Introduction

Neutrophil gelatinase–associated lipocalin (NGAL) is a protein of the lipocalin family. Lipocalins are composed of eight β-strands that form a β-barrel enclosing a calyx [1]. The calyx binds and transports low-molecular weight chemicals. These proteins interact with cell-surface receptors [2, 3]. Human NGAL consists of a polypeptide chain of 178 amino acids with a molecular mass of 25 kDa. It is expressed by neutrophils and various epithelial cells [4]. Variable degrees of NGAL gene expression is demonstrated in human tissues like uterus, prostate, salivary glands, lung, trachea, stomach, colon, and kidney [5]. Until recently, NGAL was mainly of interest to structural biologists. However, our understanding about its role in human physiology and disease is rapidly increasing. The recent findings that NGAL binds siderophores (iron-chelating molecule secreted by micro organisms) and is highly expressed in various pathological states like acute kidney injury (AKI) have prompted a large number of studies.

Mishra et al. initially in experimental [6, 7] and later in clinical setting [8] showed NGAL as a promising early biomarker for AKI. Since then, many investigators have studied its utility in AKI diagnosis and in a variety of other clinical settings. The origin of NGAL was originally proposed to be from proximal tubular epithelium [6]. However, subsequent studies found loop of Henle and collecting ducts as the major sites for NGAL synthesis in kidney [9]. The NGAL protein found in proximal tubules after ischemic injury is proposed to be derived from glomerular filtration of circulating NGAL synthesized by other organs like liver. This review discusses the role of NGAL as a biomarker in various conditions.

Physiology of NGAL

Role in iron metabolism

Siderophores are proteins produced by bacteria to scavenge iron from the extracellular space. Siderophores trap iron with high affinity and ensure continuous supply of iron necessary for survival and growth of bacteria. Enterochelin is one such siderophore produced by some strains of bacteria. The evidence for role of NGAL in iron metabolism came from crystallographic studies by Goetz et al. [10] that demonstrated enterochelin–iron complex in the NGAL calyx. NGAL is also called siderocalin as it is the first mammalian protein to bind and transport a bacterial siderophore [10].

Neutrophil gelatinase–associated lipocalin interacts with cells by specific cell-surface receptors. Hitherto, two receptors are identified; the megalin–cubilin multiscavenger complex found on the brush-border surface of renal tubular epithelial cells [11] and the 24p3R (NGAL was originally called 24p3), an organic cation transporter [12]. NGAL is also proposed to interact with some other receptors like extracellular protein kinases, hepatocyte growth factor [13], and gelatinase B [14]. These receptors seem to have a role in cellular trafficking and endocytosis of NGAL. Endocytosis occurs as NGAL protein alone (Apo-NGAL) or NGAL complexed with iron-binding siderophores (Holo-NGAL).

The cellular effects depend on the form of NGAL endocytosed. When transported as Apo-NGAL, it captures intracellular iron and transports it outside leading to depletion of intracellular iron. On the contrary, when endocytosed as Holo-NGAL, it releases iron–siderophore complex and contributes to intracellular iron pool [12].

Role in innate immunity to bacteria

As discussed earlier, by virtue of its binding with siderophores, NGAL prevents the growth of the bacterial strains that depend on siderophores for iron supply [10]. The biologic importance of this finding is examplified in genetically modified mice, deficient for NGAL genes. These animals were more sensitive to Escherichia coli infection and died of sepsis more readily than wild-type mice [15, 16].

In addition to enterochelin, NGAL has been shown to bind to other siderophores like carboxymycobactin, a soluble protein secreted by mycobacteria [17]. Thus, NGAL may potentially have an important role in defending against bacterial and mycobacterial infection through iron sequestration.

Role in kidney development

In vitro studies in rats suggest that NGAL may be involved in kidney development [18]. Administration of purified NGAL to early progenitor cells in metanephric mesenchyme produced a proliferative effect followed by epithelial differentiation of these elements and generation of nephron-shape formations expressing glomerular, proximal, and distal tubular surface cellular markers [19]. NGAL participates in kidney development by virtue of its role in iron metabolism. For mechanistic details, the reader is referred to the relevant articles [18, 19].

