Volume 85, Issue 4, Pages 909-919 (April 2014) Identification of IGFBP-7 by urinary proteomics as a novel prognostic marker in early acute kidney injury  Fabienne Aregger, Dominik E. Uehlinger, Janusz Witowski, René A. Brunisholz, Peter Hunziker, Felix J. Frey, Achim Jörres  Kidney International  Volume 85, Issue 4, Pages 909-919 (April 2014) DOI: 10.1038/ki.2013.363 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Study design. Three groups of intensive care unit (ICU) patients were analyzed. The discovery group served to identify proteins associated with prognosis. Proteomic analysis was performed in urine of day 1 of 24 acute kidney injury (AKI) patients (pts.). Eight candidate markers were identified. These markers were quantified by enzyme-linked immunosorbent assay (ELISA) and immunoturbidimetry in these 24 patients. Neutrophil gelatinase–associated lipocalin (NGAL) and insulin-like growth factor–binding protein 7 (IGFBP-7) were chosen for further validation. Following power analysis, NGAL and IGFBP-7 were determined in the independent verification group irrespective of second renal hits of 28 patients, as well as in a control group of ICU patients without AKI. Kidney International 2014 85, 909-919DOI: (10.1038/ki.2013.363) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Differentially excreted urinary proteins in patients with early versus late/non-recovery of acute kidney injury. Representative example of a two-dimensional difference gel electrophoresis (2D-DIGE). Highlighted spots in the 2D map represent significantly regulated and identified protein spots; the numbers identifying the spots correspond to the numbers given in Table 2. Horizontal axis: pH range 3-11 non-linear. Vertical axis: molecular weight range 10–180kDA. Kidney International 2014 85, 909-919DOI: (10.1038/ki.2013.363) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Urinary excretions of normalized insulin-like growth factor–binding protein 7 (IGFBP-7) and neutrophil gelatinase–associated lipocalin (NGAL) in acute kidney injury (AKI) patients with and without early recovery of renal function and in control patients. Data are given on a logarithmic scale. Values are displayed as boxplot summaries. The middle line in the box represents the median, and the whiskers cover 95% of the data, that is, the range from the 2.5 to the 97.5 percentile. There was a significant difference in urinary excretions of both proteins IGFBP-7 and NGAL between all groups (P<0.005 for IGFBP-7 and P<0.05 for NGAL, Kruskal–Wallis, N=64). When the patient with possible misclassification was excluded, IGFBP-7 discriminated better between groups (P<0.001, Kruskal–Wallis, N=63) and remained unchanged for NGAL (P<0.05, N=63). ER, early recovery; LNR, late/non-recovery. Kidney International 2014 85, 909-919DOI: (10.1038/ki.2013.363) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Logistic regression analysis of future renal recovery in acute kidney injury (AKI) patients. (a) Multivariate logistic regression model of clinical data to predict renal outcome. Addition of catecholamines did not improve the final clinical model based on urinary flow and SOFA (sequential organ failure assessment) score. (b) Addition of normalized urine insulin-like growth factor–binding protein 7 (IGFBP-7) levels to the final clinical model improved prediction of future renal recovery from 76.6 to 82.1% concordant values. (c) Addition of normalized urine neutrophil gelatinase–associated lipocalin (NGAL) levels improved the final clinical model to 78.4% concordant values. (d) Addition of normalized urine NGAL levels to the model given by the final clinical model described above and normalized urine IGFBP-7 levels did not significantly improve the model (82.2% vs. 82.1% concordant values). Kidney International 2014 85, 909-919DOI: (10.1038/ki.2013.363) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Urinary excretion of normalized insulin-like growth factor–binding protein 7 (IGFBP-7) in intensive care unit (ICU) patients without malignancy. Data are given on a logarithmic scale. Values are displayed as boxplot summaries. The middle line in the box represents the median, and the whiskers cover 95% of the data, that is, the range from the 2.5 to the 97.5 percentile. There was a significant difference in urinary excretions of normalized IGFBP-7 between all groups (P<0.0005, Kruskal–Wallis, N=48). ER, early recovery; LNR, late/non-recovery. Kidney International 2014 85, 909-919DOI: (10.1038/ki.2013.363) Copyright © 2014 International Society of Nephrology Terms and Conditions