Neutrophil gelatinase-associated lipocalin as the real-time indicator of active kidney damage
2007; Elsevier BV; Volume: 71; Issue: 10 Linguagem: Inglês
10.1038/sj.ki.5002165
ISSN1523-1755
Autores Tópico(s)Trauma, Hemostasis, Coagulopathy, Resuscitation
ResumoNeutrophil gelatinase-associated lipocalin (Ngal, 24p3, SIP24, lipocalin 2, or siderocalin) was originally purified from neutrophils, but with unknown function. Recently, it was identified that Ngal activates nephron formation in the embryonic kidney, is rapidly and massively induced in renal failure and possesses kidney-protective activity. We would like to propose that blood, urine, and kidney Ngal levels are the real-time indicators of active kidney damage, rather than one of many markers of functional nephron number (as Forest Fire Theory). Ngal is a novel iron-carrier protein exerting pleiotropic actions including the upregulation of epithelial marker E-cadherin expression, opening an exciting field in cell biology. Neutrophil gelatinase-associated lipocalin (Ngal, 24p3, SIP24, lipocalin 2, or siderocalin) was originally purified from neutrophils, but with unknown function. Recently, it was identified that Ngal activates nephron formation in the embryonic kidney, is rapidly and massively induced in renal failure and possesses kidney-protective activity. We would like to propose that blood, urine, and kidney Ngal levels are the real-time indicators of active kidney damage, rather than one of many markers of functional nephron number (as Forest Fire Theory). Ngal is a novel iron-carrier protein exerting pleiotropic actions including the upregulation of epithelial marker E-cadherin expression, opening an exciting field in cell biology. Neutrophil gelatinase-associated lipocalin (Ngal) belongs to the lipocalin superfamily, which are secreted or cytosolic proteins with barrel-like structure, carrying hydrophobic ligands (such as fatty acids, retinoids, and pheromones) in their core pocket.1.Flower D.R. North A.C. Sansom C.E. The lipocalin protein family: structural and sequence overview.Biochim Biophys Acta. 2000; 1482: 9-24Crossref PubMed Scopus (697) Google Scholar Recent works have elucidated the pathophysiological roles of lipocalins in energy homeostasis. Retinol-binding protein 4 impairs insulin signaling in muscles causing insulin resistance,2.Yang Q. Graham T.E. Mody N. et al.Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes.Nature. 2005; 436: 356-362Crossref PubMed Scopus (1672) Google Scholar whereas lipocalin-type (or brain) prostaglandin D synthase (β-trace) inhibits insulin-stimulated proliferation of vascular smooth muscle cells resulting in protection against atherosclerosis.3.Ragolia L. Palaia T. Hall C.E. et al.Accelerated glucose intolerance, nephropathy, and atherosclerosis in prostaglandin D2 synthase knock-out mice.J Biol Chem. 2005; 280: 29946-29955Crossref PubMed Scopus (105) Google Scholar Mice with a null mutation in adipocyte fatty acid-binding protein 4 or adipocyte protein 2 become obese under high-fat diet but do not develop insulin resistance.4.Hotamisligil G.S. Johnson R.S. Distel R.J. et al.Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein.Science. 1996; 274: 1377-1379Crossref PubMed Scopus (651) Google Scholar Little is known about the involvement of the ligands in these metabolic activities of lipocalins. Goetz et al.5.Goetz D.H. Holmes M.A. Borregaard N. et al.The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.Mol Cell. 2002; 10: 1033-1043Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar carried out an epoch work of the X-ray crystallography for recombinant human Ngal protein expressed in Escherichia coli. They demonstrated that the ligand for Ngal is siderophore. Siderophores are a diverse group of small (1 kDa or less) non-peptide iron (Fe3+)-binding chemicals produced in bacteria, fungi, and plants and their mammalian version remains to be identified.6.Raymond K.N. Dertz E.A. Kim S.S. Enterobactin: an archetype for microbial iron transport.Proc Natl Acad Sci USA. 2003; 100: 3584-3588Crossref PubMed Scopus (642) Google Scholar, 7.Barasch J. Mori K. Cell biology: iron thievery.Nature. 2004; 432: 811-813Crossref PubMed Scopus (51) Google Scholar, 8.Takahashi M. Nakanishi H. 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Holmes M.A. Borregaard N. et al.The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.Mol Cell. 2002; 10: 1033-1043Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar Probably outside the pocket, Ngal binds with gelatinase B (matrix metalloproteinase-9 or type IV collagenase) and with hepatocyte growth factor and modulates their activity.10.Kjeldsen L. Johnsen A.H. Sengelov H. et al.Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase.J Biol Chem. 1993; 268: 10425-10432Abstract Full Text PDF PubMed Google Scholar, 11.Yan L. Borregaard N. Kjeldsen L. et al.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. 2001; 276: 37258-37265Crossref PubMed Scopus (570) Google Scholar, 12.Gwira J.A. Wei F. Ishibe S. et al.Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro.J Biol Chem. 2005; 280: 7875-7882Crossref PubMed Scopus (136) Google Scholar Mammalian metanephric mesenchyme and ureteric bud coordinate a complicated interaction to develop themselves into mature nephrons.13.Mori K. Yang J. Barasch J. Ureteric bud controls multiple steps in the conversion of mesenchyme to epithelia.Semin Cell Dev Biol. 2003; 14: 209-216Crossref PubMed Scopus (33) Google Scholar Barasch et al.14.Barasch J. Yang J. Ware C.B. et al.Mesenchymal to epithelial conversion in rat metanephros is induced by LIF.Cell. 1999; 99: 377-386Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar established an organ culture system, where isolated rat metanephric mesenchyme converts into glomeruli and proximal tubules by stimulation with condition media prepared from a mouse ureteric bud cell line. Leukemia-inhibitory factor and Ngal were the epithelial inducers purified at the protein level using this assay.14.Barasch J. Yang J. Ware C.B. et al.Mesenchymal to epithelial conversion in rat metanephros is induced by LIF.Cell. 1999; 99: 377-386Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar,15.Yang J. Goetz D. Li J.Y. et al.An iron delivery pathway mediated by a lipocalin.Mol Cell. 2002; 10: 1045-1056Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar Genetic inactivation of Ngal (by Flo et al.) or leukemia-inhibitory factor signal transducer gp130 does not result in agenesis of the kidney, indicating a high redundancy in nephrogenesis pathways.14.Barasch J. Yang J. Ware C.B. et al.Mesenchymal to epithelial conversion in rat metanephros is induced by LIF.Cell. 1999; 99: 377-386Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar,16.Flo T.H. Smith K.D. Sato S. et al.Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.Nature. 2004; 432: 917-921Crossref PubMed Scopus (1319) Google Scholar Yang et al.15.Yang J. Goetz D. Li J.Y. et al.An iron delivery pathway mediated by a lipocalin.Mol Cell. 2002; 10: 1045-1056Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar have shown that mouse Ngal protein secreted from cultured ureteric bud cells possesses nephron-inducing activity and can also bind iron. Surprisingly, Ngal–bacterial siderophore–iron complex has much stronger activity than Ngal–siderophore (without iron) and apo-Ngal (without siderophore),17.Li J.Y. Ram G. Gast K. et al.Detection of intracellular iron by its regulatory effect.Am J Physiol Cell Physiol. 2004; 287: C1547-C1559Crossref PubMed Scopus (38) Google Scholar indicating the first example of 'siderophore-binding proteins,' in any species, dependent on the presence of iron for the biological activity.7.Barasch J. Mori K. Cell biology: iron thievery.Nature. 2004; 432: 811-813Crossref PubMed Scopus (51) Google Scholar Recapture of genetic program of embryo is often observed in tissue injuries. Devarajan et al.18.Devarajan P. Update on mechanisms of ischemic acute kidney injury.J Am Soc Nephrol. 2006; 17: 1503-1520Crossref PubMed Scopus (801) Google Scholar,19.Supavekin S. Zhang W. Kucherlapati R. et al.Differential gene expression following early renal ischemia/reperfusion.Kidney Int. 2003; 63: 1714-1724Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar have analyzed animal models of acute renal failure to screen biomarkers useful in the clinical settings and to understand the molecular mechanisms of kidney injury, to begin with by use of microarray. Within a few hours, Ngal mRNA is highly upregulated after kidney injury, such as renal ischemia-reperfusion and cisplatin nephropathy, where Ngal induction precedes the elevation of classical markers for kidney damage: serum creatinine, urinary N-acetyl glucosaminidase, β 2-microglobulin levels.19.Supavekin S. Zhang W. Kucherlapati R. et al.Differential gene expression following early renal ischemia/reperfusion.Kidney Int. 2003; 63: 1714-1724Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar,20.Mishra J. Ma Q. Prada A. et al.Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury.J Am Soc Nephrol. 2003; 14: 2534-2543Crossref PubMed Scopus (1420) Google Scholar Furthermore, Mori et al.9.Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (790) Google Scholar and other groups have reported that Ngal protein is abundantly accumulated in the blood, urine, and renal proximal and distal tubules in acute renal failure of humans: in cases associated with renal ischemia (sepsis, hypovolemia, and heart failure), nephrotoxin (antibiotics, cisplatin, bisphosphonate, nonsteroidal anti-inflammatory drugs, radio-contrast, and hemoglobinuria), kidney-parenchymal damage (glomerulonephrits, minimal change disease, focal segmental glomerulosclerosis, and diabetic nephropathy), hemolytic-uremic syndrome and posttransplant rejection. Mishra et al.21.Mishra J. Dent C. Tarabishi R. et al.Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery.Lancet. 2005; 365: 1231-1238Abstract Full Text Full Text PDF PubMed Scopus (1942) Google Scholar presented the clinical usefulness of blood and urinary Ngal as extremely early markers of acute kidney disease. As early as 1–2 h after the cardiopulmonary bypass surgery in children and in adults (with average bypass time of 2–3 h), the Ngal levels are dramatically and specifically elevated in those who are going to develop acute renal failure (diagnosed by more than 50% rise in the serum creatinine levels a few days later).21.Mishra J. Dent C. Tarabishi R. et al.Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery.Lancet. 2005; 365: 1231-1238Abstract Full Text Full Text PDF PubMed Scopus (1942) Google Scholar,22.Wagener G. Jan M. Kim M. et al.Association between increases in urinary neutrophil gelatinase-associated lipocalin and acute renal dysfunction after adult cardiac surgery.Anesthesiology. 2006; 105: 485-491Crossref PubMed Scopus (477) Google Scholar Acute worsening of chronic renal failure may be also predicted by elevated urinary Ngal levels (Nickolas T et al., submitted). In general, to evaluate the severity of renal failure, at least two aspects should be considered (Figure 1): the ratio of functional versus atrophic nephrons (or the results of kidney injury) and the severity of on-going damage. Analogy may hold true for forest fire (Forest Fire Theory). We propose here that induction of Ngal expression is a real-time indicator of active renal injury. Functional significance of Ngal upregulation in renal failure was investigated by Mori9.Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (790) Google Scholar and Mishra et al.,23.Mishra J. Mori K. Ma Q. et al.Amelioration of ischemic acute renal injury by neutrophil gelatinase-associated lipocalin.J Am Soc Nephrol. 2004; 15: 3073-3082Crossref PubMed Scopus (445) Google Scholar who analyzed separate sets of animals. Single intraperitoneal or subcutaneous injection of recombinant Ngal protein into mice (Figure 2) significantly ameliorates kidney damage after renal ischemia-reperfusion injury, if Ngal is given before ischemia or 1 h after reperfusion. Berger et al.24.Berger T. Togawa A. Duncan G.S. et al.Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury.Proc Natl Acad Sci USA. 2006; 103: 1834-1839Crossref PubMed Scopus (355) Google Scholar generated Ngal knockouts and found no difference in renal damage at 24 h after renal ischemia-reperfusion compared with wild-type mice. Although Ngal mRNA upregulation is a rapid response, the speed and amount of endogenous Ngal induction may not be sufficient to show significant protection in this setting. Experiments in more chronic and milder models may give different results. Neutrophils,25.Cowland J.B. Borregaard N. The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules.J Leukoc Biol. 1999; 66: 989-995Crossref PubMed Scopus (197) Google Scholar monocytes/macrophages,26.Meheus L.A. Fransen L.M. Raymackers J.G. et al.Identification by microsequencing of lipopolysaccharide-induced proteins secreted by mouse macrophages.J Immunol. 