Neutrophil gelatinase-associated lipocalin, or NGAL [1], belongs to the lipocalin family of proteins. These are typically small secreted proteins characterized by their ability to bind small, hydrophobic molecules in a structurally conserved pocket formed by b-pleated sheet and to form macromolecular complexes. Many lipocalins also bind to specific cell-surface receptors, but so far no NGAL receptor has been identified. NGAL has many synonyms: it is also known as neutrophil lipocalin (NL or HNL for the human form) [2], lipocalin 2, oncogene protein 24p33 or uterocalin [4] in the mouse, and neu-related lipocalin [5] or 25 kDa a2-microglobulin-related protein [6] in the rat. Human NGAL consists of a single disulfide-bridged polypeptide chain of 178 amino-acid residues with a calculated molecular mass of 22 kDa [1], but glycosylation increases its apparent molecular mass to 25 kDa. In neutrophils and urine it occurs as monomer, with a small percentage of dimer and trimer, and it also occurs as a complex with 92-kDa human neutrophil type IV collagenase, also called gelatinase B or matrix metalloproteinase-9 (MMP-9) [1,7].

Human NGAL was originally isolated from the supernatant of activated neutrophils [1], but it is also expressed at a low level in other tissues including the kidney, prostate and epithelia of the respiratory and alimentary tracts [8, 9]. It is strongly expressed in adenomas and inflamed epithelia of the bowel [10], adenocarcinomas of the breast [11] and urothelial carcinomas [12].

Because of its small molecular size and resistance to degradation, NGAL is readily excreted and detected in the urine, both in its free form and in complex with MMP-9. Urinary levels correlate with plasma or serum levels whatever the cause of increased NGAL production (own data), but particularly high urinary levels can be expected when it is released directly into the urine by the kidney tubules or urothelial carcinomas. It is uncertain how far NGAL-MMP-9 complexes from sources remote from the urinary tract are excreted as such into the urine or reform in the urine after independent excretion of NGAL and MMP-9 [7].

While the functions of NGAL are not fully understood, it appears to be upregulated in cells under "stress", e.g. from infection, inflammation, ischemia or neoplastic transformation, or in tissues undergoing involution, such as the postpartum mouse uterus and mammary glands on weaning. In relation to a possible antibacterial role, it binds enterobactin and other siderophores, depriving the microorganisms of Fe3+, an important nutritional requirement [13]. Its complex formation with MMP-9 appears to protect MMP-9 enzymatic activity from degradation [7]. The upregulation of NGAL in involuting tissues has led to the postulation of a role in apoptosis, but it appears more likely that NGAL is associated with a survival response [14]. This seems to be so in the kidney, where NGAL-siderophore-iron complex rescues the mouse kidney from ischemic injury [15].

NGAL in inflammation or infection

NGAL is released from the secondary granules of activated neutrophils [1] and plasma levels rise in inflammatory or infective conditions, especially in bacterial infections [16]. Thus the level of NGAL in plasma or serum has been proposed as a marker of infection. However, as levels of NGAL may also be raised in neoplastic conditions and renal disorders independently of any infective process, this proposed application should be treated with caution. NGAL may also be raised in infections in patients with an uncountably low number of neutrophils due to leukemia or treated leukemia, showing that the source of the raised NGAL in infections is not only the neutrophils. Indeed, serum NGAL levels correlate very poorly with the neutrophil count in unselected critically ill patients (own data). In view of the possible release of NGAL from the kidney (see below) when sepsis becomes severe enough to affect it, data on NGAL in sepsis should by reassessed to take this into account.

NGAL and neoplasia

The various types of cancer in which NGAL may be upregulated (often with MMP-9) have been referred to above. This has been shown by its expression in tumor cells and its high urinary levels, both in the free form and complexed with MMP-9 [7]. Indeed, it has been proposed that urinary NGAL-MMP-9 complexes may serve as a marker of disease status for breast cancer patients [17]. Plasma levels have not usually been measured in these cases.

