Upregulation Of Neutrophil Gelatinase-associated Lipocalin Research Articles

Evidence of kidney injury in preeclampsia: Increased maternal and urinary levels of NGAL and KIM-1 and their enhanced expression in proximal tubule epithelial cells.

Proteinuria and glomerular endotheliosis are characteristics of glomerular injury in preeclampsia, a hypertensive disorder in human pregnancy. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are biomarkers of acute/chronic renal tubule injury. To determine if tubule injury occurs in preeclampsia, we determined maternal plasma and urine NGAL and KIM-1 levels and evaluated NGAL and KIM-1 expression in kidney biopsy specimens from women with preeclampsia. Prenatal and postpartum maternal blood and urinary samples were collected from three groups of pregnant women: normal pregnancy (n = 100), preeclampsia (n = 83), and pregnancy complicated with chronic hypertension (n = 20). Plasma and urine levels of NGAL and KIM-1 were measured by ELISA. Kidney biopsy tissue sections from patients with preeclampsia (n = 5) were obtained from Pathology Archives and processed to determine NGAL and KIM-1 expression by immunostaining and high kidney solution images were assessed by electron microscopy (EM). Prenatal plasma and urine levels of NGAL and KIM-1 were significantly higher in preeclamptic than in normal controls, p < 0.01. In normal pregnancy, both plasma and urine levels of NGAL and KIM-1 at 24-48 h after delivery and 6-8 weeks postpartum were relatively comparable to that of antenatal levels. In preeclampsia, urine, but not plasma, NGAL levels were reduced at 6-8 weeks postpartum compared to the antenatal levels, p < 0.05. Although maternal and urine KIM-1 levels were reduced at 6-8 weeks postpartum compared to the antenatal levels in preeclampsia, the levels were still higher than those in normal pregnancy. Positive expression of NGAL and KIM-1 was detected in proximal tubule epithelial cells in kidney tissue specimens from preeclampsia but not in non-pregnancy controls. EM examination showed glomerular and tubular injury in preeclampsia. Our findings of increased maternal levels and urine secretion of NGAL and KIM-1, along with the upregulation of NGAL and KIM-1 expression in tubular epithelial cells in preeclampsia, provide plausible evidence that tubular injury exists in preeclampsia. The higher postpartum NGAL and KIM-1 levels in preeclamptic pregnancies indicate that tubular injury would not resolve within 2-3 months after delivery and suggest that proper follow-up and management of kidney function in women with preeclampsia would be necessary to reduce chronic kidney diseases in those women later in life.

Ameliorative effects of glycine on cobalt chloride‐induced hepato‐renal toxicity in rats

The important roles of liver and kidney in the elimination of injurious chemicals make them highly susceptible to the noxious activities of various toxicants including cobalt chloride (CoCl2 ). This study was designed to investigate the role of glycine in the mitigation of hepato-renal toxicities associated with CoCl2 exposure. Forty-two (42) male rats were grouped as Control; (CoCl2 ; 300 ppm); CoCl2 + Glycine (50 mg/kg); CoCl2 + Glycine (100 mg/kg); Glycine (50 mg/kg); and Glycine (100 mg/kg). The markers of hepatic and renal damage, oxidative stress, the antioxidant defense system, histopathology, and immunohistochemical localization of neutrophil gelatinase associated lipocalin (NGAL) and renal podocin were evaluated. Glycine significantly reduced the markers of oxidative stress (malondialdehyde content and H2 O2 generation), liver function tests (ALT, AST, and ALP), markers of renal function (creatinine and BUN), and decreased the expression of neutrophil gelatinase-associated lipocalin (NGAL) and podocin compared with rats exposed to CoCl2 toxicity without glycine treatment. Histopathology lesions including patchy tubular epithelial necrosis, tubular epithelial degeneration and periglomerular inflammation in renal tissues, and severe portal hepatocellular necrosis, inflammation, and duct hyperplasia were observed in hepatic tissues of rats exposed to CoCl2 toxicity, but were mild to absent in glycine-treated rats. The results of this study clearly demonstrate protective effects of glycine against CoCl2 -induced tissue injuries and derangement of physiological activities of the hepatic and renal systems in rats. The protective effects are mediated via augmentation of total antioxidant capacity and upregulation of NGAL and podocin expression.