Role as a growth factor

Several findings indirectly point to the role of NGAL as a growth factor. NGAL expression is markedly increased in stimulated epithelia. Its increased expression in colonic cells in areas of inflammation or neoplasia [20, 21] was recognized many years ago. Serum NGAL levels are increased in the patients with acute bacterial infections [22]. Elevated NGAL is also found in the sputum of subjects with asthma or chronic obstructive airway disease [23] and in the bronchial secretions from the patients with subclinical emphysema [24]. It is postulated that interaction between inflammatory cells and the epithelial cells leads to the upregulation of NGAL in both neutrophils and epithelium [21]. The role of NGAL is also implicated in tumor growth and progression [25, 26].

It is proposed that NGAL plays an important role in renal regeneration and repair after ischemic injury [6]. It seems to support development and de-differentiation of tubular epithelia following injury. In addition, if presented before or in early stages of AKI, it may have a protective effect. An in vitro study by Mori et al. [27] in mouse models of ischemia-reperfusion injury showed that a single dose of NGAL (10 μg) protects the kidney and decreases azotemia. This effect depends on the delivery of iron by NGAL–siderophore–iron complex. This complex rescues N-cadherin (adhesion molecule), upregulates heme oxygenase-1 (a protective enzyme), and inhibits cell death.

Methods of NGAL measurement

A variety of methods for NGAL measurement have been used in published studies. In initial studies [6], both urine and serum NGAL estimations were carried out by Western blot technique. Subsequent clinical studies have utilized immunoblotting or research ELISA-based techniques using a commercially available monoclonal NGAL antibody (Antibodyshop, Gentofte, Denmark).

A standardized point-of-care Triage® NGAL Device (Biosite Incorporated, San Diego, CA, USA) has been devised for the measurement of plasma neutrophil gelatinase–associated lipocalin (pNGAL). The assay was found to correlate well with research ELISA in a pilot study of 40 plasma samples and 12 calibrations. Its clinical application was later validated in a study of 120 patients undergoing cardiopulmonary bypass (CPB) [28]. In 45 patients who developed AKI, the diagnosis using serum creatinine concentration was delayed by 2–3 days, while pNGAL concentrations increased threefold within 2 h. At cutoff value of 150 ng/mL for the 2-h pNGAL concentration, the area under curve (AUC) for prediction of AKI was found to be 0.96 with sensitivity of 0.84 and specificity of 0.94. The test needs only microliter quantities of plasma and quantitative results are available within 15 min. Bedside testing for plasma NGAL is potentially possible using this device.

A urine NGAL assay ARCHITECT® analyzer (Abbott Diagnostics, Illinois, USA) is also available for clinical application. In a pilot study with 136 urine samples and six calibration standards, urinary neutrophil gelatinase–associated lipocalin (uNGAL) concentrations by the ARCHITECT® analyzer correlated well with research ELISA. Its clinical application was later validated in a study of 196 patients undergoing CPB [29]. The diagnosis of AKI using serum creatinine concentration was delayed by 2–3 days after CPB, while uNGAL concentrations increased by 15-fold within 2 h and by 25-fold at 4 and 6 h after CPB. At cutoff value of 100 g/L for the 2-h urine NGAL concentration, the AUC for prediction of AKI was 0.95 with sensitivity of 0.82 and specificity of 0.90 [29]. The test is found to be easy for application with no manual pre-treatment steps [30]. The test needs only 150 μL of urine and quantitative results are available within 35 min.

NGAL as a biomarker

NGAL is found to be a promising biomarker in many conditions as shown in Table 1. It has been found as a promising biomarker for early diagnosis, for predicting disease severity, for therapeutic monitoring, and for predicting clinical outcomes. The following section discusses the potential role of NGAL in a variety of renal and non-renal conditions.

Table 1 Potential uses of NGAL as a biomarker
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Acute kidney injury

The last decade has witnessed many advances in the treatment of AKI. However, morbidity and mortality have not improved significantly [31–33]. Serum creatinine is an insensitive and late marker [34] for AKI. It is important to diagnose AKI as early as possible to facilitate effective interventions [35]. This had led to an aggressive search for newer biomarkers for AKI. NGAL appears to be the most promising molecule among the many newer molecules [36].

