Role of neutrophil gelatinase-associated lipocalin for early detection of acute kidney injury.

Acute kidney injury (AKI) is characterized by abrupt or rapid decline of renal function and is usually associated with the development of serious complications as well as an independent risk of mortality in hospitalized patients. Emergency physicians play a critical role in recognizing early AKI, preventing iatrogenic injury, and reversing the course of AKI. Among the various available biomarkers for AKI, reliable and automated assay methods are commercially available for only cystatin-C and neutrophil gelatinase-associated lipocalin (NGAL). NGAL appears to be a promising marker for early detection of AKI and is likely to be adapted for wide-scale clinical use in patient management as a point-of-care test. Use of NGAL along with panel of other renal biomarkers can improve the rate of early detection of AKI. Large, multicenter studies demonstrate the association between biomarkers and hard end points such as need for renal replacement therapy (RRT), cardiovascular events, hospital stay, and death, independent of serum creatinine concentrations.

INTRODUCTION

Acute kidney injury (AKI) is characterized by abrupt or rapid decline of renal function and is usually associated with the development of serious complications as well as an independent risk of mortality in hospitalized patients.[1] Emergency physician plays a critical role in recognizing early AKI, preventing iatrogenic injury, and reversing the course of AKI. AKI develops in up to 5% of the hospitalized patients and in up to 30% patients admitted in the Intensive Care Unit. It is estimated that about 2 million people die of AKI every year. Those who survive AKI have a higher risk for later development of chronic kidney disease (CKD).[2] In developing countries, AKI usually develops in younger adults in the setting of several community-acquired diseases, affecting younger and previously healthy individuals and the spectrum depends on environmental, cultural, and socioeconomic factors.[2345] Early diagnosis and prompt treatment can significantly decrease morbidity and mortality in patients with AKI.

SIGNIFICANCE OF EARLY DIAGNOSIS OF AKI

Despite several advances in the management of critically ill patients and dialysis techniques during the last few decades, once AKI develops, the mortality has continued to remain unacceptably high. In fact, even minor elevations of serum creatinine in critically ill patients are associated with progressive increase in adverse outcomes. It has been shown that most of the preventive measures for AKI, which are efficacious in experimental settings, do not show comparable positive results in the clinical setting.[6] Hence, early diagnosis of AKI is of paramount importance. The lack of reliable biomarkers of early structural kidney injury results in an unacceptable delay in the clinical diagnosis, which severely limits prompt therapeutic approach. Improvement in clinical outcomes of the patients with acute coronary syndrome in the last few decades has been aided by the availability of a panel of biomarkers. Absence of a similar panel in AKI has been a major impediment in improving clinical outcomes. AKI is currently diagnosed by functional biomarkers, such as serum creatinine measurement and estimation of urine flow rate. However, as creatinine is primarily a marker for estimation of glomerular filtration, it cannot be considered for the estimation of kidney injury, since it is insensitive and unreliable to diagnose renal tubular injury in the absence of significant reduction in glomerular filtration rate (GFR).[7] An elevation in serum creatinine is noticed only when GFR has already reduced below 50% of normal. Consequently, more reliable biomarkers than creatinine are necessary for both an accurate evaluation of renal function and an early detection of AKI.

BIOMARKERS FOR AKI

Biomarkers suggested for an accurate and early detection of AKI are cystatin-C, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), Na+/H + exchanger isoform-3 (NHE-3), N-acetyl-b-glucosaminidase, liver-type fatty acid binding protein (L-FABP), Interleukin-18 (IL-18), etc., Among these, only for cystatin-C and NGAL, reliable and automated assay methods are commercially available.[8] The serendipitous finding that NGAL protein was easily detected in the urine soon after AKI in animal studies has prompted an explosion of translational studies to evaluate NGAL as a non-invasive biomarker in human AKI. Early diagnosis of AKI by point-of-care testing for various biomarkers in high-risk individuals can significantly help in decreasing morbidity and mortality in such patients.

