Introduction
Acute kidney injury (AKI), a common problem in intensive care units (ICUs) [
1], is associated with significantly increased mortality, hospital length of stay and medical costs [
2]. Effective treatment for established AKI other than supportive therapy including dialysis remains unknown [
3]. Therefore, it is crucially important to identify patients who are expected to develop AKI and prevent AKI if possible. For this purpose, many AKI biomarkers, including neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18) and L-type fatty acid-binding protein (L-FABP), have been investigated [
4]-[
6]. Recently, tissue inhibitor of metalloproteinase-2 (TIMP-2) was reported as an emerging biomarker for predicting severe AKI in critically ill patients [
7],[
8]. In cells of various different types, including cells in renal tubules and glomeruli, TIMP-2 is expressed constitutively [
9]. Reportedly, TIMP-2 is involved with G
1 cell cycle arrest during the early phases of cell injury [
10]. Renal tubular cells enter a short period of G
1 cell cycle arrest following renal ischemic insult [
11]. Therefore, enhanced TIMP-2 expression can be expected in the pathological condition of AKI.
A clinical evaluation revealed that urinary TIMP-2 was not inferior to any other biomarker, especially in patients with sepsis [
7]. The authors of a multinational prospective observational report described that the most frequent contributing factor to AKI is sepsis, which is observed at a rate of approximately 50% [
12]. Authors of other reports have described that 45% to 70% of all AKIs are associated with sepsis [
13]-[
15]. It is also widely recognized that patients with both sepsis and AKI have an unacceptably high mortality rate [
13]. Reportedly, inflammatory cytokine interleukin-6 (IL-6) was increased in septic AKI patients [
16]-[
18], and plasma NGAL detected septic AKI along with endotoxin activity assay [
19]. Hypoxic insult is assumed to play a crucially important role in AKI, based on findings obtained from basic research [
20]. Erythropoietin (EPO) was shown to have non-hematopoietic tissue-protective effects in animal AKI models [
21]-[
23]. Although a previous clinical trial revealed no protective effect of EPO against AKI [
24], little is known about whether the blood EPO level is useful to detect renal hypoxic injury or to monitor AKI severity.
This study was conducted to evaluate the performance of urinary TIMP-2 in an adult mixed ICU by comparison with other biomarkers used to monitor different pathways: plasma NGAL and IL-6 for inflammation, plasma EPO for hypoxia and urinary
N-acetyl-β-
d-glucosaminidase (NAG) for renal tubular epithelial injury. These comparisons of different biomarkers were expected to reveal the contributing pathophysiological pathway to AKI development and mortality. We also evaluated the influence of sepsis and the prediction of mortality in each biomarker. Although researchers in a multicenter international study previously evaluated the performance of urinary TIMP-2 with a larger population, the present study includes the important strength of comparing urinary TIMP-2 with other biomarkers that are not limited to AKI and can be used to monitor different mechanisms of diseases. Moreover, this additional validation study is the first conducted by a research group independent from the group that originally reported the performance of urinary TIMP-2 [
7],[
8].
Discussion
This study demonstrates that urinary TIMP-2 can detect severe AKI with performance as good as that of plasma NGAL and urinary NAG, with an AUC-ROC value higher than 0.80. We observed no significant impact of sepsis on urinary TIMP-2, although the authors of a previous report presented a better prediction of AKI by the combination of TIMP-2 and insulin-like growth factor-binding protein-7 (IGFBP-7) in septic subjects than in post-surgery subjects [
7]. The enrolled patients treated in a mixed ICU in the present study might have had not only AKI but also several other organ injuries. In addition to AKI detection, urinary TIMP-2 was able to predict mortality better than serum creatinine. These data, obtained with a heterogeneous ICU population in the present study, validate previous reports that demonstrated the clinical significance of measuring urinary TIMP-2 [
7],[
8],[
34] and confirmed its generalizability for clinical translation.