1993; 151: 1535-1547PubMed Google Scholar and adipocytes27.Wang Y. Lam K.S. Kraegen E.W. et al.Lipocalin-2 is an inflammatory marker closely associated with obesity, insulin resistance, and hyperglycemia in humans.Clin Chem. 2007; 53: 34-41Crossref PubMed Scopus (426) Google Scholar are cells with abundant Ngal expression (Figure 3). Importantly, immature neutrophils (myelocytes and metamyelocytes) have high expression level of Ngal mRNA, whereas mature neutrophils/granulocytes in the circulation have lost the mRNA but contain large amount of Ngal protein,25.Cowland J.B. Borregaard N. The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules.J Leukoc Biol. 1999; 66: 989-995Crossref PubMed Scopus (197) Google Scholar making it impossible to determine the involvement of neutrophil-derived Ngal in tissue injury by in situ mRNA hybridization. Ngal expression is highly induced not only in kidney injury, but also in epithelial inflammation of intestine,28.Playford R.J. Belo A. Poulsom R. et al.Effects of mouse and human lipocalin homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin on gastrointestinal mucosal integrity and repair.Gastroenterology. 2006; 131: 809-817Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar skin and airway,29.Cowland J.B. Sorensen O.E. Sehested M. et al.Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha.J Immunol. 2003; 171: 6630-6639Crossref PubMed Scopus (299) Google Scholar and in bacterial infection16.Flo T.H. Smith K.D. Sato S. et al.Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.Nature. 2004; 432: 917-921Crossref PubMed Scopus (1319) Google Scholar and cancer.30.Hanai J. Mammoto T. Seth P. et al.Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells.J Biol Chem. 2005; 280: 13641-13647Crossref PubMed Scopus (108) Google Scholar Ngal inducers so far identified in vitro are IL-1β, tissue necrosis factor α, lipopolysaccharide, basic fibroblast growth factor, prostaglandin F2 α, phorbol ester, dexamethasone, retinoic acid, serum, and hypoxia.20.Mishra J. Ma Q. Prada A. et al.Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury.J Am Soc Nephrol. 2003; 14: 2534-2543Crossref PubMed Scopus (1420) Google Scholar, 26.Meheus L.A. Fransen L.M. Raymackers J.G. et al.Identification by microsequencing of lipopolysaccharide-induced proteins secreted by mouse macrophages.J Immunol. 1993; 151: 1535-1547PubMed Google Scholar, 29.Cowland J.B. Sorensen O.E. Sehested M. et al.Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha.J Immunol. 2003; 171: 6630-6639Crossref PubMed Scopus (299) Google Scholar, 31.Liu Q. Nilsen-Hamilton M. Identification of a new acute phase protein.J Biol Chem. 1995; 270: 22565-22570Crossref PubMed Scopus (217) Google Scholar Ngal exerts a broad range of biological activities (Figure 3). The coexistence of siderophore and iron is required not only for mesenchymal–epithelial transition of embryonic kidney17.Li J.Y. Ram G. Gast K. et al.Detection of intracellular iron by its regulatory effect.Am J Physiol Cell Physiol. 2004; 287: C1547-C1559Crossref PubMed Scopus (38) Google Scholar and oncogene Ras-transformed epithelial cells by Hanai et al.,30.Hanai J. Mammoto T. Seth P. et al.Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells.J Biol Chem. 2005; 280: 13641-13647Crossref PubMed Scopus (108) Google Scholar but also for kidney protection from renal failure by Mori et al.9.Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (790) Google Scholar In contrast, Ngal–siderophore complex without iron17.Li J.Y. Ram G. Gast K. et al.Detection of intracellular iron by its regulatory effect.Am J Physiol Cell Physiol. 2004; 287: C1547-C1559Crossref PubMed Scopus (38) Google Scholar (and potentially apo-Ngal)32.Devireddy L.R. Gazin C. Zhu X. et al.A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake.Cell. 2005; 123: 1293-1305Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar can chelate iron from cells and iron deprivation is the mechanism for apoptosis of pro-B cells,32.Devireddy L.R. Gazin C. Zhu X. et al.A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake.Cell. 2005; 123: 1293-1305Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar,33.Devireddy L.R. Teodoro J.G. Richard F.A. et al.Induction of apoptosis by a secreted lipocalin that is transcriptionally regulated by IL-3 deprivation.Science. 2001; 293: 829-834Crossref PubMed Scopus (329) Google Scholar and for inhibition of bacterial growth and erythropoiesis.5.Goetz D.H. Holmes M.A. Borregaard N. et al.The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.Mol Cell. 2002; 10: 1033-1043Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar, 16.Flo T.H. Smith K.D. Sato S. et al.Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.Nature. 2004; 432: 917-921Crossref PubMed Scopus (1319) Google Scholar, 34.Miharada K. Hiroyama T. Sudo K. et al.Lipocalin 2 functions as a negative regulator of red lood cell production in an autocrine fashion.FASEB J. 2005; 19: 1881-1883PubMed Google Scholar Various other activities of Ngal are increasingly reported.12.Gwira J.A. Wei F. Ishibe S. et al.Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro.J Biol Chem. 2005; 280: 7875-7882Crossref PubMed Scopus (136) Google Scholar,28.Playford R.J. Belo A. Poulsom R. et al.Effects of mouse and human lipocalin homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin on gastrointestinal mucosal integrity and repair.Gastroenterology. 2006; 131: 809-817Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar Induction of mesenchymal–epithelial transition by Ngal is associated with upregulation of epithelial marker E-cadherin expression, but this is a slow process. In metanephric mesenchyme, morphologically distinct epithelia is observed only 7–10 days after addition of Ngal–siderophore–iron complex (unpublished observation).15.Yang J. Goetz D. Li J.Y. et al.An iron delivery pathway mediated by a lipocalin.Mol Cell. 2002; 10: 1045-1056Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar By contrast, epithelia induced by leukemia-inhibitory factor can be observed within 4–5 days.13.Mori K. Yang J. Barasch J. Ureteric bud controls multiple steps in the conversion of mesenchyme to epithelia.Semin Cell Dev Biol. 2003; 14: 209-216Crossref PubMed Scopus (33) Google Scholar,14.Barasch J. Yang J. Ware C.B. et al.Mesenchymal to epithelial conversion in rat metanephros is induced by LIF.Cell. 1999; 99: 377-386Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar In Ras-transformed mammary cells, E-cadherin protein accumulation was evaluated 48 h after treatment with the complex.30.Hanai J. Mammoto T. Seth P. et al.Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells.J Biol Chem. 2005; 280: 13641-13647Crossref PubMed Scopus (108) Google Scholar Hanai et al.30.Hanai J. Mammoto T. Seth P. et al.Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells.J Biol Chem. 2005; 280: 13641-13647Crossref PubMed Scopus (108) Google Scholar demonstrated that treatment with Ngal–siderophore–iron suppresses Raf, mitogen-activated protein kinase (MEK)-1/2, extracellular signal-regulated kinase (ERK)-1/2 pathway of mitogen-activated protein kinase and inhibits E-cadherin phosphorylation, causing decrease in E-cadherin degradation. Not only treatment with Ngal–siderophore–iron complex but also transfection with cDNA and infection with adenovirus encoding Ngal results in mesenchymal–epithelial transition of transformed cells, implying that fetal calf serum or cells themselves provide mammalian siderophores. In the case of renal ischemia-reperfusion injury, phosphorylation of MEK1/2 and ERK1/2 is biphasic (transient activation within 30 min, followed by long-term activation for several days) and the levels of their phosphorylation are not necessarily parallel with the severity of renal injury,35.Park K.M. Kramers C. Vayssier-Taussat M. et al.Prevention of kidney ischemia/reperfusion-induced functional injury MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction.J Biol Chem. 2002; 277: 2040-2049Crossref PubMed Scopus (158) Google Scholar suggesting that suppression of this pathway is not the major mechanism for Ngal-mediated kidney protection. Administration of Ngal is ineffective if given 2 h after reperfusion,9.Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (790) Google Scholar implying that Ngal is preventing the early injuries (in part by upregulating a protective enzyme heme oxygenase-1)9.Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (790) Google Scholar rather than stimulating the recovery process. In healthy adult kidneys, Ngal36.Hvidberg V. Jacobsen C. Strong R.K. et al.The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake.FEBS Lett. 2005; 579: 773-777Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar and other lipocalins (whose sizes are 17–43 kDa) including retinol-binding protein 4, α1-microglobulin37.Leheste J.R. Rolinski B. Vorum H. et al.Megalin knockout mice as an animal model of low molecular weight proteinuria.Am J Pathol. 