The Outcome of Neutrophil Gelatinase-Associated Lipocalin (NGAL)-positive Subclinical Acute Kidney Injury: A Multicenter Pooled Analysis of Prospective Studies

Abstract

Objectives

To test the hypothesis that, without diagnostic changes in serum creatinine, increased NGAL levels identify patients with subclinical acute kidney injury (AKI) and, therefore, worse prognosis.

Background

Neutrophil gelatinase-associated lipocalin (NGAL) detects subclinical AKI hours to days before increases in serum creatinine indicate manifest loss of renal function.

Methods

We analyzed pooled data from 2,322 patients with cardiorenal syndrome type 1 from ten prospective observational studies of NGAL. We used the terms NGAL(−) or NGAL(+) according to study-specific NGAL cut-off for optimal AKI prediction and the terms sCREA(−) or sCREA(+) to consensus diagnostic increases in serum creatinine defining AKI. A-priori-defined outcomes included need for renal replacement therapy (primary endpoint), hospital mortality, their combination and duration of stay in intensive care and in-hospital.

Results

Of study patients, 1,296 (55.8%) were NGAL(−)/sCREA(−), 445 (19.2%) NGAL(+)/sCREA(−), 107 (4.6%) NGAL(−)/sCREA(+) and 474 (20.4%) NGAL(+)/sCREA(+). According to the four study groups, there was a stepwise increase in subsequent renal replacement therapy initiation, (NGAL(−)/sCREA(−): 0.0015% vs. NGAL(+)/sCREA(−): 2.5% [odds ratio 16.4, 95% CI 3.6–76.9, P<0.001], NGAL(−)/sCREA(+): 7.5% and NGAL(−)/sCREA(−): 8.0%, respectively), hospital mortality (4.8%, 12.4%, 8.4%, 14.7%, respectively) and their combination (four-group comparisons: all P<0.001). There was a similar and consistent progressive increase in median number of intensive care and in-hospital days with increasing biomarker positivity: NGAL(−)/sCREA(−): 4.2 and 8.8 days; NGAL(+)/sCREA(−): 7.1 and 17.0 days; NGAL(−)/sCREA(+): 6.5 and 17.8 days; NGAL(+)/sCREA(+): 9.0 and 21.9 days; four-group comparisons: P=0.003 and P=0.040, respectively. Urine and plasma NGAL indicated a similar outcome pattern.

Conclusions

In the absence of diagnostic increases in serum creatinine, NGAL detects patients with subclinical AKI who have an increased risk of adverse outcomes. The concept and definition of AKI may need re-assessment.

INTRODUCTION

The current concept and diagnosis of acute kidney injury (AKI) are mainly based on diagnostic increases in serum creatinine indicating loss of excretory renal function. AKI is then classified according to either the RIFLE (1) or AKI Network consensus criteria (2). Although such diagnosis of AKI is of prognostic relevance (3,4), it is delayed by 24–72 hours compared to diagnosis by means of novel renal biomarkers of tubular injury like neutrophil gelatinase-associated lipocalin (NGAL) (5–7).

NGAL fulfils many characteristics for an ideal biomarker for AKI. It was discovered using unbiased transcriptomic approaches (8,9); it is rapidly induced and released from the injured distal nephron in experimental models and human disease (5,9,10); its urine and plasma concentrations increase proportionally to severity and duration of renal injury (9,11,12); its concentration rapidly decreases with attenuation of renal injury (13) and it is readily and easily measured in plasma (11) and urine (12). Finally, NGAL appears to play a key role in early AKI and local iron transport (10,14,15) providing biologic plausibility for its use as AKI biomarker.