Actually, TIMP-2 has been identified as a potential new AKI biomarker by examination of over 300 markers with a heterogeneous AKI cohort comprising sepsis, shock, major surgery and trauma [
7]. Together with TIMP-2, IGFBP-7 was also found to be the best-performing marker in the discovery study. These two molecules are reportedly involved with cell cycle arrest at G
1 phase [
10],[
35],[
36]. Therefore, the utility of TIMP-2 and IGFBP-7 suggests a crucial role of cell cycle regulation in the pathogenesis of AKI. Recently, in another independent study of urine proteome analysis using gel electrophoresis and mass spectrometry, researchers identified IGFBP-7 as a novel prognostic marker for AKI [
37]. Although urinary IGFBP-7 showed performance similar to NGAL in terms of AKI detection and reflection of AKI severity in an independent verification group of 28 patients with AKI and 12 control patients without AKI, urinary NGAL appeared to predict mortality better than IGFBP-7 did. In the present study, we did not measure urinary IGFBP-7. Further validation studies must be undertaken to confirm the utility of the combination of urinary TIMP-2 and IGFBP-7.
Especially for critically ill patients treated in ICUs for non-surgical conditions, sepsis is the most important factor affecting their prognosis. As also reported in earlier studies [
19],[
38]-[
40], NGAL was able to detect septic AKI with high AUC-ROC values, above 0.90, in the present study. The results of the present study show that IL-6 was increased not by AKI alone, but also by septic AKI, although plasma EPO was increased only by septic AKI. Urinary TIMP-2 and NAG were elevated in AKI, irrespective of sepsis complication. These distinct characteristics of the five examined biomarkers will enable discrimination of the etiologies of AKI. The 10th Acute Dialysis Quality Initiative (ADQI) Consensus Conference recommended that the etiology of AKI should be ascertained by measuring several different biomarkers that help differentiate AKI of uncertain etiology [
41]. Further studies must be conducted to determine the specificity of damage and biomarkers for individual disease states.
It is noteworthy that only urinary TIMP-2 showed better prediction of in-hospital mortality among the evaluated biomarkers compared with serum creatinine (Table
5). This feature of new AKI biomarkers has recently been addressed. One meta-analysis showed that blood and urinary NGAL can detect patients who have increased risk of adverse outcomes including mortality, even in the absence of sufficient serum creatinine increase for AKI diagnosis [
42]. Another report described a better prediction of mortality of ICU patients by urinary NGAL and L-FABP than that by serum creatinine [
40]. These observations suggest that new AKI biomarkers, including TIMP-2, can detect renal structural damage independently from functional changes shown by serum creatinine elevation and that a combination of kidney functional and damage markers enable stratification of patients with AKI at risk for poor outcomes [
43].
Several limitations might affect the results obtained from this study. First, this study was conducted at a single center. The number of patients analyzed was insufficiently large. Evaluations in multicenter ICUs with larger cohorts should be conducted to verify our findings. Second, most AKI cases (79%) were diagnosed as AKI on ICU admission, which might indicate that we were unable to enroll proper patients with an early phase of AKI, where novel biomarkers might have had more value than creatinine. Third, we evaluated AKI and sepsis, but did not evaluate their mutual cause-and-effect relationship. Although the pathophysiological mechanisms of sepsis-induced AKI have been investigated widely [
44], sepsis can be not only a cause but also a consequence of AKI in a clinical setting. In a multicenter observational study of AKI, researchers reported the clinical consequences of sepsis with AKI [
45]. Among the 611 patients with AKI, 174 patients (28%) had sepsis before AKI and 243 patients (40%) developed sepsis after AKI. The relationship of cause and effect between AKI and sepsis can affect biomarker behavior.
Conclusions
A new urine biomarker, TIMP-2, can detect severe AKI with performance as good as that of plasma NGAL and urinary NAG, with an AUC-ROC value higher than 0.80. In addition, urinary TIMP-2 was associated with mortality. Sepsis appeared to have a limited impact on urinary TIMP-2, in contrast to plasma NGAL. These distinct features of biomarkers might enable the evaluation of the contribution of sepsis to AKI development.
Acknowledgements
This study was partly supported by grants from the Tokyo Society of Medical Sciences (to KD). Alere Medical Co Ltd (Tokyo, Japan) partly supported blood sample collection and testing, but it did not contribute to the study design, data analysis or preparation of the manuscript.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
TY and KD conceived of the study, participated in its design and coordination, conducted sample collection, measured biomarkers, analyzed the data and drafted the manuscript. YH, TM and TI participated in study design and coordination, analyzed the data and drafted the manuscript. NY, MN and EN conceived of the study, participated in its design and coordination, analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.