Eur J Clin Invest 2010; 40 (3): 273–276
Neutrophil gelatinase-associated lipocalin (NGAL), a small 25 kDa stress-protein released from injured tubular cells after various damaging stimuli, is already known by nephrologists as one of the most promising biomarkers of incoming Acute Kidney Injury. Moreover, recent studies seem to suggest a potential involvement of this factor also in the genesis and progression of chronic kidney diseases. This brief review explores the new interesting involvement of NGAL in the experimental and clinical field of cardiovascular diseases, such as the pathogenesis and clinical manifestations of atherosclerosis, acute myocardial infarction and heart failure. It does not seem difficult that, in the next future, NGAL may become a new missing link between the kidney and the cardiovascular system.
As shown in recent literature, neutrophil gelatinase-associated lipocalin (NGAL) is one of the most interesting and promising nephrology biomarkers available. This small 25 kDa protein, originally known as an innate immunity antibacterial factor released by activated neutrophils, is also produced by renal tubular cells in response to different types of injury. Following the clinical observations made, NGAL is now widely considered an excellent predictor of acute kidney injury (AKI): its levels, both in plasma and urine, rise before any increase occurs in creatinine levels in response to treatments that are potentially harmful to the kidney, thus facilitating a more reliable prediction of AKI . It has been proven that in patients with chronic kidney disease (CKD) associated with polycystic kidney, glomerulonephritis or diabetic nephropathy, NGAL levels reflect the extent of kidney damage and have a close inversely proportional and independent correlation with the glomerular filtration rate [2,3]. In these, as in critically ill patients, the assessment of NGAL is of significant prognostic value; it can, in particular, predict the risk of disease progressing to more advanced CKD stages in the short to medium term [4,5]. There is therefore little doubt that NGAL measurement will play a key role in the field of nephrology. However, findings made in recent studies indicate that this protein may be used beyond the boundaries of renal physiopathology: it may, for example, play a key role in patients with cardiovascular diseases.
NGAL in vascular damage and remodelling
A turning point in the new focus on NGAL was the discovery that the vascular wall, as well as the renal tubule, responds to various types of damage by over-expressing the protein.
Using an experimental model of angioplasty in sections of aorta from rats, Bu et al. found that intimal tissue led to the release of extremely high quantities of NGAL in response to stressful manoeuvres ; this over-expression was prevented by prior tissue transfection with adenovirus strains blocking the synthesis of the nuclear factor kB (NF-kB), thus suggesting that NGAL-mediated intracellular events were closely dependent on NF-kB synthesis. The authors also showed that rat aorta smooth muscle cells incubated with high concentrations of IL-1 (a strong inflammatory mediator and NF-kB inducer), secreted large quantities of NGAL. Interestingly, NGAL was found in the medium both in its free form and as a complex with the proteolytic factor MMP9 (NGAL/MMP9). Findings made in a recent study on aortic aneurysm tissue with associated thrombotic lesions demonstrated unexpectedly high NGAL/MMP9 complex expression not only within the thrombi examined, but also on the thrombus/vessel wall interface and within the aneurysm wall itself ; in further analyses, the main source of NGAL was found to be neutrophils, probably recruited in the vascular wall by platelet activation.
A large intraluminar thrombus triggers secondary proteolytic reactions against vessel wall structural proteins, such as collagen and elastin, and can therefore be considered to incur the risk of the development of abdominal aortic aneurysms and their enlargement and rupture . Since it has a protective effect on MMP9, thus enhancing its proteolytic activity, NGAL can be considered to be an important factor indirectly contributing to the progression of aneurysm, as well as involved in the physiologic and pathologic remodelling of vessel walls. This view is further supported by the observation that a similar neutrophil NGAL/MMP9 over-expression can be found in atherosclerotic plaques, particularly those with intramural haemorrhagic debris and central necrosis [9,10]. The above evidence supports the clinical observations that high circulating leucocyte (particularly neutrophil) counts are independent predictors of recurrent ischaemic attacks. This may be explained by their presence in the necrotic core of unstable plaques and by their proteolytic activity towards atherosclerotic tissue and secondary mobilization of thrombo-embolic fragments .
NGAL and atherosclerosis: from laboratory to clinic
The large body of currently available evidence showing the close link between neutrophils, their products and the natural history of atherosclerosis and its complications has prompted a further shift ‘from the bench to the bedside’, further studies being conducted in the attempt to investigate the possible clinical utility of the newly gained patho-physiologic insight. Analogous attempts had been made in the past: for example, other neutrophil factors, such as myeloperoxidase, had been evaluated as potential markers of diffuse atherosclerosis .
In their study on 156 middle-aged patients with asymptomatic atherosclerosis, Elneihoum et al. found that NGAL levels in plasma were significantly higher in patients than in healthy controls, being directly linked to diastolic pressure (R = 0·22, P < 0·005) and age (R = 0·20, P < 0·05) . In addition, NGAL levels were found to be significantly higher in female patients with hypertension than in healthy age- and sex-matched controls. These findings suggest that hypertension itself, independent of atherosclerosis, can cause an increase in systemic NGAL production, although the mechanism underlying this change is unknown.