As mentioned earlier, Mishra et al. [6] first proposed NGAL as a novel early biomarker. In mouse models of renal ischemia-reperfusion injury subjected to 30 min of bilateral renal artery occlusion, NGAL was found to be one of the seven genes which were highly upregulated. NGAL was easily detected in the urine within 2 h following ischemia. In the same study, separate sets of mice were subjected to 5, 10, and 20 min of ischemia. Importantly, uNGAL was detected even in these mice, appearing after 6 h in mice with 5 min ischemia and after 4 h in mice with 10 and 20 min of ischemia. Thus, uNGAL was found to be a very sensitive marker of ischemic AKI and its levels correlated to the dose and duration of renal ischemia. Additionally, uNGAL was detectable after 1 day of cisplatin administration in a mouse model of nephrotoxic AKI, suggesting its sensitivity in other models of tubular injury [6].

Many clinical studies followed these important experimental observations (Table 2). In a prospective study of 71 children undergoing CPB, pNGAL and uNGAL levels were markedly increased within 2 h in patients who developed AKI [8]. The diagnosis of AKI by serum creatinine criteria was possible only after 1–3 days. uNGAL at 2 h after CPB was the most powerful independent predictor of AKI with area under curve (AUC) of 0.998. A cutoff value of 50 μ/L was 100% sensitive and 98% specific in predicting AKI. Similarly, at cutoff value of 25 μg/L pNGAL was 70% sensitive and 94% specific in predicting AKI with AUC of 0.91. In another prospective study of 91 children with congenital heart disease, both urine and plasma NGAL predicted contrast-induced nephropathy within 2 h of elective cardiac catheterization [37]. The AUC for prediction of contrast nephropathy (at 2 h) was found to be 0.92 for uNGAL and 0.91 for pNGAL.

Table 2 Major studies reporting use of uNGAL as an early biomarker in AKI
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Many other studies followed this novel observation. Zapitelli et al. [38] studied uNGAL in 140 mechanically ventilated patients. uNGAL levels increased significantly (>6 times) 2 days earlier than 50% increase in serum creatinine levels. uNGAL levels also correlated with severity of AKI. Serum NGAL was also studied in 143 critically ill children with systemic inflammatory response syndrome (SIRS) or septic shock within 24 h of admission to ICU [39]. It was found to be highly sensitive but non-specific (sensitivity 84%, specificity 39%) biomarker of AKI. Makris et al. [40] have found uNGAL as an early marker of AKI in critically ill polytrauma patients.

In a recently published study, a single measurement of uNGAL in emergency department is found to be highly sensitive and specific (sensitivity 90%, specificity 99%) in diagnosing AKI in 635 adult patients [41]. uNGAL level also helped to distinguish patients with AKI from other morbid conditions with elevated creatinine, like pre-renal azotemia and CKD. Many other studies have found NGAL as a reliable early biomarker for AKI [42–44].

In addition to being an early biomarker for AKI, uNGAL has been found to be a predictor of severity of AKI. In a study of 196 children undergoing CPB at a single center, 2-h post-operative uNGAL levels predicted requirement of renal replacement therapy, duration of hospital stay and mortality [29].

Renal transplantation

Mishra et al. [45] studied protocol biopsy specimens within 1 h of reperfusion in 13 cadaveric and 12 living-related renal allografts for NGAL expression by immunohistochemistry. The staining intensity for NGAL was found to correlate well with cold ischemia time, peak post-transplant creatinine, and requirement of dialysis. In another study involving 33 cases, uNGAL was found to be an early predictor of delayed graft function (DGF) defined as need for dialysis within the first week after transplantation [46]. uNGAL on the day of transplant surgery was predictor of DGF with AUC of 0.9. Importantly, creatinine level peaked only after 2–4 days. In a study by Kusaka et al. [47] in cadaveric transplant patients, fall in NGAL levels occurred earlier than fall in serum creatinine or improvement in urine output during recovery from DGF.

Thrombotic microangiopathy

In pediatric patients with diarrhea-associated hemolytic uremic syndrome, Trachtman et al. [48] used peak uNGAL levels within 5 days of hospitalization to divide patients into two groups (<200 and ≥200 ng/mL). Patients with higher uNGAL levels required dialysis more frequently. uNGAL served as an adjunctive early predictor of severity of the microangiopathic disease in this study. Increased uNGAL level was postulated to be secondary to associated tubular damage in HUS.

Chronic kidney disease

Polycystic kidney disease

The uNGAL levels are shown to correlate well with residual renal function, serum creatinine, and cyst enlargement in patients with autosomal dominant polycystic kidney disease [49]. In an experimental setting, addition of NGAL to the cells from PKD1 mutant mice suppressed the cyst development [50].