NGAL and the kidney

Even before NGAL had been isolated from human neutrophils, its mouse homologue 24p3 was known to be expressed by kidney cells and to undergo an early, dramatic upregulation (14- to 20-fold) in response to SV 40 viral infection [18]. A similar early and dramatic upregulation was later observed in rat proximal tubule cells after ischemia-reperfusion injury [19], and raised plasma levels of NGAL were found to be strongly correlated with decreased renal function in patients with renal damage due to systemic vasculitis [20]. The results for renal ischemia-reperfusion injury were subsequently confirmed and extended to nephrotoxic agents [21, 22, 23]. It has been suggested that urinary NGAL levels may serve as an early marker for ischemic renal injury in children after cardiopulmonary bypass [24]. Raised urinary and serum NGAL levels have also been observed in patients with established renal failure (own data) and patients with functioning renal grafts also showed urinary levels that were sufficiently raised to be readily detectable by Western blotting [12]. It is therefore apparent that a large variety of renal disorders are associated with raised plasma and urinary levels of NGAL. While plasma and urinary NGAL levels are closely correlated in acute conditions, it is to be expected that urinary NGAL levels will be particularly high after ischemic renal injury severe enough to result in acute renal failure, acute tubular necrosis or acute tubulo-interstitial nephropathy. However, the use of urinary NGAL as a potential marker for these conditions is subject to the proviso that the presence of concurrent conditions that are independently associated with raised NGAL levels must be taken into account.

NGAL quantification

In many of the above studies on NGAL, the protein has been quantified by immunoblotting. However, some research groups have developed specific ELISAs, based on polyclonal and/or monoclonal antibodies to NGAL [16, 25]. The recent development of a commercial sandwich ELISA that measures NGAL in urine, plasma or serum, should make it easier for clinical investigators to assess the potential of this interesting molecule, either as a diagnostic marker for different pathologies, or as a marker of disease progress or response to treatment. Some NGAL may be released from neutrophils during the preparation of serum, making it generally preferable to use plasma, and urine should be centrifuged to remove neutrophils in cases of urinary tract infection.

NGAL as a potential diagnostic marker

It is apparent that a variety of independent pathologies are associated with raised levels of urinary or plasma NGAL. Therefore the finding of a raised level cannot be independently diagnostic of any one of these pathologies. Other information on the patient must be taken into account in order to assess the significance of the result. As other quite effective marker molecules are available to assess inflammatory and infective states (e.g. procalcitonin as a sepsis marker), and as more specific markers are available for many of the cancers in which NGAL is raised, it seems likely that the interest in NGAL will center chiefly on its role as a marker of kidney damage, where its early and marked response to the insult (within 2 hours [24]) makes it one of the best markers hitherto studied. It is to be expected that serial rather than isolated single measurements of NGAL, whether in urine or plasma, will provide the most useful data on patients with several concurrent pathologies.

The author

Lars Otto Uttenthal, M.A., D.Phil., B.M., B.Ch., M.R.C.P. (UK)

Scientific Director, AntibodyShop A/S

This article was originally published in Clinical Laboratory international

www.cli-online.com

References

1.Kjeldsen L, Johnsen AH, Sengelov H, Borregaard N (1993) Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem 268:10425-10432.

2. Xu SY, Carlson M, Engstrom A, Garcia R, Peterson CG, Venge P (1994) Purification and characterization of a human neutrophil lipocalin (HNL) from the secondary granules of human neutrophils. Scand J Clin Lab Invest 54:365-376.

3.Flower DR, North AC, Attwood TK (1991) Mouse oncogene protein 24p3 is a member of the lipocalin protein family. Biochem Biophys Res Commun 180:69-74.

4. Liu Q, Ryon J, Nilsen-Hamilton M (1997) Uterocalin: a mouse acute phase protein expressed in the uterus around birth. Mol Reprod Dev 46:507-14.

5.Stoesz SP, GouldMN (1995) Overexpression of neu-related lipocalin (NRL) in neu-initiated but not ras or chemically initiated rat mammary carcinomas. Oncogene 11:2233-2241.

6. Triebel S, Blaser J, Reinke H, Tschesche H (1992) A 25 kDa alpha 2-microglobulin-related protein is a component of the 125 kDa form of human gelatinase. FEBS Lett 314:386-388.