BIOCHEMICAL PROPERTIES OF NGAL

NGAL (also known as human neutrophil lipocalin, lipocalin-2, siderocalin, 24p3, or LCN) is a small molecule of 25 kDa having 178 amino acids that belongs to the superfamily of lipocalins, which are proteins specialized in binding and transporting small hydrophobic molecules.[9] The lipocalins share a molecular organization comprising eight β-strands arranged in a complex β-barrel structure delineating a calyx shape, which represents their binding site. NGAL, like the other lipocalins, can bind some ligands, including the siderophores. After interaction with these receptors, NGAL is internalized inside the cell as a protein alone (Apo-NGAL) or a complex with iron-binding siderophores (Holo-NGAL). Endosomal vesicles capture Holo-NGAL inside them and transport it to the cytoplasm, where it can release the siderophore-iron complex, thus activating iron-dependent specific pathways. Conversely, Apo-NGAL, after being internalized in the cell, is able to capture cellular iron and export it to the extracellular space. This results in depletion of iron cellular pools that, under particular conditions, may even lead to apoptosis. Due to its specific binding to bacterial siderophores, NGAL can also exert a bacteriostatic effect on several strains of bacteria by its ability to capture siderophores in extracellular space and to deplete the bacterial iron supply. Therefore, NGAL represents a critical component of innate immunity to bacterial infection.

NGAL AS A PROTECTIVE AGENT IN AKI

Injured kidney tubular cells produce and secrete several biological substances associated with innate and acquired inflammatory immune responses, including NGAL. In the kidney, after ischemic injury in animal models, transcriptome profiling studies identified NGAL to be one of the most up-regulated genes, and proteomic analyses revealed NGAL to be one of the most highly induced proteins. These studies stimulated several groups of investigators to evaluate NGAL as a non-invasive biomarker in human AKI. Studies in mouse models of renal ischemia reperfusion showed NGAL production to be highly up-regulated at an early stage, and NGAL was detected in the urine within 2 hours following ischemia.[10] In response to renal tubular injury, expression of NGAL rises 1000-fold in humans and rodents, and it appears so rapidly in the urine and serum that it may be useful as an early biomarker of renal failure.

The physiological role of NGAL in this setting may be to decrease injury by reducing apoptosis and increasing the normal proliferation of kidney tubule cells. Because of its ability to act as a growth factor, NGAL has also been found to have a renoprotective effect in acute ischemic renal injury in an animal model.[11] In a murine model of renal ischemia-reperfusion injury, intravenous administration of purified recombinant NGAL administered before, during, or after ischemia resulted in marked amelioration of the morphologic and functional consequences with a reduction in the number of apoptotic tubule cells and an increase in proliferating proximal tubule cells after ischemic injury.[11]

INDICATIONS FOR MEASUREMENT OF NGAL

Results from various clinical studies suggest that NGAL measurement (both urine and plasma) might be useful in early detection of AKI. Measurement of NGAL may help in timely diagnosis and intervention of AKI to protect the kidney from further insults in numerous clinical situations in the Emergency Department and Intensive Care Unit for management of critical illness, sepsis and septic shock,[12] oliguria,[13] radio-contrast procedures,[1415] cardiopulmonary bypass (CPB),[16] polytrauma,[17] prolonged cardiovascular surgeries, etc., Measurement of NGAL might be useful in randomized control trials assessing efficacy of early intervention for AKI. Diagnosis of AKI at an early stage might improve patient selection for such trials. UNGAL level has been found to be helpful to distinguish patients with AKI from other morbid conditions with elevated creatinine such as prerenal azotemia and CKD.[18] In this study, a single measurement of uNGAL in the emergency department in 635 patients was found to be highly sensitive and specific (sensitivity 90%, specificity 99%) in diagnosing AKI. NGAL has also been found to be a useful tool in disease monitoring in other renal diseases, such as lupus nephritis,[19] IgA nephropathy,[20] and polycystic kidney disease.[21] Another relevant area for the clinical application of NGAL assays is to diagnose delayed graft function following renal transplantation earlier than rise in serum creatinine since this may be insensitive in these settings. Delayed graft function is the main risk factor for reduced allograft survival.[22]

