled by the RenalGuard system (PLC Medical Systems, Inc, Franklin, MA). 15 All the other patients (including those in the validation set) were treated with the RenalGuard system as a result of the positive results of the REMEDIAL II. Validation Set From November 2012 to December 2014, 253 patients scheduled for coronary or peripheral angiography or angioplasty with an egfr 30 ml/min per 1.73 m 2 or Mehran risk score 11 were analyzed. The egfr was calculated with the Modification of Diet in Renal Disease 17 and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. 18 The risk score for predicting CI- AKI was defined according to Mehran et al. 19 Iodixanol (Visipaque 320 mg I/mL; GE), a nonionic, iso-osmolar CM, was used in all patients. CM volume >3 egfr was considered suggestive of increased risk of CI-AKI. 20 The study was accepted by the ethical committee, and an informed consent was obtained from all patients. Table 1. Clinical Features at Baseline of Patients in the Development Set With and Without CI-AKI CI-AKI Group (n=64) No CI-AKI Group (n=394) P Value Age, y 74±9 75± Male, % 45 (70) 257 (65) 0.42 Body mass index, kg/m 2 28±5 29± Blood pressure, mm Hg Systolic 149±27 157± Diastolic 75±11 78± Mean 100±13 104± Serum creatinine (median, range) egfr, ml/min per 1.73 m ( ) 1.93 ( ) MDRD formula 28±8 31± CKD-EPI formula 31±11 35± Contrast nephropathy risk score Serum cystatin C (median, range) Left ventricular ejection fraction, % Left ventricular end diastolic pressure, mm Hg 12±2 12± ( ) 1.74 ( ) ±11 47± ±8 14± Diabetes mellitus, % 38 (60) 270 (68.5) 0.40 Peripheral vascular disease, % Drugs, % 15 (23.5) 46 (11) ACE inhibitor 27 (43) 197 (50) 0.28 Calcium channel blocker 11 (17) 106 (27) 0.12 Angiotensin II receptor inhibitor 21 (33) 126 (32) 0.88 Diuretics 40 (63) 244 (62) 1.00 Beta blockers 48 (75) 271 (69) 0.38 Statins 50 (78) 318 (80) 0.61 Performed procedure, % Coronary angiography 27 (42) 157 (40) 1.00 PCI 32 (50) 180 (46) 0.79 Coronary angiography and ad hoc PCI 3 (5) 33 (8) 0.33 Peripheral procedure 2 (3) 24 (6) 0.56 Volume of contrast media, ml 162±88 118±72 <0.001 CV/GFR ratio >3 46 (72) 1658 (42) <0.001 Prophylaxis with 33 (51.6) 277 (70.4) RenalGuard system, % ACE indicates angiotensin-converting enzyme; CI-AKI, contrast-induced acute kidney injury; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; CV/GFR >3, contrast media volume/estimated glomerular filtration rate >3; egfr, estimated glomerular filtration rate; MDRD, Modification of Diet in Renal Disease; and PCI, percutaneous coronary intervention.