Upregulation of Myocardial Neutrophil Gelatinase-Associated Lipocalin during Development of Heart Failure Caused by Volume-Overload and the Effect in Regulating Activity of Matrix Metalloproteinase-9.

In patients with heart failure (HF), circulating neutrophil gelatinase-associated lipocalin (NGAL) level is increased, which is considered to be a predictor of mortality or renal outcomes. The expression of NGAL in the heart and kidney and its role in HF remain unclear. Aortocaval fistula was created in rats as a model of volume overload (VO)-induced HF. During the development of HF, NGAL expression was upregulated in the heart but not in the kidney at both transcriptional and translational levels in the compensatory and HF phases, with a similar level in both phases. Cardiomyocytes were identified as the cell type responsible for NGAL expression. Consistent with the myocardial NGAL expression pattern, the plasma NGAL level was increased in both phases, and the level was not significantly different between both phases. We demonstrated the presence of a matrix metalloproteinase (MMP)-9/NGAL complex in cultured medium of cardiomyocytes isolated from volume-overloaded hearts by gelatin zymography. Formation of MMP-9/NGAL complex was shown to enhance the enzymatic activity of MMP-9. We found that early growth response (Egr)-1 was upregulated in the heart in both compensatory and HF phases. In neonatal cardiomyocytes, Egr-1 overexpression induced the gene expression of NGAL, which was dose-dependently suppressed by an interleukin-1 receptor antagonist. During the development of HF due to VO, NGAL was upregulated in the heart but not in the kidney in both compensatory and HF phases, with a similar expression level. Myocardial NGAL upregulation enhanced MMP-9 activity through formation of the MMP-9/NGAL complex. The expression of myocardial NGAL was regulated by Egr-1.

Hispidulin Ameliorates Endotoxin-Induced Acute Kidney Injury in Mice.

Lipopolysaccharide (LPS) is an endotoxin that plays a crucial role in septic acute kidney injury (AKI). Hispidulin is a natural flavonoid that possesses various biological activities. Recent studies have shown that hispidulin administration alleviates various inflammatory diseases in animal models. This study aimed to investigate the renoprotective effect of hispidulin on LPS-induced AKI. Male C57BL/6 mice were administered LPS (10 mg/kg) with or without hispidulin (50 mg/kg). Hispidulin administration attenuated renal dysfunction, histological alterations, and the upregulation of neutrophil gelatinase-associated lipocalin. This flavonoid also reduced cytokine production and Toll-like receptor 4 expression, inhibited nuclear factor-κB and mitogen-activated protein kinase cascades, and alleviated immune cell infiltration. The oxidation of lipids and DNA was also inhibited by hispidulin administration. This antioxidant effect of hispidulin was associated with the downregulation of NADPH oxidase 4, the activation of catalase and superoxide dismutase activities, and the restoration of glutathione levels. Moreover, hispidulin administration attenuated tubular cell apoptosis by inhibiting caspase-3 pathway. These data suggest that hispidulin ameliorates endotoxin-induced kidney injury by suppressing inflammation, oxidative stress, and tubular cell death.

Impaired Nicotinamide Adenine Dinucleotide Biosynthesis in the Kidney of Chronic Kidney Disease.

Chronic kidney disease (CKD) is a global public health problem with high morbidity and mortality. Decreased nicotinamide adenine dinucleotide (NAD+) levels were found to be associated with aging, cancer, and neurodegenerative and metabolic disorders. However, the alteration of renal NAD+ levels and biosynthesis pathways in CKD is less known. In the present study, we aimed to evaluate renal NAD+ levels and tested the expression of key enzymes in three NAD+ biosynthesis pathways in two different types of CKD rat model. CKD rat models were established by 5/6 nephrectomy (5/6 Nx) and feeding with adenine-containing feed, respectively. Renal function was assessed by serum creatinine (Scr) and blood urea nitrogen (BUN). Renal pathology was evaluated by periodic acid-Schiff (PAS) and Masson’s trichrome staining. The expression of key enzymes in three NAD+ biosynthesis pathways was determined and quantified by Western blot analysis. The results showed CKD rat models were successfully established as evidenced by increased Scr and BUN levels, upregulation of neutrophil gelatinase-associated lipocalin (NGAL), glomerular hypertrophy, and renal fibrosis. Renal NAD+ and NADH content were both declined in two CKD rat models, and NAD+ levels were negatively correlated with Scr and BUN levels in CKD rats. Three key enzymes involved in NAD+ biosynthesis were significantly downregulated in the kidney of both of the two CKD models. They were quinolinate phosphoribosyltransferase (QPRT) in the de novo pathway, nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), and NMNAT3 in the salvage pathway. Moreover, the expression of NAD+-consuming enzymes sirtuin 3 (SIRT3) and CD38 decreased significantly in CKD rats. In conclusion, NAD+ biosynthesis was significantly impaired in CKD, which may attribute to downregulation of QPRT and NMNAT 1/3.