The above hypothesis is supported by findings made in a more recent study by Malyszko et al., who  shed further light on the phenomenon. In patients with normal kidney function but with systemic hypertension and stable coronary artery disease (CAD), serum NGAL levels were significantly higher than in controls, and were also found to parallel creatininemia, hypertension duration, age and predicted GFR. The finding thus suggested the kidney as the main source of systemic NGAL secretion, the latter probably representing an early response to the onset of the hypertensive disease. In view of the underlying CAD it is, however, important to bear circulating neutrophil activity in mind. In a recent study conducted by Paulsson et al. , the levels of systemic IL-8 and NGAL/MMP9 analysed in 13 patients with CAD were significantly higher than those in healthy controls. On comparing the in vivo transmigrated neutrophils of the patients with those of controls, the tendency to secrete NGAL/MMP9 was found to be greater in patients than in controls; this suggests that the pathologic CAD-induced inflammatory reaction may play a role in the progression of local and systemic atherosclerosis, and in determining its complications.
NGAL: a novel cardiovascular risk predictor
The above consideration may be extended to other atherosclerotic-related diseases: for example, a progressive and significant increase occurs in serum NGAL following acute cerebrovascular events (ischaemic stroke, TIA), and persists for up to 1 year [16,17]. Moreover, in their prospective study conducted on 144 subjects with acute cerebrovascular disease, Falke et al. found that NGAL levels, if measured 1–3 days after the event, were a useful tool for stratifying the patients according to their mortality risk during a 4-year follow-up period . As NGAL is one of the proteins with marked hyper-expression following myocardial infarction, in both the necrotic area and the healthy surrounding tissue [10,19], it may be considered an active mediator in post-ischaemic inflammation and remodelling responses. In fact, circulating NGAL levels are markedly higher in patients with acute myocardial infarction (AMI) than in healthy subjects. Moreover, markedly high NGAL levels occurring immediately after AMI were found to be predictive of an unfavourable medium-term outcome. In patients with underlying chronic heart failure (CHF), NGAL levels changed in relation to variations found in clinical and neuro-hormonal markers of cardiac injury. Findings made in murine models confirm that NGAL hyper-expression occurs, both in vivo (damaged myocardium), and in vitro after IL-1 beta (a potent inflammatory cytokine) stimulation in cardiomyocyte cultures . In a recent study  conducted on 46 patients with CHF of different severity, Bolignano et al. reported NGAL levels that were markedly increased with respect to levels in an age-matched healthy control population. These values, moreover, presented a clear correlation with the clinical severity expressed according to the NYHA classification (R = 0·714, P = 0·0001), and with the levels of systolic pressure (R = 0·19, P = 0·05), cholesterolemia (R = 0·19, P = 0·04), albuminemia (R = −0·18, P = 0·05) and ejection fraction (R = −0·19, P = 0·05). The patients were therefore given a 2-year follow-up in order to evaluate the mortality related to cardiovascular events and, on completion of follow-up, the authors observed that the basal levels of NGAL >783 ng mL−1 (optimal cut-off by ROC analysis) were associated with a significantly higher mortality, thus confirming the potential utility of NGAL measurement in staging patients according to cardiovascular risk.
Although it appears reasonable to assume that the damaged myocardium represents the main source of the increase in NGAL levels, other organs may also significantly contribute to NGAL production, consequent to local (ischaemic?) damage following the heart injury itself. An example of this is given in a recent paper : urinary NGAL (uNGAL) excretion, significantly higher in CHF patients than in control subjects, was correlated with levels of creatininemia, GFR, albuminuria and also NT-proBNP, an important biomarker of severity of cardiac impairment.
Likewise, it was found that although patients with CHF secondary to CAD had normal renal function, they presented serum NGAL levels that were markedly higher than those in controls, the increase being independently correlated with the NYHA class and the estimated GFR value .
It is therefore of importance to establish whether an increase in systemic NGAL levels is, albeit partially, a further expression of renal injury occurring during the course of CHF.
Nephrologists are only just discovering the key role played by NGAL in the patho-physiology of different kidney diseases and its important diagnostic and predictive potential in patients with acute or chronic renal damage. Moreover, studies have now revealed that this factor is also involved in cardiovascular disease, and some of its complications. It is therefore important to ask ourselves whether NGAL represents an important, heretofore unknown, link between renal and cardiovascular function. Likewise, for other molecules, such as BNP, NGAL may be included in the panel of biomarkers of the so-called cardio-renal axis. Moreover, the predictive value of NGAL for unfavourable clinical outcomes, recently described in patients with overt heart disease, might also be extended to nephropathic patients. Further, eagerly awaited, studies should clarify these issues and may also confirm that the clinical value of NGAL measurement may be extended beyond the confines of nephrology.
Chair of Nephrology, Department of Internal Medicine, University of Messina, Messina, Italy (D. Bolignano, G. Coppolino, A. Lacquaniti, M. Buemi).