Glomerular diseases

Brunner et al. [51] in a cohort of 35 cases of childhood-onset systemic lupus erythematosus (SLE) found strong correlation of uNGAL levels with renal disease activity. In addition, uNGAL levels correlated with the activity and chronicity score on renal histology. In a similar study, Suzuki et al. found strong correlation of uNGAL levels with SLE disease activity [52]. However, plasma NGAL levels did not show such correlation in this study.

In a cohort of 70 cases of IgA nephropathy, Ding et al. [53] compared levels of uNGAL, urinary N-acetyl-β-d-glucosaminidase (uNAG) and urinary creatinine with 40 healthy controls. uNGAL levels were significantly elevated in IgA nephropathy patients compared with controls. uNGAL/creatinine ratio showed strong correlation with histological and clinical disease activity, while uNAG levels lacked such correlation.

In proteinuric patients with idiopathic glomerulonephritis, Bolignano et al. [54, 55] found significant correlation of uNGAL with serum creatinine; daily proteinuria and residual renal function over 1 year follow-up. The authors have also published their observations regarding change of uNGAL levels in 15 such patients after anti-inflammatory therapy with intravenous immunoglobulin [56]. An impressive decrease in uNGAL level was observed 1 h after the infusion. The authors hypothesized that uNGAL can serve as biomarker to assess treatment response and predict disease severity.

Other systemic conditions

Poniatowski et al. have found serum and urine NGAL as sensitive early markers of renal impairment in patients with chronic heart failure [57]. In another study, uNGAL levels were found to be significantly elevated in patients with chronic heart failure. Its level correlated directly with urine albumin excretion rate and inversely with estimated glomerular filtration rate [58]. Thus, uNGAL can be a potential biomarker for the cardio-renal syndrome.

Urinary NGAL was also found to be elevated in premature and low birth weight infants, and it correlated significantly with birth weight and gestational age [59]. A recently published study by Smith et al. [60] suggests uNGAL along with other urine metalloproteinases as the promising biomarkers for the presence of brain tumor. They propose that uNGAL level can be useful for therapeutic follow-up and early non-invasive detection of recurrence. Similarly, it can potentially serve as a marker to assess severity of inflammatory bowel diseases [61]. Fernandez et al. [62] found uNGAL–MMP9 complex as a useful biomarker for breast cancer.

Comparison of serum NGAL and uNGAL as biomarkers

The rapidly evolving knowledge about NGAL kinetics in AKI suggest that NGAL gene upregulation occurs in two major areas in early phase of AKI, namely, kidney and other systemic organs like lungs and liver [63]. It is postulated that the NGAL synthesized in the distal tubules of the kidneys is efficiently excreted in urine and that synthesized in other organs constitute the systemic pool [9, 63, 64]. Both serum and uNGAL have been found to be reliable predictors of AKI [8]. uNGAL measurement is probably more reflective of local renal injury and is non-invasive in nature reducing the need for frequent blood sampling and handling of blood lines in critically ill patients. Urine sample is also relatively free of interfering proteins [29]. On the other hand, serum NGAL measurement is clearly useful in anuric patients and does not need correction for creatinine concentration unlike urine sample. Whether one measurement is useful over the other can be known after wider clinical application of these tests. It is also possible that together they provide complementary information.

Comparison of NGAL with other newer biomarkers of AKI

In addition to NGAL, many other molecules have been studied and found useful as early biomarkers of AKI when compared with serum creatinine. Very few studies have directly compared NGAL with other molecules (Table 3). Performance of NGAL has been comparable or better than other biomarkers.

Table 3 Major studies comparing NGAL with other biomarkers
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Future directions

NGAL appears to have successfully cleared the initial hurdles of biomarker development at the experimental stage and in initial clinical studies. Newer commercial tools for its measurement will definitely help to enhance clinical application in broader population. It will likely emerge as a powerful biomarker in AKI panel akin to troponins in cardiac panel [65].

Conclusion

Urinary NGAL is rapidly emerging as a very promising early biomarker in a variety of renal and non-renal conditions. The horizons for its applications are rapidly expanding. It is likely to be used as a powerful predictive tool for many disorders, especially AKI in future. Early detection of AKI will provide a much-needed window of opportunity for quick and effective interventions. The easy and rapid detection of uNGAL using newer devices increase the feasibility of its widespread clinical application.