Several studies have found NGAL useful compared to early measurements of serum creatinine in AKI (6,7,16,17). In response to renal injury, increases in NGAL levels predict AKI 24–72 hours before diagnostic creatinine increases (5–7,11,12) and are of prognostic value (18). Despite the above characteristics, NGAL’s ability to predict the development of AKI appears imperfect (19,20). However, the predictive value of NGAL improves with increasing RIFLE class of AKI (21), suggesting that limitations in its accuracy reflect the use of an imperfect test (serum creatinine) as study endpoint. Serum creatinine requires several hours to days to accumulate, it increases in serum only after 50% or more of renal function is lost and its concentration is affected by multiple confounding factors (22). Accordingly, we hypothesized that, without diagnostic increases in serum creatinine, NGAL(+) patients might have likely subclinical AKI and, therefore, carry a worse prognosis as indicated by the need for renal replacement therapy (RRT) and other patient-centered outcomes than NGAL(−) patients. We tested this hypothesis by analyzing pooled data from multiple centers and determined the clinical outcomes of subjects classified according to their NGAL and serum creatinine concentrations.

METHODS

We performed a summary patient-level analysis using summarized data from each study (see data collection form in the Appendix). For this purpose, we used data from a previously described multicenter data pool created to study the predictive value of NGAL for AKI defined by increases in serum creatinine (18). This pool was extended by newly identified clinical studies on NGAL as biomarker of AKI. Individual patient data were not available. Figure 1 displays the results of the search and data acquisition strategies.

Figure 1.

Flow of study selection.

NGAL, neutrophil gelatinase-associated lipocalin; RRT, renal replacement therapy.

Data pool creation

Two investigators (A.H.F. and P.R.M.) independently searched PUBMED, EMBASE, CENTRAL and abstracts submitted to the Congress of the American Society of Nephrology. They identified relevant articles or abstracts and independently screened studies for inclusion (Figure 1). Selection was restricted to published prospective cohort studies in humans investigating the diagnostic and prognostic ability of NGAL for AKI, need for renal replacement therapy and in-hospital mortality. Each independent biomarker study was approved by a local Institutional Review Board and all participants gave written informed consent. All studies originally were designed to examine the diagnostic accuracy of NGAL level, prospectively enrolled consecutive patients, had clearly defined enrolment and exclusion criteria and laboratory/research personnel and clinicians were blinded. All studies enrolled representative patient cohorts who might receive the test in clinical practice. Included studies had similar sample collection and processing and provided full datasets.

NGAL measurement and AKI definition

For the purpose of this study, we included urine and plasma NGAL values and reported those in a combined fashion and separately. We asked each author to use the measurement performed at least 24–48 hours before the diagnosis of AKI when NGAL was measured more than once or when no serum creatinine increase occurred to use NGAL measured at intensive care unit admission or 2–6 hours after any new renal insult. We defined AKI according to the RIFLE classification (1) as an increase in serum creatinine ≥50% from baseline to peak value within seven days of admission to the intensive care unit basing on daily serum creatinine measurement (RIFLE class R or worse). We choose RIFLE over the AKI Network classification (2) because of its greater sensitivity (23). Baseline serum creatinine was defined as the concentration obtained at outpatient departments or at hospital admission in cardiac surgery patients or at intensive care unit admission in critically ill patients when previous values were not available. Chronic kidney disease was defined as an estimated glomerular filtration rate <60 mL/min/1.73m² calculated using the simplified Modification of Diet in Renal Disease (MDRD) study formula (24) in adults or the simplified Schwartz formula in children (25). Renal replacement therapy (RRT) was initiated based on center- and physician-specific practice and no patient received RRT for non-renal indications.

We applied the term “NGAL(−)” or “NGAL(+)” to indicate the absence or presence of tubular injury, as defined by each study-specific NGAL cut-off value for the optimal combination of sensitivity and specificity for AKI prediction (18). The early ‘research-based’ urine or plasma NGAL ELISA assays were internally valid but their cut-off values were not transferable to other ELISA assays or immunoblots used. We used the term serum creatinine “sCREA(−)” or “sCREA(+)” to indicate the absence or presence of manifest AKI as defined by RIFLE criteria.

Patients were classified as follows:

1. NGAL(−) / sCREA(−)

2. NGAL(+) / sCREA(−)

3. NGAL(−) / sCREA(+)

4. NGAL(+) / sCREA(+)

Patient outcomes

For the present study, we invited each investigator to analyze data from their individual patient cohort and return a specifically designed data collection form (see Appendix) recording demographic data, information on co-morbidities, and a-priori defined patient outcomes including renal replacement therapy initiation (primary endpoint), in-hospital mortality, the combination of both and the length of intensive care unit and hospital stay and according to patients’ NGAL and serum creatinine states as defined above.

