Urinary Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) • Insulin-Like Growth Factor-Binding Protein 7 (IGFBP7) Predicts Adverse Outcome in Pediatric Acute Kidney Injury

Background The G1 cell cycle inhibitors tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) have been identified as promising biomarkers for the prediction of adverse outcomes including renal replacement therapy (RRT) and mortality in critically ill adult patients who develop acute kidney injury (AKI). However, the prognostic value of urinary TIMP-2 and IGFBP7 in neonatal and pediatric AKI for adverse outcome has not been investigated yet. Methods The product of the urinary concentration of TIMP-2 and IGFBP7 ([TIMP-2]•[IGFBP7]) was assessed by a commercially available immunoassay (NephroCheck™) in a prospective cohort study in 133 subjects aged 0–18 years including 46 patients with established AKI according to pRIFLE criteria, 27 patients without AKI (non-AKI group I) and 60 apparently healthy neonates and children (non-AKI group II). AKI etiologies were: dehydration/hypovolemia (n = 7), hemodynamic instability (n = 7), perinatal asphyxia (n = 9), septic shock (n = 7), typical hemolytic-uremic syndrome (HUS; n = 5), interstitial nephritis (n = 5), vasculitis (n = 4), nephrotoxic injury (n = 1) and renal vein thrombosis (n = 1). Results When AKI patients were classified into pRIFLE criteria, 6/46 (13%) patients fulfilled the criteria for the category “Risk”, 13/46 (28%) for “Injury”, 26/46 (57%) for “Failure” and 1/46 (2%) for “Loss”. Patients in the “Failure” stage had a median 3.7-fold higher urinary [TIMP-2]•[IGFBP7] compared to non-AKI subjects (P<0.001). When analyzed for AKI etiology, highest [TIMP-2]•[IGFBP7] values were found in patients with septic shock (P<0.001 vs. non-AKI I+II). Receiver operating characteristic (ROC) curve analyses in the AKI group revealed good performance of [TIMP-2]•[IGFBP7] in predicting 30-day (area under the curve (AUC) 0.79; 95% CI, 0.61–0.97) and 3-month mortality (AUC 0.84; 95% CI, 0.67–0.99) and moderate performance in predicting RRT (AUC 0.67; 95% CI, 0.50–0.84). Conclusions This study shows that urinary [TIMP-2]•[IGFBP7] has a good diagnostic performance in predicting adverse outcomes in neonatal and pediatric AKI of heterogeneous etiology.


