Urinary Biomarkers in the Early Detection of Acute Kidney Injury after Cardiac Surgery

Patient Selection

All adult patients undergoing cardiac surgery at Columbia University Medical Center (New York, NY) were eligible for enrollment. A total of 90 patients were prospectively studied from June 25, 2005 to January 11, 2006. Exclusion criteria for the study were patients who depended on chronic dialysis support and patients who died within the first 24 h after surgery. Postoperative AKI was defined by using modified Stage 1 and Stage 2 categories of AKIN criteria, which is an absolute increase in Scr of ≥0.3 mg/dl from baseline or an absolute increase in Scr of 2- to 3-fold from baseline within the first 72 h after cardiac surgery, respectively (9). In a subgroup analysis, early postoperative AKI was defined as an absolute elevation in Scr of ≥0.3 mg/dl from baseline within the first 24 h after operation. Late postoperative AKI was defined as absolute elevation in Scr of ≥0.3 mg/dl from baseline between 24 and 72 h after operation.

To evaluate the effect of handing of urine sample at the time of collection, protease inhibitor, prolonged freezing, and repeated freeze-thaw cycles before biomarker measurement, additional urine samples were collected from 15 established AKI patients seen for renal consultation at Brigham and Women's Hospital and Thomas Jefferson University Hospital (TJUH).

The preoperative GFR was estimated using the method described by Cockcroft and Gault (27). A total 20 patients (12 non-AKI and 8 AKI patients) had underlying chronic kidney disease with estimated GFR ≤60 ml/min, according to the National Kidney Foundation Kidney Disease Outcomes Quality Initiative guidelines (28). ICU- and hospital-free days within 30 d were defined as the number of days a patient was outside of the ICU or hospital within the first 30 postoperative days and were zero if the patient died in the ICU or in the hospital, respectively. The institutional review boards of TJUH, Brigham and Women's Hospital, and Columbia University approved the study.

Urine and Serum Sample Collections and Storage

Ten milliliters of urine were collected at the following time points: (1) preoperative; (2) immediately after cardiopulmonary bypass (CPB), or, in case of off-pump coronary artery bypass grafting, after reperfusion of the last bypass graft; and (3) 3, 18, and 24 h later. A total of 429 urine samples were collected from 36 patients with AKI and 54 patients without AKI. The urine samples were centrifuged at 1000 g for 5 min and the supernatants were stored at −20°C for up to 20 mo, then stored at −80°C. Scr was measured at baseline, immediately postoperative, and then at least daily during the hospital stay.

Urinary NGAL was measured within the first 3 mo without repeated freeze-thaw cycles. The time between initial urine collection and assays for NAG and KIM-1 at TJUH was about 1.5 yr. Urine samples were subjected to two freeze-thaw cycles before assays for KIM-1 and NAG at TJUH.

Stability of Urinary Biomarkers

Thirty milliliters of freshly voided urine samples from established AKI patients were collected and centrifuged at 1000 g for 5 min. The supernatant was aliquoted into ten eppendorf tubes and processed in varying manners for comparisons. The reference eppendorf tubes were frozen at −80°C within the first 20 min after collection. Other urine samples were allowed to sit at room temperature or refrigerated at 4°C for 2, 4, 6, and 24 h before freezing at −80°C. Biomarkers were measured within 2 wk after initial urine collection and then remeasured after prolonged freezing at −80°C for 1 and 2 yr with and without repeat freeze-thaw cycles. To determine the need for protease inhibitor, reference eppendorf tubes with and without addition of a protease inhibitor cocktail tablet (complete mini, Roche, Mannheim, Germany) were frozen at −80°C soon after collection.

In addition, we performed comparative stability studies to evaluate the effect of prolonged storage at −20 versus −80°C using duplicate urine samples that were stored at −80 and −20°C for 2 yr, respectively.

Measurement of Urinary KIM-1, NAG, and NGAL

Urinary soluble KIM-1 protein was quantified by a modified ELISA system using polyclonal KIM-1 antibodies as described previously (14). Fifty microliters of urine sample were used for detection of urinary KIM-1 protein. Urinary NAG was measured by colorimetric assay by using a commercially available kit (Roche Applied Science, Indianapolis, IN) (14) according to the manufacturer's protocol. Urinary NGAL concentration was measured using a commercially available ELISA kit (Antibody Shop, Gentofte, Denmark). Urine samples were diluted to 1:50 and 1:5000 dilutions before performing an ELISA assay to fit the concentrations of respective NGAL protein in the linear range of the standard curve. The measured values were normalized to the urinary creatinine concentration. The inter- and intraassay coefficients of variation for KIM-1, NAG, and NGAL were <10%. The measurements were made in duplicate and in a blinded fashion.

Serum and Urinary Creatinine Determination

Scr and urine creatinine concentrations were measured by the Jaffé assay using Roche/Hitachi 917 systems (Roche Diagnostics, Indianapolis, IN) and Creatinine Companion (Exocell, Philadelphia, PA) (29), respectively.