Statistical Analysis

All analyses followed a preset statistical analysis plan, which included the above a-priori defined hypothesis. Data were tested on normality using histograms. When data were normally distributed, analysis-of-variance was used to compare numerical data of patients according to the defined groups. Otherwise, non-parametric testing was used (Kruskal-Wallis test for four-group comparison, Mann-Whitney test for two-group comparison). Fisher’s exact test or the chi-square test was applied for comparison of categorical values as appropriate. Analysis was repeated after weighing of the study endpoints according to the relative sample size of each study. We used SPSS Version 16.0 (SPSS Inc, Chicago, IL). A two-sided P-value of <0.05 was considered to be statistically significant.

RESULTS

Ten (6,7,11,12,19,20,26–29) out of 22 authors contacted returned complete data sets. The reasons for exclusion are reported in Figure 1.

We obtained data on 2,322 patients from America, Europe and Australia (Table 1) with the majority having cardiorenal syndrome type 1 (30). The incidence of AKI ranged from 15% to 49%. Patients’ characteristics and baseline peak NGAL levels are shown in Table 2. The majority of patients were NGAL(−)/sCREA(−), while 25% of patients developed AKI based on the RIFLE definition, 3% required renal replacement therapy and 8% died in-hospital. In patients with diabetes, NGAL(+)/sCREA(−) status was less common than NGAL(−)/sCREA(+). Chronic kidney disease was similarly common among NGAL(−)/sCREA(−) (11.0%) and NGAL(+)/sCREA(−) (10.6%) patients.

Table 1.

Characteristics of studies.

Study	Population Type	AKI Etiology	AKI Incidence	No. of patients*		NGAL Measurement,† hrs
				Urine NGAL	Plasma NGAL
Dent et al. (11)	Children	Cardiac surgery	36%	-	125	48
Bennett et al. (12)	Children	Cardiac surgery	49%	194	-	48
Krawczeski et al. (29)	Children	Cardiac surgery	35%	189	358	48
Zappitelli et al. (27)	Adults	Critically ill	30%	33	-	36
Cruz et al. (6)	Adults	Critically ill	28%	-	279	24
Haase-Fielitz et al. (7)	Adults	Cardiac surgery	23%	-	100	48
Koyner et al. (26)	Adults	Cardiac surgery	16%	52	51	36
Wagener et al. (19)	Adults	Cardiac surgery	15%	383	-	36
Martensson et al. (28)	Adults	Critically ill	34%	64	64	48
Siew et al. (20)	Adults	Critically ill	19%	430	-	48

^*With NGAL measured at least 24 to 48 hours before the diagnosis of AKI or at ICU admission several hours after new renal insult and excluding patients with urinary tract infection.

^†Preceding the diagnosis of AKI based on RIFLE class R or worse (1).

AKI, acute kidney injury. NGAL, Neutrophil Gelatinase-associated Lipocalin.

Table 2.

Baseline characteristics of patients (N=2,322).

	NGAL(−) / sCREA(−)	NGAL(+) / sCREA(−)	NGAL(−) / sCREA(+)	NGAL(+) / sCREA(+)	P Value
Number of patients, n	1,296 (55.8%)	445 (19.2%)	107 (4.6%)	474 (20.4%)	-
Age, y	47.2 (4.5–61.6)	53.5 (3.6–60.7)	59.0 (4.4–67.3)	50.4 (3.6–66.8)	0.89
Female, n	475 (36.7%)	181 (40.7%)	42 (39.3%)	210 (44.3%)	0.028
Chronic kidney disease*, n	143 (11.0%)	47 (10.6%)	16 (15.0%)	81 (17.1%)	0.030
Diabetes mellitus, n	113 (8.7%)	78 (17.5%)	28 (26.2%)	29 (6.1%)	<0.001
Congestive heart failure, n	67 (5.2%)	51 (11.5%)	10 (9.4%)	14 (3.0%)	<0.001
Peak NGAL, ng/mL	59 (20–97)	213 (117–1,124)	69 (21–118)	354 (208–1,888)	<0.001

Linear values denote median (25^th to 75^th percentiles).