3 3 Quintavalle et al NGAL and Contrast-Induced Acute Kidney Injury Figure 1. Baseline neutrophil gelatinase associated lipocalin (NGAL) and cystatin C levels versus estimated glomerular filtration rate (egfr). Spearman s correlation analysis (r) for baseline urine, serum, and normalized NGAL levels and cystatin C. CI indicates confidence interval; Cys C, cystatin C; sngal, neutrophil gelatinase associated lipocalin; ungal, urine neutrophil gelatinase associated lipocalin; and ungal/ucr, normalized urine NGAL/urine creatinine ratio. Biomarkers of Kidney Function and Injury Blood urea nitrogen, scr, and cystatin C were measured the day before and 2, 6, 12, 24, and 48 hours after administration of the CM; additional measurements were performed on deterioration of baseline renal function. ungal and sngal were measured the day before and 2, 6, 24, and 48 hours after CM administration. Urine was collected and stored at 80 C until analysis with the automated immunoassay ARCHITECT platform (Abbott Diagnostic, Abbott Park, IL). Intra- and interassay variabilities were 3.5% and 10%, respectively. Urine creatinine (ucr), measured by quantitative colorimetric microplate assay kit, was used to normalize ungal for changes in urine concentration (ungal/ucr or normalized ungal). Blood samples were centrifuged at 4000 rpm (1400g) for 20 minutes, and the serum harvested and stored in aliquots at 80 C until analysis with the Human NGAL Rapid ELISA Kit (BioPorto Diagnostics A/S, Gentofte, Demnark). Intra- and interassay variabilities were 3.4% and 7%, respectively. All NGAL measurements were performed in a central core laboratory (Federico II University) by independent and blinded operators. Events Definition CI-AKI was defined as an increase in the scr concentration 0.3 mg/dl from the baseline at 48 hours after CM administration or the need for dialysis. 21 Major adverse events (MAE) at 1 year included all-cause death, dialysis, nonfatal myocardial infarction, myocardial revascularization, and sustained kidney injury (ie, a persistent 25% egfr reduction compared with baseline at the last follow-up available value). The length of in-hospital stay (LOS) was calculated as the sum of the number of days since admission until discharge from the hospital. LOS was collected to assess (1) its difference in patients with versus those without CI-AKI and (2) the impact of the current recommendation for the diagnosis of CI-AKI in patients who did not developed CI-AKI. 2 One-year follow-up was available in all patients in the development set and in all the 141 of the 253 (56%) patients Figure 2. Urine NGAL (A), serum NGAL (B), normalized urine NGAL (urine NGAL/urine creatinine ratio; C), and cystatin C (D) at various time points in patients who developed (closed symbol) or not (open symbol) contrast-induced acute kidney injury (CI-AKI). P value computed by mixed model with repeated measures. *P<0.05; **P<0.010; ***P< NGAL indicates neutrophil gelatinase associated lipocalin.

4 4 Quintavalle et al NGAL and Contrast-Induced Acute Kidney Injury Table 2. Cut-Off Values for the Prediction of CI-AKI sngal in the validation set enrolled within December All events were adjudicated by a Clinical Events Committee, who was blinded to both scr and NGAL levels. Statistical Analysis Continuous variables are given as mean±sd or median (interquartile range) as appropriate. Normality assumption was verified graphically (ie, quantile-quantile plot) and was confirmed using the Shapiro Wilk test. Categorical variables are expressed in percentages. Comparisons between groups were performed by Student s t test or χ 2 test. Spearman correlation test was used to evaluate the relation between different biomarkers. To test the predictive value of absolute and normalized ungal and the value of sngal for CI-AKI, we used mixed model for repeated measures after transforming absolute ungal, ucr, and sngal levels into natural logarithm, and so overcoming the problem of the non-normal distribution. In this model, the NGAL values and time period interaction were considered as fixed effects and patients as a random effect. Mann Whitney U test was performed to assess potential time-related changes in ungal, sn- GAL, and ungal/ucr ratio in relation to the occurrence of CI-AKI or MAE. Receiver operating characteristic curves were generated, and the area under the curve (AUC) was calculated to evaluate the diagnostic sensitivity and specificity of the biomarkers at different time points (baseline, 2, 6, and 24 hours) for CI-AKI and 1-year MAE. Optimal cut-offs were identified by the Youden index. 