Over expressions of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in human uterine cervical neoplasms enhance tumor invasion

Objective: Accumulating evidence has demonstrated upregulation of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in multiple malignancies which play a critical role in tumor progression. However, up to now little is known about the role played by NGAL in human uterine cervical carcinomas. In this retrospective study we aimed to evaluate the role of tissue expressions of NGAL and KIM-1 in a spectrum of uterine invasive and noninvasive cervical neoplasms. Methods: Immunohistochemical NGAL and KIM-1 expressions were investigated in a total of 107 formalin-fixed, paraffin-embedded uterine cervical tumor specimens and their association with different clinicopathologic parameters was evaluated. Results: In this series, cases with 30 low- and 29 high- grade cervical squamous intraepithelial lesion (SIL), 27 squamous cell carcinoma (SCC), 15 adenosquamous carcinoma (ASC) and 6 adenocarcinoma (ACs) were detected. Positive NGAL expression was detected in only inflammatory cells of cases, whereas KIM-1 expression was detected in tumor cells. Statistically it was determined that the positivity rate of strong NGAL and KIM-1 expression was prominently higher in invasive carcinomas when compared with non-invasive squamous cell neoplasms (P &lt; 0.01). KIM-1 expressions were not detected in any of the few cases with adenocarcinoma. Conclusion: Our findings have showed the presence of a correlation between membranous and cytoplasmic expressions of NGAL and KIM-1 and the invasive characteristics of uterine cervical neoplasms. As a result, expressions of NGAL and KIM-1 may be important in foreseeing the invasion and tumor progression.

Effect of Polyethylene-glycolated Carboxyhemoglobin on Renal Microcirculation in a Rat Model of Hemorrhagic Shock.

Primary resuscitation fluid to treat hemorrhagic shock remains controversial. Use of hydroxyethyl starches raised concerns of acute kidney injury. Polyethylene-glycolated carboxyhemoglobin, which has carbon monoxide-releasing molecules and oxygen-carrying properties, was hypothesized to sustain cortical renal microcirculatory PO2 after hemorrhagic shock and reduce kidney injury. Anesthetized and ventilated rats (n = 42) were subjected to pressure-controlled hemorrhagic shock for 1 h. Renal cortical PO2 was measured in exposed kidneys using a phosphorescence quenching method. Rats were randomly assigned to six groups: polyethylene-glycolated carboxyhemoglobin 320 mg · kg, 6% hydroxyethyl starch (130/0.4) in Ringer's acetate, blood retransfusion, diluted blood retransfusion (~4 g · dl), nonresuscitated animals, and time control. Nitric oxide and heme oxygenase 1 levels were determined in plasma. Kidney immunohistochemistry (histologic scores of neutrophil gelatinase-associated lipocalin and tumor necrosis factor-α) and tubular histologic damages analyses were performed. Blood and diluted blood restored renal PO2 to 51 ± 5 mmHg (mean difference, -18; 95% CI, -26 to -11; P < 0.0001) and 47 ± 5 mmHg (mean difference, -23; 95% CI, -31 to -15; P < 0.0001), respectively, compared with 29 ± 8 mmHg for hydroxyethyl starch. No differences between polyethylene-glycolated carboxyhemoglobin and hydroxyethyl starch were observed (33 ± 7 mmHg vs. 29 ± 8 mmHg; mean difference, -5; 95% CI, -12 to 3; P = 0.387), but significantly less volume was administered (4.5 [3.3-6.2] vs. 8.5[7.7-11.4] ml; mean rank difference, 11.98; P = 0.387). Blood and diluted blood increased the plasma bioavailability of nitric oxide compared with hydroxyethyl starch (mean rank difference, -20.97; P = 0.004; and -17.13; P = 0.029, respectively). No changes in heme oxygenase 1 levels were observed. Polyethylene-glycolated carboxyhemoglobin limited tubular histologic damages compared with hydroxyethyl starch (mean rank difference, 60.12; P = 0.0012) with reduced neutrophil gelatinase-associated lipocalin (mean rank difference, 84.43; P < 0.0001) and tumor necrosis factor-α (mean rank difference, 49.67; P = 0.026) histologic scores. Polyethylene-glycolated carboxyhemoglobin resuscitation did not improve renal PO2 but limited tubular histologic damages and neutrophil gelatinase-associated lipocalin upregulation after hemorrhage compared with hydroxyethyl starch, whereas a lower volume was required to sustain macrocirculation.