NGAL IN NON-RENAL CONDITIONS

NGAL has been found to be a biomarker in many other non-renal conditions such as brain tumor,[23] inflammatory bowel disease,[24] and pre-eclampsia.[25] It has also been seen that NGAL rises in response to prolonged hypobaric hypoxia and demonstrates a relationship to the presence and severity of acute mountain sickness.[26]

NGAL as a predictor of disease severity and outcome

Several clinical studies and systematic reviews indicate that NGAL should be considered a reliable diagnostic and prognostic biomarker for kidney injury. Some of the studies[27282930] have been summarized in Table 1. When compared with the conventional biomarker such as creatinine, NGAL assay can detect renal injury at an early stage. A recent multicenter pooled analysis of published data on 2322 critically ill children and adults revealed the surprising finding that approximately 20% of patients display early elevations in NGAL concentrations but never develop increases in serum creatinine.[31] Importantly, this sub-group of “NGAL-positive creatinine-negative” subjects encountered a substantial increase in adverse clinical outcomes, including mortality, dialysis requirement, ICU stay, and overall hospital stay.[31] NGAL assay may be useful as a prognostic tool with regard to the prediction of renal replacement therapy (RRT) initiation and in-hospital mortality. It has been found to be a useful diagnostic and prognostic biomarker in the specific setting of cardiorenal syndrome including patients with chronic or acute heart failure.[32]

Table 1

Summary of studies demonstrating the utility of NGAL in point-of-care testing for AKI

Few studies have evaluated the utility of NGAL for risk stratification and prognosis. Dent et al.[33] found the 2-hour postoperative pNGAL level a reliable predictor of duration of AKI and length of hospital stay, while the 12-hour pNGAL level was a predictor of mortality. Similarly, Bennett et al.[34] found 2-hour uNGAL level a reliable predictor of severity and duration of AKI, length of hospital stay, requirement for RRT, and mortality in 196 children undergoing CPB. Single uNGAL measurement in the Emergency Department was found to predict the need for nephrology consultation. Zappitelli et al.[35] studied uNGAL in 140 critically ill mechanically ventilated patients. A significant rise (16 times) in levels of uNGAL occurred 2 days earlier than a 50% increase in serum creatinine levels. uNGAL levels increased in a stepwise fashion with worsening RIFLE class.

Estimation of NGAL

The first analytical procedures set up for measurement of NGAL in blood or urine samples were based on enzyme-linked immunosorbent assay (ELISA) or immunoblotting systems.[16] In general, these are manual methods, which are not standardized and not recommended for clinical practice, but only for research studies. Their use is also limited by their cost and feasibility. The measurement of NGAL in serum, plasma and urine samples can also be performed by means of commercially available ELISA kits using both manual procedures and several auto-analyzers. Pedersen et al. have recently evaluated the analytical performance of this ELISA kit, using the manual procedure for the measurement of NGAL in both urine and plasma samples and found that these can be used with acceptable precision for plasma and urine.[36] A commercially available point-of-care test (POCT) method is a fluorescence-based immunoassay for a rapid measurement (approximately 30 min) of NGAL in whole blood or plasma samples. This POCT method has a detection limit at 60 ng/mL and the upper limit of the working range at 1300 ng/mL. More recently, a chemiluminiscent microparticle immunoassay (CMIA) method has become commercially available, using the automated platform for the measurement of NGAL in urine samples.

Both urine and blood samples have their advantages and limitations. Blood NGAL measurements are invasive and may potentially reflect the effect of extrarenal disease on NGAL concentrations. However, samples are readily available and the measurement can be performed rapidly on whole blood or plasma (15–20 min) on a point-of-care device. Urine sampling is non-invasive and there are less potentially interfering proteins present than in blood specimens. However, many critically ill patients may be oligo-anuric. In addition, urinary NGAL concentrations may be affected by the status of hydration and diuretic treatment.

FACTORS INFLUENCING NGAL MEASUREMENT

In a study on 426 adults undergoing CPB,[37] UNGAL levels were significantly elevated in all patients (with or without AKI), suggesting that CPB may initiate inflammation and activation of neutrophils leading to increased NGAL levels. In a recent in vitro study by Bobek et al.,[38] NGAL was found to be ultrafiltered and adsorbed by polysulfone membranes, reducing its blood levels. As many patients with AKI undergo hemodialysis, this finding may be a potentially confounding factor in the estimation of NGAL.