^*As defined by estimated glomerular filtration rate <60 mL/min/1.73m² using the MDRD study formula (24,25).

NGAL, Neutrophil Gelatinase-associated Lipocalin. sCREA, serum creatinine. MDRD, modification of diet in renal disease.

Figure 2A shows the proportion of patients in each study according to NGAL/sCREA status with about 20% of all patients being NGAL(+)/sCREA(−). Overall, 43% of patients diagnosed with AKI by means of NGAL would have been missed using creatinine criteria alone (Figure 2B).

Figure 2.

Figure 2A Proportion of patients according to biomarker states.

NGAL, neutrophil gelatinase-associated lipocalin; sCREA, serum creatinine; u, urine; p, plasma.

Figure 2B. Proportion of NGAL(+) / sCREA(−) patients in relation to the proportion of patients diagnosed to have AKI by conventional creatinine-based criteria (RIFLE classification [1]) and NGAL positivity.

Neutrophil Gelatinase-associated Lipocalin, NGAL; u, urine; p, plasma.

The formula used was:

$$\frac{(\text{NGAL}+/\text{sCREA}-)=\text{renal}\text{impairment}\text{identified}\text{by}\text{NGAL}}{(\text{NGAL}+/\text{sCREA}-)+(\text{NGAL}-/\text{sCREA}+)+(\text{NGAL}+/\text{sCREA}+)=\text{all}\text{renal}\text{impairment}}$$

Patient outcomes

Treatment with RRT increased from 0.0015% in biomarker-negative patients to 2.5% in NGAL(+)/sCREA(−) patients, 7.5% in NGAL(−)/sCREA(+) patients and 8.0% in NGAL(+)/sCREA(+) patients (four-group comparison: P<0.001) (Figure 3). Specifically, more patients with NGAL+/sCREA- status needed RRT initiation than patients with NGAL-/sCREA- status; OR 16.4 [95% CI 3.6–76.9], P<0.001. Additional endpoints comparing these patient groups are shown in Table 3.

Figure 3.

Incidence of renal replacement therapy (RRT) initiation, in-hospital morality and a combination of both according to Neutrophil Gelatinase-associated Lipocalin (NGAL) and serum creatinine. There was a stepwise increase in all outcomes.

Table 3.

The value of NGAL complements prognosis in patients without diagnostic creatinine increase.

Outcome	NGAL-/sCREA- vs. NGAL+/sCREA-
Need for RRT initiation, n	OR 16.4 (95% CI 3.6–76.9), P<0.001
In-hospital mortality, n	OR 2.8 (95% CI 1.9–4.1), P<0.001
Need for RRT initiation / In-hospital mortality, n	OR 3.6 (95% CI 2.5–5.2), P<0.001
ICU stay*	2.9 days; P=0.026
Hospital stay*	8.2 days; P=0.16

^*Median difference.

Hospital mortality doubled from biomarker negative patients to NGAL(+)/sCREA(−) status and more than tripled in patients positive for both biomarkers (four-group comparison: P<0.001) (Figure 3). More NGAL(+)/sCREA(−) patients (69/445; 15.5%) developed the composite endpoint of RRT initiation or in-hospital mortality when compared to biomarker negative patients (63/1,296; 4.9%), P<0.001. A greater proportion of NGAL(+)/sCREA(+) patients (84/474; 17.7%) reached the combined endpoint of RRT initiation and in-hospital mortality compared to patients negative for both biomarkers (odds ratio: 4.2 [95% CI 3.0–6.0]; P<0.001).