Statistical Analyses

Continuous variables were compared using the t test or the nonparametric Mann–Whitney U test, as appropriate. For analysis of single biomarkers, receiver-operating characteristic (ROC) curves were generated and the areas under the curve (AUCs) calculated and compared using the method of Hanley and McNeil for comparison for a single time point (30). Two-tailed P values <0.05 were considered statistically significant. For joint analysis of multiple biomarkers, a fitted multiple logistic regression model was used to give maximum sensitivity and specificity (Table 1). Statistical analyses were performed using SAS Version 9.1 (SAS Institute, Cary, NC) and MedCalc Version 9.3.1 (MedCalc, Inc., Mariakerke, Belgium).

Stability of Urinary KIM-1, NAG, and NGAL Proteins

Urinary KIM-1, NAG, and NGAL proteins did not degrade significantly up to 24 h at 4°C and at room temperature, and up to 1 yr in prolonged storage at −80°C with repeat freeze and thaw cycles (Table 2A). An addition of protease inhibitor was not necessary to prevent degradation of urinary KIM-1, NAG, and NGAL proteins. In addition, urinary NAG remained stable even after 2 yr of prolonged storage at −80°C with repeat freeze-thaw cycles. There was no significant degradation of urinary NAG and KIM-1 proteins after prolonged storage at −20°C for 2 yr (Table 2B), but urinary NGAL protein degraded significantly.

Patient Characteristics for Cardiac Surgery

The clinical and demographic characteristics of 90 adult patients are reported in Table 3. Thirty-six patients developed postoperative AKI (40%), of which 32 patients developed Stage 1 and the remaining four patients with Stage 2 AKIN criteria AKI. Early AKI was diagnosed in 16 patients within the first 24 h after surgery. Late AKI was diagnosed in 15 patients between 24 and 48 h and in the remaining 5 patients between 48 and 72 h after surgery. There were 26 (72%) of 36 AKI patients who maintained persistent elevations of Scr over 48 h after an absolute increase in Scr of ≥0.3 mg/dl from baseline. There were 14 (88%) early and 12 (60%) late postoperative AKI patients who maintained persistent elevations of Scr over 48 h after an absolute increase in Scr of ≥0.3 mg/dl from baseline. No differences were noted between patients with and without AKI with respect to age or sex. CPB time was significantly longer in patients with AKI (P < 0.001). The increases in Scr on postoperative days were statistically significantly different from the baseline and non-AKI groups (P < 0.05). One early AKI patient required continuous renal replacement therapy. Five patients with postoperative AKI died in the hospital.

Quantitation of Serial Urinary KIM-1, NAG, and NGAL Proteins

Comparisons of median and mean biomarker levels among patients with and without postoperative AKI are shown in Table 4 and Figure 1. Urinary KIM-1 levels were significantly elevated immediately after surgery and 3 h later (P < 0.005) and remained elevated 24 h later (P < 0.05) in the early postoperative AKI group. However, urinary KIM-1 levels were not higher in the late postoperative AKI group compared with the non-AKI group. Urinary NGAL and NAG levels peaked immediately after operation and then decreased in both AKI and non-AKI groups at later time points. Urinary NGAL levels were higher in the late postoperative AKI group (P < 0.05), but not in the early postoperative AKI group. Urinary NAG levels were elevated without statistical significance in both early and late postoperative AKI groups.

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Figure 1.

Pattern of urinary biomarker expression. Graphs show mean normalized urinary kidney injury molecule-1 (KIM-1), N-acetyl-β-D-glucosaminidase (NAG), and neutrophil gelatinase associated lipocalin (NGAL) concentrations at multiple time points after cardiac surgeries. Error bars are SEM. AKI, acute kidney injury; early AKI, development of acute kidney injury within first 24 h after operation; late AKI, development of acute kidney injury between 24 and 72 h after operation; Ucr, urine creatinine concentration.

We observed a decrease of Scr immediately after operation in most AKI and non AKI patients. None of the AKI patients had an absolute increase in Scr of ≥0.3 mg/dl (or ≥30% delta Scr) from baseline immediately after surgery.

ROC Analysis of Urinary NAG, KIM-1, and NGAL Independently and Combined as Early Biomarkers for Detection of Postoperative AKI

The performance of KIM-1, NAG, and NGAL in diagnosing postoperative AKI is illustrated in Table 5. Immediately after surgery and 3 h after the operation, the AUCs for the diagnosis of postoperative AKI using KIM-1 were 0.68 and 0.65; 0.61 and 0.63 for NAG; 0.59 and 0.65 for NGAL; and 0.75 and 0.78, respectively, for the combination of the three biomarkers. Subgroup analysis of patients with early postoperative AKI, immediately and 3 h after operation, showed AUCs for the diagnosis of AKI using KIM-1 were 0.79 and 0.73; 0.60 and 0.59 for NAG; 0.51 and 0.58 for NGAL; and 0.80 and 0.84, respectively, for the combination of the three biomarkers. Table 6 demonstrates the sensitivity, specificity, and positive and negative likelihood ratio immediately and 3 h after operation for diagnosis of AKI.

The performance difference between the three combined biomarkers and some of the individual biomarkers by AUC for diagnosis of early AKI was statistically significantly different at immediately and 3 h after operation (P < 0.05), but there was no added value for diagnosis of late AKI.