Introduction
Acute kidney injury (AKI) is a complex disorder that constitutes an independent risk factor for morbidity and mortality in adult and pediatric patients [1,2]. Approximately 10% of all children admitted to a pediatric intensive care unit (PICU) develop AKI, and this rate exceeds 80% with increasing severity of illness [3,4]. In critically ill children, the reported mortality from AKI is still as high as 60% [5]. The frequency and spectrum of AKI etiologies differ between pediatric and adult patients. Of note, pediatric AKI epidemiological data revealed a shift from primary renal diseases to renal involvement secondary to other systemic diseases, particularly in hospitalized children [6,7]. Furthermore, AKI survivors appear to be at significant shortand long-term risk for complications such as chronic kidney disease (CKD) [8].
Changes in serum creatinine (SCr) and/or urine output form the basis of diagnostic and staging criteria for AKI [9]. However, SCr is a late and unspecific marker of reduced glomerular filtration rate and is insensitive to acute changes in kidney function [10]. Neither the cause and location of the renal disease (e.g. prerenal versus intrinsic; affected renal tubule segment; nephrotoxic versus ischemic AKI) nor the extent of renal damage are adequately reflected by SCr concentrations [9]. Eventually, SCr is influenced by several non-renal factors such as muscle mass, medications, diet and tubular secretion, thus provoking inaccuracies in AKI diagnosis. Hence, current research aims at providing better diagnostic tools for detection, specification and prognosis of AKI. This includes (i) early diagnosis of kidney damage before renal function has severely diminished, (ii) differentiation of functional (prerenal) versus intrinsic AKI, (iii) identification of AKI patients at risk for severe and long-lasting kidney damage and for other adverse outcomes [11,12]. Over the last decade, significant progress has been made in the identification and validation of novel biomarkers for AKI [12]. Several of them also proved valuable for use in pediatric AKI [13]. Recent studies reported on the discovery and validation of two urinary G1 cell cycle arrest biomarkers for early diagnosis of kidney damage: tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) [14][15][16]. Following injury, renal tubular cells enter a period of G1 cell-cycle arrest that is assumed to protect cells from dividing once DNA damage has occurred [17,18]. In consequence, this will lead to either cellular repair including reconstitution of genomic integrity, cell death or cellular senescence [19][20][21][22]. Urinary [TIMP-2]•[IGFBP7] performed better than any other biomarker reported to date for predicting the development of moderate or severe AKI according to Kidney Disease Improving Global Outcomes (KDIGO) criteria in patients at high risk for AKI from multiple causes [23]. Furthermore, the risk for death, dialysis or persistent renal dysfunction in AKI patients increased with higher [TIMP-2]•[IGFBP7] values [16]. Recently, a secondary analysis from the Sapphire study in adults demonstrated that [TIMP-2]•[IGFBP7] determined early in the setting of critical illness identified AKI patients at increased risk for mortality or requirement for renal replacement therapy (RRT) within 9 months following AKI [24].
The performance of urinary [TIMP-2]•[IGFBP7] in neonatal and pediatric AKI for the prediction of adverse outcomes has never been tested. This prompted us to investigate the value of urinary [TIMP-2]•[IGFBP7] in an etiologically heterogenous cohort of patients with established AKI according to pRIFLE criteria for the prediction of adverse clinical outcomes.

Ethics Statement
The study was approved by the local ethics committee of the Medical Faculty of Heidelberg. Written informed consent was obtained by legal guardian of each patient with assent from the patient when appropriate. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.

Study Design and Participants
We performed a prospective cohort study at the University Children's Hospital Heidelberg. Patients aged 0 to 18 years referred to our clinic with established AKI and patients who developed AKI during hospitalization were consecutively enrolled in the study from October 2011 to September 2014. AKI was classified according to the pediatric-modified Risk, Injury, Failure, Loss, and Endstage renal disease (pRIFLE) AKI criteria [3]. Exclusion criteria were prematurity, hereditary nephropathies and postrenal AKI as examined by initial renal ultrasound. Etiological assignments of AKI were established by three independent physicians. Noteworthy, two AKI patients of our study cohort suffered from CKD stage 2 and presented with prerenal AKI ("acute on chronic") due to hypovolemia. One patient was kidney-transplanted due to congenital renal hypoplasia and dysplasia, suffered from chronic allograft nephropathy and also presented with prerenal AKI due to hypovolemia. One patient was excluded from our study cohort, as he was stem cell transplanted and received a graft versus host disease (GVHD) prophylaxis that included cyclosporine which has been shown previously to increase the urinary TIMP-2/creatinine ratio [25].
Control subjects without AKI were taken from two different cohorts: the first group (non-AKI group I, total: n = 27) consisted of non-AKI patients either admitted to our neonatal or pediatric intensive-care unit (NICU/PICU) (n = 17) or who presented for out-patient treatment with minor medical conditions such as constipation (n = 10). Diagnoses of NICU/PICU non-AKI patients were: postoperative care (n = 15), seizures (n = 1), pneumonia (n = 1). In order to identify a potential impact of non-renal illnesses on urinary [TIMP-2]•[IGFBP7] in non-AKI group I, we investigated another control group consisting of apparently healthy children aged 0-18 years (non-AKI group II, n = 60). Exclusion criteria were: (a) Any known or suspected acute illness at the time of enrollment; (b) any known or suspected chronic disease; (c) any surgery within the last 6 months; (d) any blood product transfusion within the previous 2 months.