SIGNIFICANCE OF TESTING PANEL OF BIOMARKERS FOR AKI

Of the many new biomarkers available, NGAL appears to be the most promising but it is likely to be most useful when combined with other markers such as cystatin C or KIM-1 to form an “AKI panel”.[394041] Several studies have examined the use of such biomarkers in combination.[4243] In a study of 100 adult cardiac surgical patients measuring NGAL and cystatin C as well as urea and creatinine, the new biomarkers were superior to the conventional measures in predicting AKI. Blood NGAL and cystatin C were independent predictors of AKI.[42] Another study measured KIM-1, NAG, and NGAL in 90 adults undergoing cardiac surgery.[44] The AUCs for KIM-1 to predict AKI immediately and 3 hours after operation were 0.68 and 0.65; for NAG were 0.61 and 0.63; and for NGAL were 0.59 and 0.65. Combining the three biomarkers enhanced the sensitivity of early detection of postoperative AKI compared with individual biomarkers: The combined AUCs for the three biomarkers were 0.75 and 0.78.

In a large prospective multicenter study of a panel of nine biomarkers to predict clinical outcomes in 971 emergency department patients with suspected sepsis, plasma NGAL emerged as the strongest predictor of shock and death.[45] In a secondary analysis of this cohort, an elevated plasma NGAL level at the time of presentation to the emergency department predicted severe AKI with an AUC of 0.82.[46] In a study examining biomarkers for the prediction of AKI following elective cardiac surgery, urinary NGAL concentrations measured at the time of admission to the ICU predicted the subsequent development of AKI with an AUC of 0.773, and outperformed other biomarkers including α1-microglobulin and cystatin C.[47] In a similar analysis of multiple urinary biomarkers following cardiac surgery, the 6-hour post-operative NGAL best predicted severe AKI with an AUC of 0.88.[48] Serial measurements of multiple urinary biomarkers after pediatric cardiac surgery have revealed a sequential pattern for the appearance of AKI biomarkers,[9] with NGAL and L-FABP being the earliest responders (with 2–4 hours after initiation of cardiopulmonary bypass) and KIM-1 and IL-18 representing the intermediate responders (increased 6–12 hours after surgery). Hence, use of a panel of biomarkers for AKI is likely to improve the early detection of AKI.

LIMITATIONS OF NGAL AS A BIOMARKER FOR AKI

Despite the optimism in the field, there are important limitations that exist in the published AKI biomarker literature that must be acknowledged. First, majority of studies reported were from single centers and from homogenous patient populations. Second, most studies did not include patients with chronic kidney disease. Third, only a few studies have investigated biomarkers for the prediction of AKI severity, morbidity, and mortality. Fourth, use of single biomarker is likely to have a lower yield; hence, biomarker combinations are likely to improve our ability to predict AKI and its outcomes, and these studies are only beginning to surface. Large multicenter studies are required for further validation of the use of NGAL in heterogenous patient populations and for defining cut-off values for diagnosis and outcomes of AKI. In addition, various other factors such as RRT and underlying CKD may influence its measurement as discussed earlier.

CONCLUSION

NGAL appears to be a promising marker for early detection of AKI and is likely to be adapted for wide-scale clinical use in patient management as a point-of-care test. Early NGAL measurements can identify patients with subclinical AKI who have an increased risk of adverse outcomes, even in the absence of diagnostic increases in serum creatinine. Its ability to detect AKI early may allow clinicians to take corrective and restorative measures before the kidney damage becomes established and irreversible. NGAL appears to be an exciting marker of AKI, but more studies need to be conducted to confirm its utility in routine clinical practice and to fine tune the choice of appropriate cut offs for different clinical settings and populations. Use of NGAL along with a panel of other renal biomarkers can improve the rate of early detection of AKI. Large, multicenter studies demonstrate the association between biomarkers and hard end points such as need for RRT, cardiovascular events, hospital stay, and death, independent of serum creatinine concentrations.