22 Multiple logistic regression analysis was performed to determine whether the biomarkers were independent predictors of CI-AKI, adjusting for several clinical variables, selected according to data in the literature and significance (P<0.1) at univariate analysis. To overcome the risk of collinearity between NGAL and egfr, the regression model was run with and without egfr in the model. To assess the independent predictive power of both functional (scr) and injury (NGAL) biomarkers, 3 groups were defined and analyzed: (1) group without scr increase 0.3 mg/dl or sngal 179 ng/ml (group without kidney damage or functional change); (2) group with sngal 179 ng/ml alone (group with kidney damage); and (3) group with scr increase 0.3 mg/dl, irrespective to the sngal values (group with functional change). The Kaplan Maier method was used to calculate event-free survival, and comparison between groups was performed by using the Threshold AUC (95% CI) Sensitivity, % Specificity, % LR+/LR ROC analysis 179* 0.62 ( ) /0.58 Quartile 149* 0.59 ( ) /0.36 Quintile 182* 0.60 ( ) /0.55 ungal ROC analysis 20* 0.61 ( ) /0.51 Quartile 8* 0.56 ( ) /0.47 Quintile 21.6* 0.60 ( ) /0.50 Normalized ungal ROC analysis 1.5* 0.54 ( ) /0.78 Quintile 1* 0.53 ( ) /0.68 scystatin C ROC analysis 10% 0.66 ( ) /0.35 AUC indicates area under the curve; CI, confidence interval; CI-AKI, contrast-induced acute kidney injury; LR, likelihood ratio; ROC, receiver operating characteristic; sngal, serum neutrophil gelatinase associated lipocalin; and ungal, urine neutrophil gelatinase associated lipocalin. *At 6 h. At 24 h after contrast media exposure. log-rank statistic. Cox proportional hazards model was performed to provide hazard ratios with 95% confidence intervals and adjustment for previously selected risk factors, as reported for the multiple logistic regression analysis. To correct for multiple testing, 1000 bootstrap iterations were computed. For all tests, P<0.05 was considered statistically significant. Data were analyzed with STATA software (version11/se, College Station, TX). Results CI-AKI and NGAL Baseline features of the patients in the development set, stratified for CI-AKI, are reported in Table 1. The characteristics of the patients enrolled in the REMEDIAL II (n=280) versus those in the Non-REMEDIAL II (n=178) are reported in the Table I in the Data Supplement. Patients who experienced CI- AKI had more severe chronic kidney disease, had a higher rate of peripheral vascular disease, received a greater volume of CM, and were less often treated by the RenalGuard system. LOS was longer in patients who developed CI-AKI (10±6 versus 6±4 days; P<0.001). In patients who did not developed CI-AKI, LOS stay was prolonged for 1 day only for ruling out the occurrence of CI-AKI. Indeed, in these patients, LOS would have been 5±6 days instead of 6±4 days (P<0.001). Furthermore, the in-hospital death rate was higher (although not statistically significant) in patients who developed CI-AKI (4/64 [6%] versus 9/394 [2.5%]; P=0.076). At baseline, NGAL (urine, serum, and normalized) and cystatin C were negatively correlated with egfr (Figure 1). Correlations between NGAL and scr are reported in Figure I in the Data Supplement. As represented in Figure 2, values of ungal, sngal, ungal/ucr ratio, and cystatin C at the different time points began to diverge significantly between CI-AKI group and non CI-AKI group at 2 hours after CM

5 5 Quintavalle et al NGAL and Contrast-Induced Acute Kidney Injury Table 3. Univariate and Multivariate Predictors of CI-AKI Biomarker Confounding Factor Univariate Model Multivariate Model* OR CI (95%) P Value OR CI (95%) P Value ungal 20 ng/ml CV/GFR > LVEDP Cystatin C 10% egfr Age Mehran score PVD RenalGuard system PCI sngal 179 ng/ml Normalized ungal 1.5 Cystatin C 10% CV/GFR > LVEDP egfr Age Mehran score PVD RenalGuard system Baseline sngal PCI CV/GFR > LVEDP Cystatin C 10% egfr Age Mehran score PVD RenalGuard system PCI CI indicates confidence intervals; CI-AKI, contrast-induced acute kidney injury; CV/GFR >3, contrast media volume/ estimated glomerular filtration rate >3; egfr, estimated glomerular filtration rate; LVEDP, left ventricular end-diastolic pressure; OR, odds ratio; PCI, percutaneous coronary intervention; PVD, peripheral vascular disease; sngal, serum neutrophil gelatinase associated lipocalin; and ungal, urine neutrophil gelatinase associated lipocalin. *Bootstrap sampling method (1000 reps) was used. injection (Figure 2). Cut-offs of ungal, sngal, ungal/ ucr ratio, and cystatin C for predicting CI-AKI are reported in the Table 2 and Table II in the Data Supplement. The best overall combination of sensitivity and specificity for ungal, sngal, and ungal/ucr ratio was observed at 6 hours after CM exposure and at 24 hours for cystatin C (Table 2; Figures II and III in the Data Supplement). Multiple logistic regression for ungal, sngal, and ungal/ucr ratio cutoffs for CI-AKI is reported in Table 3. Consistent adjusted odds ratios (OR) were obtained for sngal 179 ng/ml and ungal 20 ng/ml, but not for ungal/ucr ratio 1.5. Based on this result, sngal 179 ng/ml and ungal 20 ng/ml at 6 hours and cystatin C increase 10% at 24 hours were regarded the best thresholds for prediction of CI-AKI.