NGAL(+)/sCREA(−) patients had a >70% longer stay in the intensive care unit compared to patients negative for both biomarkers; (P=0.026) (Figure 4A). There was a gradual increase in median length of stay in the Intensive Care Unit with the shortest duration (days) for NGAL(−)/sCREA(−) patients (4.2 [2.2–6.4]), low intermediate duration for NGAL(−)/sCREA(+) patients (6.5 [3.0–11.7]), high intermediate duration for NGAL(+)/sCREA(−) patients (7.1 [5.4–10.3]) and longest duration for NGAL(+)/sCREA(+) patients (9.0 [8.0–14.0]); P=0.003 (four-group comparison).

Figure 4.

Length of stay (LOS) stratified by Neutrophil Gelatinase-associated Lipocalin (NGAL) and serum creatinine in the Intensive Care Unit (ICU) (A) and in-hospital (B). There was a stepwise increase in median LOS in ICU and in-hospital.

NGAL(+)/sCREA(−) patients spent twice as long in hospital compared to patients negative for both renal biomarkers; (P=0.16) (Figure 4B). The shortest hospital stay was for NGAL(−)/sCREA(−) patients (8.8 days [7.7–19.0]) with low intermediate values for NGAL(+)/sCREA(−) patients (17.0 [8.4–24.2]), high intermediate values for NGAL(−)/sCREA(+) patients (17.8 [5.1–26.4]) and longest stay for NGAL(+)/sCREA(+) patients (21.9 [15.8–29.9]); P=0.040 (four-group comparison).

Table 4 presents outcomes according to urine versus plasma NGAL confirming a similar outcome pattern independent of the biological material used to measure NGAL.

Table 4.

Outcome according to urine NGAL versus plasma NGAL.

	NGAL(−) / sCREA(−)	NGAL(+) / sCREA(−)	NGAL(−) / sCREA(+)	NGAL(+) / sCREA(+)
Urine NGAL (N=1,345)	N=758 (56.4%)	N=307 (22.8%)	N=63 (4.7%)	N=217 (16.1%)
Need for RRT initiation, n	2 (0.003%)	9 (2.9%)	2 (3.2%)	16 (7.4%)
In-hospital mortality, n	35 (4.6%)	37 (12.1%)	3 (4.8%)	30 (13.8%)
ICU stay, days	4.2 (2.1–8.5)	7.6 (5.7–14.5)	6.5 (3.0–11.6)	10.4 (7.7–14.8)
Hospital stay, days	8.8 (7.2–18.8)	17.0 (8.2–24.6)	18.3 (5.2–36.0)	21.8 (11.3–24.1)
Plasma NGAL (N=977)	N=538 (55.1%)	N=138 (14.1%)	N=44 (4.5%)	N=257 (26.3%)
Need for RRT initiation, n	0 (0%)	2 (1.5%)	6 (13.6%)	22 (8.6%)
In-hospital mortality, n	27 (5.0%)	18 (13.0%)	6 (13.6%)	40 (15.6%)
ICU stay, days	4.0 (2.2–5.4)	6.5 (4.3–9.0)	5.1 (3.0–12.0)	8.6 (8.0–11.9)
Hospital stay, days	9.7 (7.3–20.3)	14.6 (8.1–22.7)	11.1 (4.5–21.1)	25.5 (18.0–33.9)

Linear values denote median (25^th to 75^th percentiles).

NGAL, Neutrophil Gelatinase-associated Lipocalin. RRT, renal replacement therapy; ICU, Intensive Care Unit.

DISCUSSION

Key study findings

We conducted a multicenter analysis of pooled data to explore the prognostic value of AKI detected by NGAL. We hypothesized that, without diagnostic increase in serum creatinine, NGAL(+) patients might have likely subclinical AKI and carry a worse prognosis than NGAL(−) patients. We found that a positive NGAL finding carried a similar risk of adverse outcome than a positive creatinine finding. We also found that NGAL(+)/sCREA(−) tests identified approximately 40% more AKI cases than sCREA(+) alone and that these patients were at greater risk of longer ICU and hospital stay, RRT and death compared to controls. As expected, NGAL(+)/sCREA(+) patients had the greatest risk of adverse outcomes. A smaller group of patients were NGAL(−)/sCREA(+), implying loss of renal function without evidence of acute tubular injury. These patients’ outcome was intermediate in severity. Finally, NGAL in the urine or plasma showed a similar pattern for the outcomes assessed.