Sample and data collection
Urine samples were obtained immediately after admission to our hospital or-in case of anuria -after restoration of diuresis. Urine samples were centrifuged and the supernatant was frozen, stored in aliquots at −80°C and thawed prior to analysis. SCr, C-reactive protein (CrP) and urinalysis were measured as part of the routine clinical chemistry testing. Measurement of SCr was performed using an IDMS-traceable enzymatic method. The estimated creatinine clearance (eCCl) for children was calculated by using the revised Schwartz formula: (k = 0.413 × height/serum creatinine) [26]. For determination of baseline eCCl, the lowest value of SCr in the 3 months prior to study enrollment was obtained from the hospital database. In case of unknown baseline serum creatinine and absence of previous kidney injuries in the case history, the patient was assumed to have normal renal function and assigned a baseline eCCl of 120 ml/min per 1.73 m 2 [27]. In addition, for term neonates who developed AKI within the first days of life, i.e. before SCr reached baseline levels, an eCCl of 40 ml/min per 1.73 m 2 was assigned [28][29][30]. eCCl was used for pRIFLE CCl classification with "Risk" defined as eCCl decrease of 25% from baseline, "Injury" defined as eCCl decrease of 50%, "Failure" defined as eCCl decrease of 75% or absolute value <35 ml/min per 1.73 m2 and "Loss" as persistent renal failure > 4 weeks. Of note, neonates were diagnosed as "Failure" only if eCCl decreased by at least 75% and not based on an absolute eCCl value <35 ml/min per 1.73 m 2 . In addition, pRI-FLE uo stage was determined by urinary output criteria [3]. For this, urine output was systematically and continuously evaluated every 2 hours during the initial period by age-appropriate techniques. As newborns can be oliguric during the first 24 hours of life, a reduction in urinary output was not considered a criterion in this time period. The maximum score was taken for pRIFLE stage assignment.

Laboratory analysis
The respective concentrations of TIMP-2 and IGFBP7 were measured in spot urine samples using the NephroCheck TM Test (Astute Medical, San Diego, CA, USA). Urine TIMP-2 and IGFBP7 are simultaneously measured with this point-of-care test. [TIMP-2]•[IGFBP7] indicates the product (by multiplication) of the respective urinary concentration of both biomarkers that is automatically calculated by the ASTUTE140 1 Meter. The product is divided by 1,000 to report a single numerical test result with a unit of (ng/ml) 2 /1000, the unit for all [TIMP-2]•[IGFBP7] values in this report.

Statistical analysis
Distribution of numerical data was analyzed by the Kolmogorov-Smirnov Test. Not normally distributed data are presented as median and interquartile range. Comparison of not normally distributed numerical data was performed by the Mann-Whitney U-test or the Kruskal-Wallis-test with post-hoc Dunn's Test, respectively (age, SCr on study enrollment, eCCl on study enrollment, proteinuria, urine protein-to-creatinine ratio, CrP, length of ICU stay, length of hospital stay, [ value maximizing the Youden index was used to derive sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), assuming sensitivity and specificity were equally important. The composite outcome of death or dialysis within 3 months was assessed using Kaplan-Meier estimates and log-rank test for comparison between the groups. Descriptive statistics, statistical tests, ROC curve and Kaplan-Meier analyses were performed using IBM 1 SPSS 1 Statistics Version 22.