An early-screening biomarker of endometrial carcinoma: NGAL is associated with epithelio-mesenchymal transition.

neutrophilgelatinase-associated lipocalin is currently one of the most interesting and enigmatic proteins involved in the development of malignancies. In this study, we found that the expression of neutrophilgelatinase-associated lipocalin was up-regulated in endometrial cancer tissues and cell lines, significantly increased in early-grade ones, suggesting it may serve as a biomarker for early-stage screening for endometrial carcinoma. Moreover, neutrophilgelatinase-associated lipocalin was up-regulated in Ishikawa cells under going epithelio-mesenchymal transition induced by epidermal growth factor (5 ng/ml). Up-regulation of neutrophilgelatinase-associated lipocalin may correlate with the down-regulation of E-cadherin expression, up-regulation of Vimentin expression, enhanced cell migration, invasion and proliferation, which are the typical hallmarks of epithelio-mesenchymal transition processes. neutrophilgelatinase-associated lipocalin may play a dual role during tumorigenetic and developmental processes of endometrial carcinoma. These results suggested neutrophilgelatinase-associated lipocalin to be a potential molecular target in the early diagnosis and treatment of endometrial carcinoma. Further studies are warranted to clarify the molecular mechanisms behind the expression and function of neutrophilgelatinase-associated lipocalin and epithelio-mesenchymal transition.

0268 : Implication of the Neutrophil Gelatinase-Associated Lipocalin (NGAL) from immune cells in aldosterone induced cardiovascular remodeling

Background Inadequate activation of the Mineralocorticoid Receptor (MR) promotes hypertension, inflammation and fibrosis. Neutrophil Gelatinase- Associated Lipocalin (NGAL), a pro-inflammatory/fibrotic glycoprotein, is a direct target of the MR in cardiovascular cells, and is increased in immune cells during inflammation. Recently, we demonstrated that NGAL is crucial for the hypertensive and pro-fibrotic effects of the nephrectomy-aldosteronesalt (NAS) challenge in mice. However, the specific cell types that modulate NGAL production during aldosterone-dependent hypertension are still unknown. The aim of this study was to characterize NGAL expression in mouse immune cells, and to study the effect of MR activation on NGAL and pro-inflammatory cytokines expression in dendritic cells (DCs). Methods Male C57Bl6 mice were submitted to 28 days of NAS challenge (200μg/kg/d). CD4 + , CD8 + T cells, B cells, DCs and Macrophages (Mo) were sorted from spleen. DCs and Mo were cultured from WT and NGAL-KO mice and treated with aldosterone (100nM) for 24h. NGAL and cytokines mRNAs abundance was measured by RT-qPCR. Results NAS treatment induced a selective increase in the recruitment of activated-CD8 + cells, B cells and granulocytes in lymph nodes. NGAL abundance was higher in PBMC, DCs and Mo, and further increased in NAS mice ( In vitro MR activation by aldosterone in DCs induced an upregulation of NGAL and cytokines involved in the adaptive immune response (TGF-β1, IL-23). Interestingly, the absence of NGAL in DCs prevented this increase. Conclusion MR activation and subsequent NGAL induction in DCs could play a pivotal role in the inflammation observed during aldosterone-dependent hypertension. The author hereby declares no conflict of interest