6 6 Quintavalle et al NGAL and Contrast-Induced Acute Kidney Injury Table 4. Clinical Features at Baseline of Patients in the Development Set With and Without 1-Year Major Adverse Events MAE Group, (n=121) Event-Free Group, (n=337) P Value Age, y 76±8 74± Male, % 82 (67.5) 217 (64.5) 0.85 Body mass index, kg/m 2 28±5 29± Serum creatinine (median, range) egfr, ml/min per 1.73 m ( ) 1.91 ( ) MDRD formula 29±9 31± CKD-EPI formula 32±12 36± Figure 3. Receiver operating characteristic curves of urine NGAL (A) and serum NGAL (B), in the validation set assessed at 6 hours after administration of contrast medium, for the diagnosis of CI-AKI. AUC indicates area under the curve; CI-AKI, contrastinduced acute kidney injury; and NGAL, neutrophil gelatinase associated lipocalin. In detail, the positive predictive value (PPV) and the negative predictive value (NPV) for CI-AKI of sngal, ungal, and cystatin C were 20% and 93%, 27% and 97%, and 27% and 91%, respectively. The characteristics of patients in the validation set were similar to those in the development set, besides the lower CM volume and the systematic use of the RenalGuard system (Table III in the Data Supplement). CI-AKI occurred in 29 of 253 (11.5%) patients in the validation set. ungal 20 ng/ml and sngal 179 ng/ml at 6 hours were observed in 139 (55%) and 130 (51%) patients, respectively. Although all the 29 patients who developed CI-AKI had high NGAL level at 6 hours, the overall PPV was low (ungal 20 ng/ml, 21%; sngal 179 ng/ml, 22%). On the contrary, the NPV was 100%, indeed no patients with sngal <179 ng/ml and ungal <20 ng/ml at 6 hours developed CI-AKI (Figure 3). Cystatin C increase 10% at 24 hours showed a PPV of 21% and an NPV of 90%. MAE and NGAL In the development set, 1-year MAE occurred in 121 (26.4%) patients. In detail, 67 patients (14.6%) died, 17 (3.5%) patients needed dialysis (3 of whom eventually died), 11 (2.4%) had nonfatal myocardial infarction, 29 (6.3%) had myocardial revascularization, and 19 (4%) had sustained GFR reduction. Serum cystatin C (median, range) Left ventricular ejection fraction, % 2.00 ( ) 1.73 ( ) ±10 47± Diabetes mellitus, % 78 (65) 230 (68) 0.58 Peripheral vascular disease, % 23 (19) 48 (13) 0.24 Drugs, % Beta blockers 82 (68) 230 (68) 0.91 Statins 98 (81) 270 (80) 0.87 Performed procedure, % Coronary angiography 48 (39.5) 136 (40.5) 0.91 PCI 62 (51) 150(44.5) 0.24 Coronary angiography and ad hoc PCI 6 (5) 30 (9) 0.23 Peripheral procedure 6 (5) 21 (6) 0.82 Contrast nephropathy risk score 13±2 12± Volume of contrast media, ml 128±83 121± CM/GFR ratio >3 86 (71%) 198 (59%) Prophylaxis with RenalGuard system, % 80 (66) 232 (69) 0.54 CI-AKI, % 34 (28) 30 (9) <0.001 Stage 1 21 (17) 28 (8) Stage 2 2 (1.5) 2 (0.5) Stage 3 11 (9) 0 Need for renal replacement therapy, % 14 (11.5%) 0 <0.001 Length of in-hospital stay, days 10±8 6±4 <0.001 CI-AKI indicates contrast-induced acute kidney injury; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; CM, contrast media; egfr, estimated glomerular filtration rate; GFR, glomerular filtration rate; MAE, major adverse events; MDRD, Modification of Diet in Renal Disease; and PCI, percutaneous coronary intervention. Death was cardiac in 67% of patients, noncardiac in 29.5% of patients, and unknown in 3.5% of patients. All the 19 patients who had sustained GFR reduction experienced also additional MAE at follow-up: indeed, 17 patients needed dialysis (3 of whom eventually died), 5 had nonfatal myocardial infarction,