Subject characteristics
One-hundred thirty-three subjects were enrolled in the study including 46 AKI patients, 27 patients without AKI (non-AKI group I) and 60 apparently healthy control children (non-AKI group II). The epidemiological data, etiologies of AKI, pRIFLE stages and renal laboratory parameters are presented in Table 1. There was no statistically significant difference regarding age, gender distribution and proportion of neonates between the three groups. The distribution of AKI causes reflects the typical spectrum of a PICU in a tertiary care University Children's Hospital ( Table 1). The median duration from the onset of AKI to study enrollment was 2.0 (1.0 to 5.0) days. SCr on study enrollment was significantly higher in the AKI group compared to the non-AKI group I and eCCl on study enrollment was markedly decreased in the AKI group compared to the non-AKI group I (Table 1). In addition, the AKI group displayed significantly higher values for CrP, urinary protein concentration and urinary protein-to-creatinine ratio compared to the non-AKI group I (P < 0.001). The length of the ICU stay was 4-fold and the length of hospitalitation 1.7-fold higher in the AKI group compared to the non-AKI group I ( Table 1). Sixteen of 46 AKI patients (34.8%) received RRT, 14 (87.5%) hemodialysis and 2 (12.5%) peritoneal dialysis. Seven patients (15.2%) deceased within 3 months following study enrollment, six of them (13.0%) within the first 30 days. Causes of death were septic shock with coexisting AKI (n = 5), cerebral incarceration in perinatal asphyxia (n = 1) and low-cardiac output (n = 1). On day of demise two of the patients received RRT, and all suffered from AKI according to pRIFLE criteria. At the time of study enrollment, 6 patients (13.0%) fullfilled the "Risk", 13 patients (28.3%) the "Injury", 26 (56.5%) the "Failure" and 1 patient (2.2%) the "Loss" criteria. No patient developed end-stage renal disease. During the clinical course, 4 patients progressed to the next higher pRIFLE stage.

Urinary [TIMP-2]•[IGFBP7] stratified for age in subjects with or without AKI
Median urinary [TIMP-2]•[IGFBP7] was numerically, but not significantly (P = 0.41) higher in the non-AKI group II compared to the non-AKI group I (Table 1) and significantly increased in the AKI group (P<0.001 vs. non-AKI group I; P = 0.001 vs. non-AKI-group II). As shown in Fig 1 and S5   As shown in Fig 1 and S5
To reduce the risk of a sampling bias in the neonatal population by application of the pRI-FLE classification that was originally developed in children above the age of one month [3], this age-group was additionally stratified for the neonatal modified KDIGO AKI definition [31]. As shown in S9 Table,