0214 : Mineralocorticoid Receptor (MR) activation induces the expression of Neutrophil Gelatinase-Associated Lipocalin (NGAL) in dendritic cells in vitro and during the aldosterone-dependent hypertension

Inadequate activation of the Mineralocorticoid Receptor (MR) promotes hypertension, inflammation and fibrosis. Neutrophil Gelatinase- Associated Lipocalin (NGAL), a pro-inflammatory/fibrotic glycoprotein, is a target of MR-genomic upregulation in cardiovascular cells, and is increased in immune cells during inflammation. Recently, we have demonstrated that NGAL is crucial for hypertensive effects of aldosterone-salt (NAS) challenge in mice. The specific cell types that modulate the NGAL production during Aldosterone (Aldo)-dependent hypertension are unknown. The aim was to characterize the NGAL expression in mouse immune cells, and to study the effect of MR activation on the NGAL and pro-inflammatory cytokines expression in dendritic cells (DCs). Male C57Bl6 mice were treated in groups Sham and NAS (200μg/kg/d, 28 days). Peripheral blood mononuclear cells (PBMC) were isolated, and CD4 + , CD8 + T cells, B cells, DCs and Macrophages (Mø) were sorted from spleen. DCs and Mø were cultured from WT and NGAL-KO mice and treated with Aldo (100nM) or vehicle for 24hrs. NGAL and cytokines mRNAs abundance was measured by qRT-PCR. NAS mice presented high systolic blood pressure (123 mmHg vs. Sham 101±6 mmHg, p<0.05), cardiac and renal hypertrophy. Additionally, NAS treatment induced a selective increase in the recruitment of activated- CD8 + cells, B cells and granulocytes in lymph nodes. NGAL abundance was higher in PBMC, DCs and Mø, which were further increased in NAS mice (<3-fold vs. Sham, p<0.05 mRNA). In vitro MR activation by Aldo in DCs, but not in Mø, induced an upregulation of NGAL and of cytokines involved in the adaptive immune response: TGF-β1 and IL-23p19 (n=4, p<0.05). Interestingly, the NGAL absence in DCs prevented this overexpression. The MR activation and their subsequent NGAL induction in DCs could play a pivotal role in the inflammation observed during the Aldodependent hypertension.

Neutrophil Gelatinase-Associated Lipocalin Levels in Patients with Thalassemia and Sickle Cell Disease: Correlation with Renal Injury