7 7 Quintavalle et al NGAL and Contrast-Induced Acute Kidney Injury and 7 patients had myocardial revascularization. Clinical, angiographic, and procedural features of patients with MAE (MAE group) and those without MAE (event-free group) are summarized in Table 4. The MAE group had higher percentages of individuals with greater age, lower egfr, higher contrast nephropathy risk score, and CI-AKI than did the event-free group. Table 5. sngal Cut-Off Values for the Prediction 1-Year Major Adverse Events Changes in ungal, sngal, and ungal/ucr ratio in relation to the occurrence of MAE are represented in the Figure 4. sngal 179 ng/ml at 6 hours after CM exposure was the threshold predicting 1-year MAE (Figure 4 and Table 5; Table IV in the Data Supplement). This sngal value was observed in 53% of patients, with, respectively, 14% and 93% PPV and NPV for MAE. Univariate and multivariate Cox Threshold AUC (95% CI) Sensitivity, % Specificity, % LR+/LR ROC analysis 179* 0.67 ( ) /0.43 Quartile 149* 0.62 ( ) /0.15 Quintile 182* 0.63 ( ) /0.49 ungal ROC analysis 20* 0.57 ( ) /0.71 Quartile 8* 0.54 ( ) /0.66 Quintile 21.6* 0.57 ( ) /0.69 Normalized ungal ROC analysis 1.5* 0.51 ( ) /0.79 Quintile ( ) /0.89 scystatin C Figure 4. Urine NGAL (A), serum NGAL (B), and normalized urine NGAL (urine NGAL/urine creatinine ratio; C) at various time points in patients who developed (closed symbol) or not (open symbol) 1-year major adverse events. *P<0.05; **P< NGAL indicates neutrophil gelatinase associated lipocalin. ROC analysis 10% 0.60 ( ) /0.59 AUC indicates area under the curve; CI, confidence interval; LR, likelihood ratio; ROC, receiver operating characteristic; sngal, serum neutrophil gelatinase associated lipocalin; and ungal, urine neutrophil gelatinase associated lipocalin. *At 6 h. At 24 h after contrast media exposure.

8 8 Quintavalle et al NGAL and Contrast-Induced Acute Kidney Injury proportional hazards models were used to provide corrected hazard ratio for each parameter, using the same confounding factors considered in the CI-AKI analysis (Table 6). According to the defined cut-offs of kidney damage (ie, sngal 179 ng/ml at 6 hours) and functional change (ie, scr 0.3 mg/ dl at 48 hours), MAE occurred in 27/198 (13.5%) patients in the group without kidney damage or functional change, in 57/193 (29.5%) patients in the group with kidney damage alone (unadjusted OR versus group 1 =2.83; 95% confidence interval ; P=0.011), and in 37/67 (55%) patients in the group with functional change (unadjusted OR versus group 1 =6.64; 95% confidence interval ; P<0.001; Figure 5). By logistic regression analysis, kidney damage alone and kidney functional change were independent predictors of 1-year MAE (Table 7). MAE at 1 year occurred in 31 of 141 (22%) patients in the validation set. sngal 179 ng/ml at 6 hours was observed in 75 (53%) patients. The overall PPV was 34.5%, and the NPV was 92.5%. Only 6 of the 68 (9%) patients in the group without kidney damage or functional change had 1-year MAE. On the contrary, 17/57 (30%) patients in the group with kidney damage alone (unadjusted OR versus group 1 =4.33; 95% confidence interval ; P<0.001) and 8/16 (50%) patients in the group with functional change (unadjusted OR versus group 1 =7.03; 95% confidence interval ; P<0.001) experienced 1-year MAE. Table 6. Univariate and Multivariate Cox Regression for 1-Year Major Adverse Events Prediction Biomarker Confounding Factor Univariate Model Multivariate Model* HR CI (95%) P Value HR CI (95%) P Value ungal 20 ng/ml Cystatin C 10% CV/GFR > egfr Age Mehran score PVD RenalGuard system LVEDP PCI sngal 179 ng/ml Baseline sngal Cystatin C 10% CV/GFR > egfr Age Mehran score PVD RenalGuard system LVEDP PCI Normalized ungal egfr Cystatin C 10% CV/GFR > Age CI-AKI score PVD RenalGuard system LVEDP PCI CI indicates confidence intervals; CI-AKI, contrast-induced acute kidney injury; CV/GFR >3, contrast media volume/ estimated glomerular filtration rate >3; egfr, estimated glomerular filtration rate; HR, hazard ratio; LVEDP, left ventricular end-diastolic pressure; PCI, percutaneous coronary intervention; PVD, peripheral vascular disease; sngal, serum neutrophil gelatinase associated lipocalin; and ungal, urine neutrophil gelatinase associated lipocalin. *Bootstrap sampling method (1000 reps) was used.

