Description
NGAL (neutrophil gelatinase-associated lipocalin) belongs to the lipocalin family of proteins. These are small secreted proteins characterized by their ability i) to bind small hydrophobic molecules in a structurally conserved pocket formed by β-pleated sheet, ii) to bind to specific cell-surface receptors, and iii) to form macromolecular complexes. NGAL has many synonyms: perhaps the most widely used is lipocalin 2 (LCN 2); more recently the name siderocalin has been used to express NGAL's ability to bind bacterial siderophores. As NGAL was first reported as oncogene 24p3 in the mouse, this name is still occasionally used to identify NGAL in other species.
The NCBI reference sequence XM_001927681 from genomic sequencing of Sus scrofa (chromosome 1) suggests that pig NGAL consists of a single disulfide-bridged polypeptide chain of 179 aminoac-id residues with a calculated molecular mass of 20.35 kDa. By analogy with NGAL from other mammalian species, glycosylation is expected to increase its ap-parent molecular mass on SDS-PAGE to about 24-25 kDa. The N-glycosylation site at residue 65 is intact and is in fact conserved in all the NGAL sequences known in placental mammals.
Because pig NGAL has not yet been isolated and no antibodies or immunochemical detection methods have hitherto been developed to detect or measure it, there are no published data on the physiological or pathological functions of this protein in the pig. However, it is likely that some features of the biosynthesis, location and release of pig NGAL will be similar to those of its homologues in other species. The present kit has in fact been developed to extend NGAL research and the use of NGAL as a marker molecule, especially for kidney injury, to the pig. In the following, some pathophysiological features of NGAL in other mammalian species will be mentioned, because similar features may be observed in the pig.
As with other mammalian NGALs, it is likely that in certain situations pig NGAL may be co-expressed and form complexes with matrix metalloproteinase-9 (MMP-9). Pig NGAL may also prove to form homodimers and higher oligomers with itself. As pig NGAL, unlike its human homologue but like all other mammalian NGALs known, does not possess a third cysteinyl residue capable of forming an intermolecular disulffde bridge, any complex formation detected will probably depend on non-covalent forces, at least initially.
In other mammals, NGAL is expressed in neutrophil polymorphonuclear leucocytes and at lower basal levels in various other cell types, tissues or organs. These include epithelial cells of the uterus, mammary gland, lung, spleen, vagina and epididymis. It may be expressed in additional cell types during embryonic development and in response to various stimuli. This applies to certain kidney cells (see below), LPS-stimulated macrophages, dexamethasone-stimulated L-cells, and basic ffbroblast growth factor-stimulated fibroblasts. In the mouse, 24p3 is a type-1 acutephase protein, being secreted by hepatocytes in the in-vivo turpentine model or in response to TNF-α or dexamethasone in vitro.
In disease, NGAL may be moderately raised in bacterial infections because of release from activated neutrophils or from epithelial cells. It may also be moderately raised in atheroma and the ailing myocardium, and in certain epithelial cancers such as those of the mammary gland, gastrointestinal tract or urothelium.
NGAL AND ACUTE KIDNEY INJURY
Apart from the expression of mammalian NGAL that may occur in the above situations, 24p3 undergoes an early and dramatic upregulation in mouse kidney cells after infection with SV40 or polyoma virus. This was the ffrst indication of NGAL's early response to kidney cell injury.
Years later this ffnding was extended to post-ischemic and nephrotoxic kidney injury. The marked upregulation of NGAL mRNA and protein levels in the early post-ischemic rat kidney was detected predominantly in proximal tubule cells and NGAL was easily detected by Western blotting of urine after ischemia reperfusion and cisplatin-induced nephrotoxicity in the mouse.
Urinary NGAL has also been found to be raised in mouse models of diabetic and obstructive nephropathy. However, there is a general paucity of quantitative data on NGAL levels in non-human urine, plasma or serum, whether in the basal state or after kidney injury, and there are as yet no published data from the pig. It is the purpose of the present kit to allow such data to be obtained.
In man, it is now widely recognized that NGAL, whether measured in urine or plasma, is the earliest responding marker of acute kidney injury known, with the further advantage that its response is high and easy to measure while retaining proportionality with the severity of injury. It is therefore to be hoped that the determination of urinary or plasma NGAL may become a convenient end-point in pig models of renal injury, nephrotoxicity and other nephropathies