Background and Aims: Neutrophil gelatinase-associated lipocalin (NGAL) is a protein belonging to the lipocalin superfamily initially found in activated neutrophils, in accordance with its role as an innate antibacterial factor. However, it subsequently was shown that many other types of cells, including in the kidney tubule, may produce NGAL in response to various injuries. The increase in NGAL production and release from tubular cells after harmful stimuli of various kinds may have self-defensive intent based on the activation of specific iron-dependent pathways, which in all probability also represent the mechanism through which NGAL promotes kidney growth and differentiation. NGAL levels clearly correlate with severity of renal impairment, probably expressing the degree of active damage underlying the chronic condition. For all these reasons, NGAL may become one of the most promising next-generation biomarkers in clinical nephrology and beyond. We aimed to investigate the clinical significance of NGAL levels and its correlation with renal function in patients with hemoglobinopathies.Patients and Methods: 117 adult patients with hemoglobinopathies were included in the study divided in 3 groups. Group A: 30 patients with transfusion-dependent thalassemia major (TM); Group B: 29 patients with Non-Transfusion-Dependent Thalassemia (TI) and Group C: 58 patients with HbS/βthal disease, while 20 apparently healthy individuals served as controls (Group D). In patients and controls along with standard blood and urine chemistry. Measurements of serum Cystatin C and NGAL were performed by means of immunonephelometric and immunoenzymatic techniques, (Siemens Healthcare Diagnostics, Liederbach, Germany and R&D Systems, Minneapolis, MN, USA), respectively. Estimated Glomerular Filtration Rate (eGFR) values were calculated with an unadjusted for body surface Cystatin C based equation: eGFR (mL/min)=77.24(Cys C)-1.2623.Results: The main results of the study showed that: a) NGAL levels were significantly higher in all the groups of patients compared to controls: Group A 95.0±45.0µg/L, Group B 139.1±86.1µg/L, Group C 117.8±37.3µg/L vs Group D 50.3±11.3µg/L (p<0.001), b) Cystatin C levels were significantly higher in patients of Group A 0.94±0.34mg/L and Group C 1.04±0.50mg/L compared to controls 0.75±0.09mg/L (p<0.01), while no significant difference was found between Group B 0.72±0.13mg/L and controls (p>0.300), c) NGAL levels and eGFR values (Group A: 96.9±39.8, Group B: 117.0±26.0, Group C: 86.2±27.8 and group D: 109.6±15.0mL/min, respectively) correlated significantly in patients of Group A and Group C (r=-0.739, p<0.001 and r=-0.735, p<0.001, respectively), while NGAL values are independent from eGFR values in patients of Group B.Conclusions: These findings illustrate the tubular-glomerular activation feedback mirrored by NGAL in patients with transfusion-dependent thalassemia major and HbS/βthal disease, who suffer from renal injuries, indicating that tubular damage precedes GFR reduction. Upregulation of NGAL in patients with non-transfusion-dependent thalassemia independently of renal injuries may reflect the compensatory, protective role of NGAL in response to diverse cellular stresses, including inflammation and oxidative stress. However, recent reports have implicated NGAL upregulation as a mechanism that contributes to anemia in the setting of chronic low grade inflammation. In experimental models, systemic and medullary NGAL has been demonstrated to induce inhibition of erythropoiesis through induction of apoptosis and arrest of differentiation of erythroid progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Neutrophil gelatinase-associated lipocalin/lipocalin2, derived from gut crypt cells, exerts intestinal antimicrobial effect via bacterial stimulation of Toll-like receptor 4 and 9

Neutrophil gelatinase-associated lipocalin (NGAL)/ lipocalin2, known as a sensitive biomarker of acute kidney injury, prevents bacterial iron uptake, resulting in the inhibition of its overgrowth [1]. We previously demonstrated that this protein was discharged into gut lumen from crypt cells in septic conditions, and inhibited the growth of Escherichia coli [2]. However, it remains unclear which pathway is associated with the upregulation of NGAL. We therefore designed the present study to reveal whether the pattern-recognition receptor of bacteria, the Toll-like receptor (TLR) family, plays a pivotal role for NGAL secretion from gut crypt cells.

Neutrophil Gelatinase-Associated Lipocalin (NGAL) as a Biomarker for Acute Kidney Injury

Extensive pre-clinical studies have established the biological role, plausibility, and proof of concept of neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute kidney injury (AKI). In AKI, there is a rapid and massive upregulation of NGAL in the kidney. The biological role of NGAL induction is attenuation of apoptosis and an enhanced proliferative response, as well as defense of the urinary system from bacteria. These actions are mediated by chelation of siderophores, removal of iron from extracellular environments and redirection to intracellular sites. In AKI, both urine and plasma NGAL are derived largely from the damaged nephron. NGAL expression in the kidney, urine, and plasma is directly proportional to the duration and severity of kidney injury, temporally correlated with the inciting stimulus, and specific to intrinsic AKI. These findings have now been successfully translated to multiple human studies, and NGAL has emerged as an excellent biomarker in the urine and plasma for early diagnosis, risk stratification, severity prediction, differential diagnosis, and outcomes prediction in several common clinical AKI scenarios. The deployment of standardized clinical platforms has further facilitated the validation of urine and plasma NGAL as an AKI biomarker. Some relevant patents are also summarized in this review.