Relation to previous studies

AKI may affect 20% to 30% of hospitalized patients; it carries significant costs and is independently associated with increased morbidity and mortality (31,32). Our study is consistent with other reports on the prognostic importance of serum creatinine increase (31,32). Similarly, NGAL has been repeatedly shown to predict the need for RRT and mortality in AKI (7,12,16). However, previous studies have also shown that NGAL failed to reliably predict changes in serum creatinine, even though its predictive value improved with increasing AKI severity (21).

Until now, the above findings have been interpreted as reflecting NGAL’s shortcomings as a biomarker (20). An alternative explanation, however, is that the limitation lies with serum creatinine as the diagnostic standard and the different nature of the signal provided. Thus, NGAL indicates tubular injury which precedes renal functional loss by several days and serum creatinine indicates subsequent loss of renal excretory function. Our findings suggest that a state of AKI likely exists when NGAL is increased, independent of serum creatinine increases.

Previous work supports the biological plausibility of the above notion and suggests that serum creatinine is a delayed, low-sensitivity and misleading biomarker of AKI (5,7), affected by many confounding factors (22). In this regard, several studies (33,34,35) suggest an analogy between the troponin/creatine kinase and the NGAL/creatinine relationship with a novel more sensitive biomarker identifying previously undetected organ injury. This increased diagnostic sensitivity of troponin is clinically relevant (33) and, in the field of cardiology, has altered the definition, diagnosis, and management of acute myocardial infarction. This concept may similarly apply to NGAL.

Significance of study findings

Potential explanations of study findings are given in Table 5. Our study suggests that acute tubular damage may occur without detectable loss of excretory function (and vice versa) and may predict worse clinical outcome and that NGAL and serum creatinine reflect distinct pathophysiological events. A changed view of AKI may change clinical practice and its treatment. Detection of elevated NGAL may enable more rapid conventional interventions or introduction of novel therapies to prevent or effectively treat such otherwise undetected cardiorenal syndrome type 1 (30). Novel renal biomarkers might facilitate standardization of early diagnosis and treatment. On the other hand, a normal NGAL result may inform clinical decision-making leading to improved use of hospital resources.

Table 5.

Possible combinations of NGAL and serum creatinine status.

Diagnostic increase of NGAL (subclinical AKI, days before diagnostic serum creatinine increase)	Diagnostic increase of serum creatinine (manifest AKI)	Patient outcomes*	Potential explanations of study findings
Positive	Positive	Worse	Subclinical AKI, manifest AKI
Positive	Negative	Worse	Subclinical AKI
Negative	Positive	Worse	False negative NGAL / Glomerular impairment without tubular injury (prerenal azotemia) / NGAL negative because measurement was days before serum creatinine increase
Negative	Negative	Better	No subclinical AKI, no manifest AKI

NGAL, Neutrophil Gelatinase-associated Lipocalin; AKI, acute kidney injury as defined by the RIFLE criteria (1) or AKI Network classification (2).

^*As there is no consent for such outcomes: e.g. need for renal replacement therapy (RRT) initiation; mortality, death during RRT, prolonged length of stay in in the Intensive Care Unit or in the hospital.

For the first time, we identified a substantial group of patients who do not fulfil current creatinine-based consensus criteria for AKI yet are likely to have acute tubular injury. We further demonstrated that these patients have a higher risk of adverse outcomes including death. These patients may benefit from medical attention. Such medical attention may carry a greater likelihood of success because changes in NGAL levels are rapid (hours) and changes in serum creatinine slow (days). By analogy with acute myocardial infarction (34), patients with AKI may now receive early intervention (13,36,37). In addition, our findings suggest that the definition of AKI should be refined by the application of criteria that include NGAL.

Since NGAL positive AKI, with or without the development of sCREA(+), is associated with poor patient outcomes, serum creatinine fails to identify likely AKI in some patients who are at increased risk of death. This observation does not imply that serum creatinine should be discarded as a marker of AKI. In fact, in most study patients, subclinical tubular injury preceded detectable decreases of renal function and when both occurred together clinical outcomes were worse. Thus, knowledge of NGAL levels modifies prognostic assessment not only in patients without an increase in serum creatinine but also in patients with creatinine-based consensus criteria for AKI. In these patients, however, AKI could only be diagnosed late, making any treatment less likely to succeed.