9 9 Quintavalle et al NGAL and Contrast-Induced Acute Kidney Injury Figure 5. Kaplan Meier curves of 1-year major adverse events in patients without kidney damage or functional change (dotted line), with kidney damage alone (serum neutrophil gelatinase associated lipocalin [sngal] 179 ng/ml at 6 hours and serum creatinine [scr] <0.3 mg/dl at 48 hours; thinner continuous line), and with functional change (scr 0.3 mg/dl at 48 hours irrespective of the NGAL values; broken line). *P<0.05; **P<0.010; ***P< Discussion Biomarkers for CI-AKI Diagnosis The results of the current study suggest that the following: 1. NGAL is a reliable marker for ruling out CI-AKI. This finding may allow physicians to discharge earlier 50% of patients at risk, thus avoiding unnecessary prolonged hospitalization with its associated practical and economic advantages. 2,3 2. High NGAL levels (ungal 20 mg/dl and sngal 179 mg/dl) at 6 hours occurred in >50% of patients. This confirms that, in high-risk patients, AKI after CM exposure occur more often than detected by scr criteria. The observation that NGAL starts to increase within 2 hours after CM administration is consistent with the experimental Table 7. Events Variable Independent Predictors of 1-Year Major Adverse Univariate Model Multivariate Model* HR CI (95%) P Value HR CI (95%) P Value Group with kidney damage alone Group with kidney < functional change CI indicates confidence interval; egfr, estimated glomerular filtration rate; HR, hazard ratio; scr, serum creatinine; and sngal, serum neutrophil gelatinase associated lipocalin. *Confounding factors are age, cystatin C, contrast media volume and egfr, severity of coronary artery disease, previous myocardial infarction, left ventricular ejection fraction, and diabetes mellitus. sngal 179 ng/ml at 6 h, and scr increases <0.3 mg/dl at 48 h. scr increases 0.3 mg/dl at 48 h irrespective to the sngal at 6 h. Patients with functional change alone and those with both kidney damage and functional change were combined because of the limited number (n=8) in the group with functional change alone. finding that the cytotoxic effects of the CM occur within minutes to hours from exposure ungal 20 ng/ml and sngal 179 ng/ml at 6 hours after CM exposure anticipate a significant scr increase in only 20% of cases. 12,24 This low PPV limits the clinical relevance of NGAL assessment in the setting of CI-AKI. Several reasons have been proposed to explain this inconsistency of NGAL performance in AKI: (1) heterogeneity in baseline renal function, 25 (2) different definition and severity of AKI, (3) different clinical settings, 9 and (4) the current diagnosis of AKI, which relies on the assessment of scr, rather than a marker of AKI. 13,28,29 4. NGAL performance is superior to that of serum cystatin C in ruling out CI-AKI. We previously observed that a serum cystatin C increase <10% at 24 hours is a reliable marker for ruling out CI-AKI. 17 Although confirmed in the present study, the clinical performance of this serum cystatin C threshold was inferior to that of NGAL. Both biomarkers, however, showed a similar low PPV. The only difference is the more rapid diagnosis (6 versus 24 hours) observed with NGAL compared with cystatin C. At present, it is unknown whether this earlier (6 versus 24 hours) diagnosis might translate into a clinical advantage. Future studies are warranted to address whether this early diagnosis of AKI may guide new hydration or pharmacological approaches preventing the progression from kidney damage to functional change. 3 NGAL and Prognosis High ( 179 ng/ml) sngal at 6 hours after CM exposure is an independent predictor of unfavorable outcome. This supports the concept that AKI likely exists when NGAL is increased, independently of any increase in scr. 9,13 Therefore, NGAL and scr reflect distinct pathophysiological events. 