Neutrophil gelatinase–associated lipocalin is instrumental in the pathogenesis of antibody‐mediated nephritis in mice

The mechanism by which anti-DNA antibodies mediate lupus nephritis has yet to be conclusively determined. Previously, we found that treatment of mesangial cells with anti-DNA antibodies induced high expression of neutrophil gelatinase-associated lipocalin (NGAL), an iron-binding protein up-regulated in response to kidney injury. We undertook this study to determine whether NGAL is instrumental in the pathogenesis of nephritis, is induced as part of repair, or is irrelevant to damage/repair pathways. To investigate the role of NGAL in antibody-mediated nephritis, we induced nephrotoxic nephritis by passive antibody transfer to 129/SyJ and C57BL/6 mice. To determine if NGAL up-regulation is instrumental, we compared the severity of renal damage in NGAL wild-type mice and NGAL-knockout mice following induction of nephrotoxic nephritis. We found that kidney NGAL expression, as well as urine NGAL levels, were significantly increased in mice with nephrotoxic nephritis as compared to control-injected mice. Tight correlations were observed between NGAL expression, renal histopathology, and urine NGAL excretion. NGAL-knockout mice had attenuated proteinuria and improved renal histopathology compared to wild-type mice. Similarly, following nephritis induction, NGAL injection significantly exacerbated nephritis and decreased survival. NGAL induced apoptosis via caspase 3 activation and up-regulated inflammatory gene expression in kidney cells in vitro and when injected in vivo. We conclude that kidney binding of pathogenic antibodies stimulates local expression of NGAL, which plays a crucial role in the pathogenesis of nephritis via promotion of inflammation and apoptosis. NGAL blockade may be a novel therapeutic approach for the treatment of nephritis mediated by pathogenic antibodies, including anti-glomerular basement membrane disease and lupus nephritis.

8523 POSTER Up-regulation of Neutrophil Gelatinase-associated Lipocalin in Oral Squamous Cell Carcinoma – Relation to Cell Differentiation

8523 POSTER Up-regulation of Neutrophil Gelatinase-associated Lipocalin in Oral Squamous Cell Carcinoma – Relation to Cell Differentiation

Up-regulation of neutrophil gelatinase-associated lipocalin in oral squamous cell carcinoma: Relation to cell differentiation

Neutrophil gelatinase-associated lipocalin (NGAL, also known as lipocalin2, LCN2) is a secreted glycoprotein with increased expression in solid tumors. The expression and functions of NGAL in oral cancer, however, remain unclear. We investigated the expression of NGAL in oral cancer tissues and oral cancer cell lines. By immunohistochemical examinations, NGAL expression was strongly up-regulated in well-differentiated OSCC tissues and moderately to weakly up-regulated in moderately to poorly differentiated OSCC tissues. In contrast, NGAL expression was weak or very weak in normal mucosa and leukoplakia. By western blot analysis, NGAL expression levels positively correlated with cell morphology patterns and loss of E-cadherin. In addition, the enzymatic activity of the NGAL/MMP-9 complex significantly correlated with the results obtained by zymographic analysis. In conclusion, NGAL expression is high in well-differentiated cancer, suggesting that NGAL may be a useful diagnostic marker of tumor-cell differentiation.

Induction of Neutrophil Gelatinase-associated Lipocalin Expression by Co-stimulation with Interleukin-17 and Tumor Necrosis Factor-α Is Controlled by IκB-ζ but neither by C/EBP-β nor C/EBP-δ

Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding antimicrobial protein that is up-regulated in epithelial tissues during inflammation. We demonstrated previously that the gene encoding NGAL (LCN2) is strongly up-regulated by interleukin (IL)-1β in an NF-κB-dependent manner but not by tumor necrosis factor (TNF)-α, another potent activator of NF-κB. This is due to an IL-1β-specific synthesis of the NF-κB-binding co-factor IκB-ζ, which is essential for NGAL induction. We demonstrate here that NGAL is strongly induced by stimulation with TNF-α in the presence of IL-17, a pro-inflammatory cytokine produced by the newly discovered subset of CD4+ T helper cells, TH-17. In contrast to the murine NGAL orthologue, 24p3/lipocalin 2, we found no requirement for C/EBP-β or C/EBP-δ for NGAL induction by IL-17 and TNF-α as neither small interfering RNAs against the two C/EBP mRNAs nor mutation of the C/EBP sites in the LCN2 promoter abolished IL-17- and TNF-α-induced up-regulation of NGAL. NGAL induction is governed solely by NF-κB and its co-factor IκB-ζ. This was demonstrated by a pronounced reduction in the amount of NGAL mRNA and NGAL protein synthesized in cells treated with small interfering RNA against IκB-ζ and a total lack of activation of an LCN2 promoter construct with a mutated NF-κB site. As IL-17 stimulation stabilizes the IκB-ζ transcript, we propose a model where TNF-α induces activation and binding of NF-κB to the promoters of both NFKBIZ and LCN2 genes but induce only transcription of IκB-ζ. Co-stimulation with IL-17 leads to accumulation of IκB-ζ mRNA and IκB-ζ protein, which can bind to NF-κB on the LCN2 promoter and thus induce NGAL expression.