Discussion
This is the first study investigating urinary [TIMP-2]•[IGFBP7] in neonatal and pediatric primary and secondary AKI of heterogeneous etiology other than the likewise homogeneous setting of cardiac surgery with cardiopulmonary bypass [32]. Our findings show that once-only measurement of [TIMP-2]•[IGFBP7] in patients with AKI has a good prognostic performance in predicting adverse outcomes such as mortality and a moderate performance in predicting the requirement for RRT.
While in recent years a major focus has been the identification of novel biomarkers for prediction of incipient AKI, less attention was paid to the search for informative biomarkers predictive of AKI outcome, including renal recovery, need for RRT and patient death [33]. In a proteomic approach in a small cohort of critically ill adult patients, IGFBP7 and neutrophil gelatinase-associated lipocalin (NGAL) were identified as best predictors of renal recovery; subsequent validation confirmed the prognostic value of IGFBP7 and NGAL in predicting mortality (IGFBP7: AUC 0.68; NGAL: AUC 0.81), recovery (IGFBP7: AUC 0.74; NGAL: AUC 0.70) and severity of AKI (IGFBP7: AUC 0.77; NGAL: AUC 0.69) [34]. Kashani et al. demonstrated that the risk for major adverse kidney events including death, RRT or persistent renal dysfunction within 30 days increased considerably for [TIMP-2]•[IGFBP7] above 0.3 and doubled when values were > 2.0 [16]. Interestingly, a recently published secondary analysis of the Sapphire study revealed that a single measurement of [TIMP-2]•[IGFBP7] early in the setting of critical illness is able to identify adult patients with AKI at increased risk for mortality or receipt of RRT over the subsequent 9 months [24]. Our data demonstrate that an once-only urinary [TIMP-2]•[IGFBP7] measurement is a good predictor for 30 day-(AUC 0.79) and 3 month-mortality (AUC 0.84) and a fair predictor for RRT (AUC 0.67) in a heterogeneous neonatal and pediatric cohort of established primary and secondary AKI according to pRIFLE criteria. The lower performance of urinary [TIMP-2]•[IGFBP7] for the prediction of RRT may be due to the fact that the indication for RRT is physician-dependent and not coherently defined. In addition, by Kaplan-Meier and cox proportional hazard model analyses for the composite end point "death or dialysis" within 3   Age-related reference values for neonates and children are indispensable to interprete presumably pathological results, yet have not been evaluated for urinary [TIMP-2]•[IGFBP7]. We therefore analyzed [TIMP-2]•[IGFBP7] in two different control groups, namely one group consisting of patients without AKI and another group consisting of apparently healthy neonates and children. Reference values were largely stable among the different age groups and comparable to adults [16], but tended to be lower in neonates and younger children. There was no statistically significant difference between both control groups, arguing against an effect of extrarenal disease or other hospital-associated conditions on [TIMP-2]•[IGFBP7] values in the non-AKI patient group (non-AKI group I). Hence, in addition to adults [16], our data from neonates and children emphasizes that urinary [TIMP-2]•[IGFBP7] appears to be specific for AKI, in contrast to several other biomarkers [16]. For some age groups (0-28 days, 2-5 years, 6-11 years) apparently healthy neonates and children showed moderately higher urinary [TIMP-2]•[IGFBP7] values compared to the non-AKI patients. As urine samples were in most cases obtained a few hours before centrifugation and transported to our clinic by the parents, pre-analytical sample handling might be a source of error. values were significantly associated with the "Failure" stage (P<0.001). Lower patient numbers in the "Risk" and "Injury" group and a higher percentage of short-term renal injuries (e.g. hypovolemia/dehydration and hemodynamic instability) are potential explanations for the only moderate increase of [TIMP-2]•[IGFBP7] for the "Risk" and "Injury" stage. When analyzed for the different AKI etiologies, the strongest increase in [TIMP-2]•[IGFBP7] was found in septic shock. All septic shock patients with AKI fulfilled "Failure" criteria according to the pRIFLE classification. Both intrarenal hemodynamic changes as well as nonhemodynamic mediators of injury (e.g. inflammatory cytokines, oxidative stress, arachidonic acid metabolites, vasoactive substances) contribute to renal damage in sepsis and septic shock [35]. Thus, the multitude of cascades involved in tubular damage might explain markedly elevated [TIMP-2]•[IGFBP7] values when compared to other AKI causes. That septic AKI is rather a protracted than a short-term process could be another explanation for high [ There are several limitations of our study. First, this is a single-center study in a relatively small patient cohort, which requires validation in a larger patient population. Second, our study cohort includes both neonates and children. Due to varying degrees of creatinine reabsorption in the proximal tubules, overall lower but rapidly increasing GFRs, inter-individual maturational differences and interference with maternal creatinine values within the first days of life, a SCr/eCCl-based definition of AKI in neonates has to be regarded with caution. This is especially the case when using the pRIFLE classification and eCCl estimates for neonates with missing baseline SCr values. Eventually, an estimated baseline eCCl of either 40 ml/min per 1.73 m 2 for neonates or 120 ml/min per 1.73 m 2 for children aged > 1 month might lead to misclassification. In addition, the pRIFLE classification was originally developed in children > 1 month of age [3]. Therefore, we performed subanalyses for the group of neonates and the group of children, which independently confirmed the main findings of our study. Of note, the accuracy of [TIMP-2]•[IGFBP7] in predicting 30-day and 3-month mortality further increased after exclusion of the neonates. To minimize the risk of a sampling bias in the neonatal group owing to AKI assignment based on the pRIFLE criteria, this group was additionally classified using the increasingly accepted neonatal modified KDIGO AKI definition [31]. In spite of a reduced number of newborns classified as AKI, the prognostic accuracy for the prediction of adverse outcomes remained largely unchanged (S9 Table). Considering the characteristics of neonatal renal function and the difficulties regarding clinical diagnosis and prognosis using current laboratory values (i.e. SCr), the search for new biomarkers of kidney injury is of particular interest for this age group. A third limitation of our study is the heterogeneity of the patient population, which comprised different subtypes of AKI. Fourthly, as several patients were transferred to our clinic with already established AKI, the time interval between onset of AKI and study enrollment was variable. As the respective half-life of these biomarkers is short, we might have missed earlier increases in [TIMP-2]•[IGFBP7]. Supporting Information S1