7.Yan L, Borregaard N, Kjeldsen L, Moses MA (2001) The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL. J Biol Chem 276:37258-37265.

8. Cowland JB, Borregaard N (1997) Molecular characterization and pattern of tissue expression of the gene for neutrophil gelatinase-associated lipocalin from humans. Genomics 45:17-23.

9. Friedl A, Stoesz SP, Buckley P, Gould MN (1999) Neutrophil gelatinase-associated lipocalin in normal and neoplastic human tissues. Cell type-specific pattern of expression. Histochem J 31:433-441.

10. Nielsen BS, Borregaard N, Bundgaard JR, Timshel S, Sehested M, Kjeldsen L (1996) Induction of NGAL synthesis in epithelial cells of human colorectal neoplasia and inflammatory bowel diseases. Gut 38:414-420.

11. Stoesz SP, Friedl A, Haag JD, Lindstrom MJ, Clark GM, Gould MN (1998) Heterogeneous expression of the lipocalin NGAL in primary breast cancers. Int J Cancer 79:565-572.

12.Monier F, Surla A, Guillot M, Morel F (2000) Gelatinase isoforms in urine from bladder cancer patients. Clin Chim Acta 299:11-23.

13. Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN, Strong RK (2002) The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 10:1033-1043.

14. Tong Z, Wu X, Ovcharenko D, Zhu J, Chen CS, Kehrer JP (2005) Neutrophil gelatinase-associated lipocalin as a survival factor. Biochem J 391:441-448.

15. Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J, Schmidt-Ott KM, Chen X, Li JY, Weiss S, Mishra J, Cheema FH, Markowitz G, Suganami T, Sawai K, Mukoyama M, Kunis C, D'Agati V, Devarajan P, Barasch J (2005) Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest 115:610-621.

16. Xu SY, Pauksen K, Venge P (1995) Serum measurements of human neutrophil lipocalin (HNL) discriminate between acute bacterial and viral infections. Scand J Clin Lab Invest 55:125-131.

17. Fernandez CA, Yan L, Louis G, Yang J, Kutok JL, Moses MA (2005) The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients. Clin Cancer Res 11:5390-5395.

18. Hraba-Renevey S, Turler H, Kress M, Salomon C, Weil R (1989) SV40-induced expression of mouse gene 24p3 involves a post-transcriptional mechanism. Oncogene 4:601-608.

19. Matthaeus T, Schulze-Lohoff E, Ichimura T, Weber M, Andreucci M, Park KM, Alessandrini A, Bonventre JV (2001) Co-regulation of neutrophil gelatinase-associated lipocalin and matrix metalloproteinase-9 in the postischemic rat kidney. J Am Soc Nephrol 12:787A.

20. Ohlsson S, Wieslander J, Segelmark M (2003) Increased circulating levels of proteinase 3 in patients with anti-neutrophilic cytoplasmic autoantibodies-associated systemic vasculitis in remission. Clin Exp Immunol 131:528-535.

21. Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, Barasch J, Devarajan P (2003) Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 14:2534-2543.

22. Amin RP, Vickers AE, Sistare F, Thompson KL, Roman RJ, Lawton M, Kramer J, Hamadeh HK, Collins J, Grissom S, Bennett L, Tucker CJ, Wild S, Kind C, Oreffo V, Davis JW 2nd, Curtiss S, Naciff JM, Cunningham M, Tennant R, Stevens J, Car B, Bertram TA, Afshari CA (2004) Identification of putative gene based markers of renal toxicity. Environ Health Perspect 112:465-479.

23. Mishra J, Mori K, Ma Q, Kelly C, Barasch J, Devarajan P (2004) Neutrophil gelatinase-associated lipocalin: a novel early urinary biomarker for cisplatin nephrotoxicity. Am J Nephrol 24:307-315.

24. Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, Ruff SM, Zahedi K, Shao M, Bean J, Mori K, Barasch J, Devarajan P (2005) Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 365(9466):1231-1238.

25. Kjeldsen L, Koch C, Arnljots K, Borregaard N (1996) Characterization of two ELISAs for NGAL, a newly described lipocalin in human neutrophils. J Immunol Methods 198:155-164.