Abstract 262: Upregulation of neutrophil gelatinase-associated lipocalin by ErbB2 through NF-κB activation

Abstract ErbB2 (HER2, neu) is a receptor tyrosine kinase overexpressed in about 25% of invasive breast carcinomas. Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted glycoprotein expressed in a variety of cancers including breast carcinomas. NGAL can inhibit erythroid cell production leading to anemia. Anemia usually occurs in cancer patients and negatively impacts quality of life. However, current treatment for cancer-related anemia has potential complications. ErbB2, NGAL, and anemia have all been associated with increased metastasis and poor prognosis in breast cancer patients, although the relationship between ErbB2 and NGAL expression is not clear. Here, using breast cancer cell lines in vitro and transgenic mice carrying the activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV-neu) in vivo, we demonstrate that ErbB2 overexpression leads to NGAL upregulation, which is dependent on nuclear factor kappa B (NF-κB) activity. MMTV-neu transgenic mice developed anemia after tumor onset, and anemia progression could be partially arrested by an NF-κB inhibitor and an ErbB2-targeted therapy. Taken together, upregulation of NGAL by ErbB2 through NF-κB activation is involved in cancer-related anemia, and ErbB2, NF-κB, NGAL pathway may serve as potential therapeutic targets for cancer-related anemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 262.

Upregulation of Neutrophil Gelatinase–Associated Lipocalin by ErbB2 through Nuclear Factor-κB Activation

Abstract ErbB2 (HER2, neu) is a receptor tyrosine kinase overexpressed in about 25% of invasive breast carcinomas. Neutrophil gelatinase–associated lipocalin (NGAL) is a secreted glycoprotein expressed in a variety of cancers, including breast carcinomas. NGAL can inhibit erythroid cell production, leading to anemia. Anemia usually occurs in cancer patients and negatively affects quality of life. However, current treatment for cancer-related anemia has potential complications. ErbB2, NGAL, and anemia have all been associated with increased metastasis and poor prognosis in breast cancer patients, although the relationship between ErbB2 and NGAL expression is not clear. Here, using breast cancer cell lines in vitro and transgenic mice carrying the activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV-neu) in vivo, we show that ErbB2 overexpression leads to NGAL upregulation, which is dependent on nuclear factor-κB (NF-κB) activity. MMTV-neu transgenic mice developed anemia after tumor onset, and anemia progression could be partially arrested by a NF-κB inhibitor and ErbB2-targeted therapy. Taken together, upregulation of NGAL by ErbB2 through NF-κB activation is involved in cancer-related anemia, and the ErbB2, NF-κB, and NGAL pathways may serve as potential therapeutic targets for cancer-related anemia. [Cancer Res 2009;69(24):9163–8]

Urinary NGAL Marks Cystic Disease in HIV-Associated Nephropathy

Nephrosis and a rapid decline in kidney function characterize HIV-associated nephropathy (HIVAN). Histologically, HIVAN is a collapsing focal segmental glomerulosclerosis with prominent tubular damage. We explored the expression of neutrophil gelatinase-associated lipocalin (NGAL), a marker of tubular injury, to determine whether this protein has the potential to aid in the noninvasive diagnosis of HIVAN. We found that expression of urinary NGAL was much higher in patients with biopsy-proven HIVAN than in HIV-positive and HIV-negative patients with other forms of chronic kidney disease. In the HIV-transgenic mouse model of HIVAN, NGAL mRNA was abundant in dilated, microcystic segments of the nephron. In contrast, urinary NGAL did not correlate with proteinuria in human or in mouse models. These data show that marked upregulation of NGAL accompanies HIVAN and support further study of uNGAL levels in large cohorts to aid in the noninvasive diagnosis of HIVAN and screen for HIVAN-related tubular damage.