3 Haase et al 13 and Nickolas et al 9 demonstrated that, in the absence of a diagnostic increase in scr, NGAL-positive patients might still have AKI and carry a worse prognosis than NGAL-negative patients. Increased NGAL was also found highly predictive of clinical outcome, including nephrology consultation, renal replacement therapy, and admission to the intensive care unit. 28 In the current study, sngal but not ungal resulted as an independent predictor of 1-year MAE. A recent systematic review and meta-analysis support that the diagnostic accuracy of plasma NGAL/sNGAL is similar to that of ungal. 12 However, ungal is more representative of the renal pool, whereas sngal represents the systemic pool. 30 In fact, ungal is mainly produced by the distal nephron after injury and is immediately secreted into the urine. 24 In contrast, although sngal probably arises predominantly from injured thick ascending tubules and collecting ducts, it is a product of multiple sources and might be a good biomarker of inflammation. 13,24 Indeed, sngal may reveal the effect of toxins that injure multiple organs other than kidney. 24 Furthermore, sngal measurements may be affected by several coexisting variables, such as chronic kidney disease, hypertension, systemic infections, inflammatory conditions, anemia, and hypoxia. Study Limitations The present study refers to a high-risk population with a mean egfr of 30 ml/min per 1.73 m 2 ; therefore, the results should not be extended for patients with normal or

10 10 Quintavalle et al NGAL and Contrast-Induced Acute Kidney Injury moderately depressed kidney function. Moreover, patients who experienced MAE at follow-up had higher percentages CI-AKI, greater age, lower egfr, and higher contrast nephropathy risk score. Therefore, additional studies are necessary to (1) confirm these findings in a larger testing population and (2) assess whether the early diagnosis of CI-AKI through NGAL dosage is cost-effective and associated with better clinical outcome. Conclusions Low NGAL (ungal <20 mg/dl and sngal <179 mg/dl) at 6 hours after CM exposure is a reliable marker for ruling out CI-AKI. On the contrary, high NGAL (ungal 20 mg/dl and sngal 179 mg/dl) at 6 hours is a poor predictor of CI- AKI. Furthermore, sngal 179 mg/dl at 6 hours predicts 1-year MAE. Sources of Funding This work was partially supported by funds from: Associazione Italiana Ricerca sul Cancro, AIRC (grant n.ro 10620) to GC, MERIT (RBNE08E8CZ_002) to GC, PON01_02342 to CB. None. 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12 Neutrophil Gelatinase Associated Lipocalin and Contrast-Induced Acute Kidney Injury Cristina Quintavalle, Chiara Viviani Anselmi, Francesca De Micco, Giuseppina Roscigno, Gabriella Visconti, Bruno Golia, Amelia Focaccio, Bruno Ricciardelli, Enzo Perna, Laura Papa, Elvira Donnarumma, Gerolama Condorelli and Carlo Briguori Circ Cardiovasc Interv. 2015;8: doi: /CIRCINTERVENTIONS Circulation: Cardiovascular Interventions is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2015 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: An erratum has been published regarding this article. Please see the attached page for: /content/8/10/e full.pdf Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation: Cardiovascular Interventions can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Circulation: Cardiovascular Interventions is online at:

13 Correction In the article by Quintavalle et al (Quintavalle C, Anselmi CV, De Micco F, Roscigno G, Visconti G, Golia B, Focaccio A, Ricciardelli B, Perna E, Papa L, Donnarumma E, Condorelli G, Briguori C. Neutrophil gelatinase associated lipocalin and contrast-induced acute kidney injury. Circ Cardiovasc Interv. 2015;8:e DOI: /CIRCINTERVENTIONS ), which published online September 2, 2015, and appears in the September 2015 issue of the journal, a correction was needed. On page 1, the institutional affiliation for Elvira Donnarumma, PhD, SDN Foundation, has been changed to read, IRCCS SDN, Naples, Italy. The institutional affiliation for Laura Papa, PhD, Institute for Endocrinology and Experimental Oncology, National Research Council, Naples, Italy, has been changed to read, Institute of Genetics and Biomedical Research, Milan Unit, Milan, Italy and Humanitas Research Hospital, Rozzano, Italy. The authors regret this error. This correction has been made to the article, which is available at (Circ Cardiovasc Interv. 2015;XXX: DOI: /HCV ) 2015 American Heart Association, Inc. Circ Cardiovasc Interv is available at DOI: /HCV

14 Supplemental Material 1

15 Table S1. Clinical features of REMEDIAL II versus Non-REMEDIAL II patients. REMEDIAL II group (n = 280) Non-REMEDIAL II group (n = 178) P Age (years) Male (%) 183 (65.5) 119 (67) 0.76 Body-mass Index (kg/m 2 ) Blood Pressure (mmhg) Systolic Diastolic Mean Serum creatinine (median, range) 1.80 ( ) 1.95 ( ) 0.25 egfr (ml/min/1.73 m 2 ) 32 ± 8 30 ± Contrast nephropathy risk score 12 ± 3 12 ± Serum cystatin C (median, range) 1.80 ( ) 1.81 ( ) 0.81 Left ventricular ejection fraction (%) Left ventricular end diastolic pressure (mmhg) Diabetes Mellitus (%) 189 (70) 119 (67) 0.62 Peripheral chronic artery disease (%) 38 (14) 23 (13) 0.78 Drugs (%) ACE inhibitor Calcium channel blocker Angiotensin II receptor inhibitor Diuretics Beta blockers Statins 135 (50) 70 (25) 84 (31.5) 173 (64.5) 196 (70) 223 (80) 89 (50) 47 (26.5) 63 (36) 111 (62) 123 (69) 145 (81.5) Performed procedure (%) Coronary angiography PCI Coronary angiography and ad hoc PCI Peripheral procedure 110 (39) 123 (44) 24 (8.8) 17 (6) 74 (41.5) 89 (50) 12 (7) 10 (5.5) Volume of contrast media (ml) 140±77 138± Prophylaxis with Renalguard system (%) 132 (47) 178 (100) <0.001 egfr: estimated glomerular filtration rate; ACE: angiotensin-converting-enzyme; PCI: percutaneous coronary intervention. 2

16 Table S2. Cut-off values for the prediction of CI-AKI. Biomarker sngal ungal Normalized ungal Time* (hours) AUC (95% CI) 0.60 Threshold Sensitivity (%) Specificity (%) LR+/LR- ( ) / ( ) / ( ) / ( ) / ( ) / ( ) /0.78 3

17 Table S3. Clinical features of the patients in the development and the validation sets Development set (n = 458) Validation set (n = 253) P Age (years) Male (%) 302 (66) 158 (62.5) 0.37 Body-mass Index (kg/m 2 ) Blood Pressure (mmhg) Systolic Diastolic Mean Serum creatinine (median, range) 2.05 ( ) 2.17 ( ) 0.25 egfr (ml/min/1.73 m 2 ) 29 ± 8 28 ± Contrast nephropathy risk score 12 ± 2 12 ± Serum cystatin C (median, range) 1.92 ( ) 2.06 ( ) 0.31 Left ventricular ejection fraction (%) Left ventricular end diastolic pressure (mmhg) Diabetes Mellitus (%) 308 (67) 164 (65) 0.56 Peripheral chronic artery disease (%) 61 (13) 45 (18) 0.13 Drugs (%) ACE inhibitor Calcium channel blocker Angiotensin II receptor inhibitor Diuretics Beta blockers Statins 224 (49) 117 (25.5) 147 (32) 284 (62) 319 (69.5) 368 (80) 127 (50) 67 (26.5) 91 (36) 138 (62) 138 (69) 207 (82) Performed procedure (%) Coronary angiography PCI Coronary angiography and ad hoc PCI Peripheral procedure 184 (40) 212 (46) 36 (8) 26 (5.5) 119 (47) 117 (46) 7 (3) 10 (4) Volume of contrast media (ml) 140±85 101±65 <0.020 Prophylaxis with Renalguard system (%) 310 (68) 253 (100) <0.001 egfr: estimated glomerular filtration rate; ACE: angiotensin-converting-enzyme; PCI: percutaneous coronary intervention. 4