1999; 155: 1361-1370Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar and probably also liver-type fatty acid-binding protein 1 and prostaglandin D synthase are freely filtrated in the glomeruli, bound to multiligand scavenger-receptor megalin (expressed abundantly and specifically on the brush borders of proximal tubules) and taken up efficiently by endocytosis (as well as albumin and β2-microglobulin).38.Moestrup S.K. Verroust P.J. Megalin- and cubilin-mediated endocytosis of protein-bound vitamins, lipids, and hormones in polarized epithelia.Annu Rev Nutr. 2001; 21: 407-428Crossref PubMed Scopus (152) Google Scholar Insufficient tubular reabsorption (owing to specific saturation in the endocytic pathway or general malfunctioning of proximal tubules) should contribute in part to urinary Ngal. Devireddy et al.32.Devireddy L.R. Gazin C. Zhu X. et al.A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake.Cell. 2005; 123: 1293-1305Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar identified brain-type organic cation transporter as Ngal receptor in pro-B cells. Iron-free siderophore-like activity (which assists the binding of Ngal and Fe3+) is detectable in the normal urine of humans, mice, rats, and dogs.9.Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (790) Google Scholar,39.Schmidt-Ott K.M. Mori K. Kalandadze A. et al.Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia.Curr Opin Nephrol Hypertens. 2006; 15: 442-449Crossref PubMed Scopus (206) Google Scholar Tear lipocalin, a major protein component specifically found in human tears, also binds with siderophores.40.Fluckinger M. Haas H. Merschak P. et al.Human tear lipocalin exhibits antimicrobial activity by scavenging microbial siderophores.Antimicrob Agents Chemother. 2004; 48: 3367-3372Crossref PubMed Scopus (162) Google Scholar Lipocalin 12, found specifically in mouse epididymis, is also structurally related to Ngal and may have binding capacity for siderophores.41.Holmes M.A. Paulsene W. Jide X. et al.Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration.Structure. 2005; 13: 29-41Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar Ngal induction is a rapid event detectable within a few hours, characterizing Ngal as one of immediate early genes or acute-phase reactants such as IL-6 and C-reactive protein. Fold induction of Ngal mRNA and protein is log order of magnitude, reaching 1000-fold in most severe cases of renal injury.9.Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (790) Google Scholar,39.Schmidt-Ott K.M. Mori K. Kalandadze A. et al.Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia.Curr Opin Nephrol Hypertens. 2006; 15: 442-449Crossref PubMed Scopus (206) Google Scholar Therefore, normalization for urinary creatinine level is not necessary to evaluate urinary Ngal.21.Mishra J. Dent C. Tarabishi R. et al.Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery.Lancet. 2005; 365: 1231-1238Abstract Full Text Full Text PDF PubMed Scopus (1942) Google Scholar Indeed, a patient with urinary Ngal level of 40 μg/ml died of multiorgan failure 12 h after urine collection9.Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (790) Google Scholar (Mori K et al., unpublished observation). There are a number of siderohpre-dependent and -independent activities reported for Ngal. The role of endogenously expressed Ngal in these activities should be better verified using Ngal-deficient mice. During renal ischemia-reperfusion injury, the liver also starts to express Ngal mRNA.39.Schmidt-Ott K.M. Mori K. Kalandadze A. et al.Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia.Curr Opin Nephrol Hypertens. 2006; 15: 442-449Crossref PubMed Scopus (206) Google Scholar Therefore, the source of Ngal protein in the blood and urine during renal injury can be a complex from the kidney, liver, and white blood cells (in the injured tissues and in circulation). Tissue-specific disruption of Ngal gene will make it possible to investigate what is the predominant source of Ngal, as a biomarker or as a protective mechanism for renal failure. Purification and identification of mammalian siderophores and to learn their metabolism and regulation are also important steps. The reported method to recognize siderophores is only available for iron-free molecules.9.Mori K. Lee H.T. Rapoport D. et al.Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.J Clin Invest. 2005; 115: 610-621Crossref PubMed Scopus (790) Google Scholar A new way to detect iron-loaded siderophores must be invented. Characterization of Ngal receptors, their downstream intracellular signaling, and subcellular localization of events will help elucidate the detailed molecular mechanism underlying the actions of Ngal, siderophores, and iron. Ngal is a very unique protein endowed with iron-carrying activity and diagnostic and therapeutic utilities for renal failure, which is acquiring an explosion of attention by researchers and clinicians beyond nephrology.
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