There may be alternative explanations for the NGAL(+)/sCREA(−) syndrome: Some of these patients might develop loss of renal function after the consensus period of seven days and NGAL identified these patients more than one week before such loss of function. A small number of patients might have died soon after NGAL testing, possibly too early to manifest sCREA(+) AKI. Finally, in a minority of patients, NGAL might have simply acted as a marker of inflammation, one of the most important risk factors for AKI.

We also identified a very small subgroup of patients with no biomarker evidence of tubular injury but loss of excretory function (NGAL(−)/sCREA(+), 4.6%). As NGAL, in contrast to serum creatinine, was not consistently serially measured, this observation might represent a false negative finding. However, ‘pre-renal azotemia’ (loss of function without tubular injury) may also explain such findings (38). Therefore, if NGAL was truly negative, these patients may represent a subgroup currently referred to as having pre-renal azotemia (39). In our study, NGAL(−)/sCREA(+) status was more common in elderly and diabetic patients and was still associated with a two-fold increase in risk of developing the composite outcome of death and renal replacement therapy compared to patients negative for both biomarkers (NGAL-/sCREA-). This is consistent with recent reports associating even transient azotemia with a greater risk of death (40). NGAL might now help identify some of these patients.

Although it is known that NGAL concentration is somewhat increased in patients with diabetic nephropathy (41–43), there is no information on acute NGAL responsiveness. On the other hand, it remains unknown whether patients in the NGAL(−)/sCREA(+) subgroup did not have active tubular damage as no biopsies were performed. However, there is evidence from animal experiments that prerenal azotemia does not translate into tubular damage or detection of NGAL in the urine or plasma (44,45).

Finally, NGAL detected approximately 40% of patients with probable AKI who were missed by consensus criteria. This proportion is similar to that identified by troponin in subjects with myocardial injury missed by conventional biomarkers (30).

This study cannot determine the source of NGAL, but the kidney should be the major source of NGAL. If plasma NGAL was produced outside the kidney and then filtered, the proximal renal tubules would completely reabsorb NGAL with no or minimal urine NGAL levels (10). Also, the decrease in glomerular filtration rate seen in AKI would decrease NGAL clearance resulting in decreased urine NGAL and increased plasma NGAL. Therefore, NGAL measured in the urine should either result largely from injured and not reabsorbing proximal tubules or arise from the injured distal nephron. Indeed, experimental evidence supports this view that NGAL in plasma might predominantly arise from the injured thick ascending tubules and the collecting ducts via back-leak from injured renal tissue - again reflecting renal damage. Basing on these results and our study findings with urine and plasma NGAL indicating a very similar patient outcome pattern, NGAL should be considered as a vigorous outcome marker in AKI.

Strengths and limitations of the study

This study has several strengths. It is multicentre and involved more than 2,000 patients at risk of AKI; it used data collected independently in different countries, assessed patients with diverse conditions and used different commercially available assays. However, it is retrospective in design with all the inherent imperfections of such studies and, because no individual patient data were obtained, meaningful meta-regression analysis was not possible. Our results might be affected by selection based on voluntary data contribution. The direction and the magnitude of this potential bias is unknown. Nonetheless, the findings show strength of association, temporality, consistency, biological plausibility and gradient, coherence with previous studies and are analogous to other fields of biomarker investigations. These features make it probable that the findings reflect a biological phenomenon (46). Future prospective studies should confirm histopathological agreement of NGAL with tubular injury, shown in animal experiments, and explore the prognostic value of other structural AKI biomarkers beyond NGAL (47), independent of serum creatinine. More importantly, they should test whether NGAL-based early diagnosis of AKI leads to the more successful and more timely deployment of therapies which, until now, could only be delivered late in the course of AKI (48,49) and improved outcomes.

Conclusions

The study findings show that NGAL complements serum creatinine in AKI diagnosis and prognosis. In a significant proportion of patients at renal risk, acute tubular damage may occur without loss of excretory function. Such NGAL(+) patients are at greater risk of adverse outcomes, including death and renal replacement therapy, both in the presence or absence of an increase in serum creatinine. These patients may be reasonably classified as having AKI, even though they do not fulfil current AKI consensus criteria. Their detection and the size of their cohort justify re-assessment of the concept and definition of AKI in patients with cardiorenal syndrome type 1.

ABBREVIATIONS

AKI

acute kidney injury

NGAL

neutrophil gelatinase-associated lipocalin

RIFLE

renal risk (R), injury (I), failure (F), loss of renal function (L), end stage renal disease (E) classification

RRT

renal replacement therapy

sCREA

serum creatinine

Appendix

Setting of AKI	_________(1–4)
Cardiac surgery (1)
Emergency department (2)	Citation of publication:
Contrast-induced nephropathy (3)
General ICU/Critical Care patients (4)
Sample size (Use NGAL concentration with best combination of sensitivity and specificity for prediction of RIFLE AKI: increase in serum creatinine >50% from baseline to peak value which was based on a rolling time window of seven days basing on daily serum creatinine measurement [=class R or worse])	NGAL-positive AND no AKI: n=_________ NGAL-negative AND no AKI: n=_________ NGAL-positive AND AKI: n=_________ NGAL-negative AND AKI: n=_________
Age (years, SD)	NGAL-positive AND no AKI: yr NGAL-negative AND no AKI: yr NGAL-positive AND AKI: yr NGAL-negative AND AKI: yr
Sex	NGAL-positive AND no AKI: Female n= NGAL-negative AND no AKI: Female n= NGAL-positive AND AKI: Female n= NGAL-negative AND AKI: Female n=
Renal impairment (eGFR <60mL/min/1.73m2 by simplified MDRD formula or Schwartz formula)	NGAL-positive AND no AKI: n=_________ NGAL-negative AND no AKI: n=_________ NGAL-positive AND AKI: n=_________ NGAL-negative AND AKI: n=_________
Diabetes mellitus (on medication)	NGAL-positive AND no AKI: n=_________ NGAL-negative AND no AKI: n=_________ NGAL-positive AND AKI: n=_________ NGAL-negative AND AKI: n=_________
Chronic heart failure (NYHA III/IV OR LVEF < 40%)	NGAL-positive AND no AKI: n=_________ NGAL-negative AND no AKI: n=_________ NGAL-positive AND AKI: n=_________ NGAL-negative AND AKI: n=_________
Peak NGAL concentration (ng/mL, SD)
NGAL-positive AND no AKI:	ng/mL
NGAL-negative AND no AKI:	ng/mL
NGAL-positive AND AKI:	ng/mL
NGAL-negative AND AKI:	ng/mL
Length of stay in ICU	NGAL-positive AND no AKI: median______ (95% CI______ -______ ), days NGAL-negative AND no AKI: median______ (95% CI______ -______ ), days NGAL-positive AND AKI: median______ (95% CI______ -______ ), days NGAL-negative AND AKI: median______ (95% CI______ -______ ), days
Length of stay in hospital	NGAL-positive AND no AKI: median (95% CI), days NGAL-negative AND no AKI: median (95% CI), days NGAL-positive AND AKI: median (95% CI), days NGAL-negative AND AKI: median (95% CI), days
Number of patients with RRT initiation
NGAL-positive AND no AKI:	n=________________
NGAL-negative AND no AKI:	n=________________
NGAL-positive AND AKI:	n=________________
NGAL-negative AND AKI:	n=________________
In-hospital mortality
NGAL-positive AND no AKI:	n=________________
NGAL-negative AND no AKI:	n=________________
NGAL-positive AND AKI:	n=________________
NGAL-negative AND AKI:	n=________________
RRT initiation and in-hospital mortality
NGAL-positive AND no AKI:	n=________________
NGAL-negative AND no AKI:	n=________________
NGAL-positive AND AKI:	n=________________
NGAL-negative AND AKI:	n=________________