Neutrophil Gelatinase-associated Lipocalin mRNA Levels Research Articles

Expression of NGAL-specific cells and mRNA levels correlate with inflammation in the salivary gland, and its overexpression in the saliva, of patients with primary Sjögren’s syndrome

Salivary gland involvement is a characteristic feature of primary Sjögren’s syndrome (pSS), where tissue destruction is mediated by infiltrating immune cells, and may be accompanied by the presence of adipose tissue. Optimally diagnosing this multifactorial disease requires the incorporation of additional routines. Screening for disease-specific biomarkers in biological fluid could be a promising approach to increase diagnostic accuracy. We have previously investigated disease biomarkers in saliva and tear fluid of pSS patients, identifying Neutrophil gelatinase-associated lipocalin (NGAL) as the most upregulated protein in pSS. In the current study, we aimed to explore for the first time NGAL expression at the site of inflammation in the pSS disease target organ. Immunohistochemical staining was conducted on minor salivary gland biopsies from 11 pSS patients and 11 non-SS sicca subjects, targeting NGAL-specific cells. Additional NGAL/PNAd double staining was performed to study NGAL expression in high endothelial venules, known as specialised vascular structures. Moreover, NGAL mRNA expression was measured utilising quantitative real-time polymerase chain reaction (qRT-PCR) on minor salivary gland biopsies from 15 pSS patients and 7 non-SS sicca individuals that served as tissue controls. Our results demonstrated NGAL expression in acinar and ductal epithelium within the salivary gland of pSS patients, where significantly greater levels of acinar NGAL were observed in pSS patients (p < .0018) when compared to non-SS subjects. Also, acinar expression positively correlated with focus score values (r 2 = 0.54, p < .02), while ductal epithelial expression showed a negative such correlation (r 2 = 0.74, p < .003). Some PNAD+ endothelial venules also expressed NGAL. An increase in NGAL staining with increased fatty replacement was also observed in pSS patients. Concurringly, a 27% increase in NGAL mRNA levels were also detected in the minor salivary glands of pSS patients when compared to non-SS tissue control subjects. In conclusion, there is a positive association between increase in NGAL expression and inflammation in the pSS disease target organ, which also coincides with its previously demonstrated upregulation in the saliva of pSS patients. Additional functional analyses are needed to better understand the immunological implications of this potential biomarker.

Interleukin-1β as a driver of renal NGAL production

Neutrophil gelatinase-associated lipocalin (NGAL) is increasingly regarded as a biomarker of acute kidney injury, or kidney injury in general, but the stimuli responsible for its production are incompletely understood. This study tested the relationship between the pro-inflammatory cytokine interleukin-1β (IL-1β) and both circulating and renal NGAL, using chronic subcutaneous infusion of IL-1β in mice and tissue culture of renal cell lines. Following a 14-day subcutaneous infusion of vehicle or IL-1β (10ng/h) in male C57Bl/6 mice, a striking positive correlation (r2=0.94; P<0.01) was observed between plasma IL-1β and NGAL concentrations. NGAL was markedly increased in the kidneys of IL-1β-infused mice compared with vehicle-treated mice, both at the protein and mRNA level, indicating increased local as well as systemic production of NGAL. Immunohistochemical staining revealed prominent increases of NGAL in the proximal tubular epithelium of IL-1β infused mice. These effects occurred in the absence of overt renal injury, with plasma creatinine concentration not significantly different between groups. Further showing that IL-1β has a direct effect on NGAL production by tubular epithelial cells, exposure of a proximal tubular cell line (HK-2 cells) and a cortical collecting duct principal cell line (mpkCCD cells) to IL-1β for 24h produced a significant increase of NGAL mRNA levels (>30-fold). These data indicate IL-1β serves as a powerful stimulus for renal production of NGAL.

The expression and significance of matrix metalloproteinases and neutrophil gelatinase-associated lipocalin in serum and placental tissue of preeclampsia maternal

Objective To investigate expression and significance of matrix metalloproteinase-9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL) in serum and placental tissue of preeclampsia maternal.Methods 74 cases of preeclampsia pregnant women as research subjects,which 39 cases of severe preeclampsia,35 cases of mild preeclampsia,selected 30 cases of normal late maternal as the control group.Three groups of maternal serum levels of MMP-9 and of NGAL were detected by enzyme-linked immunosorbent assay.RT-PCR was used to test placenta MMP-9 mRNA,and NGAL mRNA level.Results Preeclampsia maternal serum levels of MMP-9 and NGAL level were significantly higher than those of severe preeclampsia pregnant women serum level of MMP-9 and NGAL were significantly higher than mild preeclampsia (t =3.127,2.846,3.508,3.284,all P ＜ 0.05).Preeclampsia placentas of MMP-9 mRNA and NGAL mRNA levels were significantly lower than the control group (t =-2.842,-2.728,-3.532,-3.276,(a)ll P ＜ 0.05),severe preeclampsia placentas of MMP-9 and NGAL were significantly higher than mild preeclampsia(t =2.732,2.741,all P ＜ 0.05),severe preeclampsia placentas of MMP-9 mRNA and NGAL mRNA levels were significantly lower than in patients with mild preeclampsia (t =-2.681,-2.587,all P ＜ 0.05).Preeclampsia maternal serum levels of MMP-9 and NGAL was positively correlated with placental tissue mRNA levels of MMP-9 and NGAL mRNA level was positively correlated (P ＜ 0.05).Conclusion Preeclampsia maternal serum MMP-9 and NGAL were significantly elevated,placental MMP-9 mRNA and NGAL mRNA levels were significantly decreased,suggested that they may be involved in the pathogenesis of preeclampsia. Key words: Pre-eclampsia; Matrix metalloproteinases; Neutrophil gelatinase-associated lipocalin

749: The impact of obesity on the disease course of preeclampsia

disease course of preeclampsia Emily Miller, Alan Peaceman Northwestern University, Maternal Fetal Medicine, Chicago, IL OBJECTIVE: Obesity is a well-established risk factor for the development of preeclampsia. Various possible causal etiologies have been proposed to explain this relationship. Insofar as these pathways toward preeclampsia may differ from the pathophysiology of preeclampsia in non-obese parturients, the clinical ramifications of the disease may differ. This study was designed to assess whether there is a difference in the clinical presentation of preeclampsia between obese and non-obese women. STUDY DESIGN: This is a retrospective cohort study of women who received a diagnosis of pre-eclampsia. Women were categorized as obese (pre-pregnancy BMI 30.0 kg/m2) or non-obese (pre-pregnancy BMI 30.0 kg/m2). Clinical data pertaining to their preeclampsia were abstracted from the chart, including gestational age at delivery, neonatal birth weight, blood pressure ranges, and lab values (creatinine, liver function tests, uric acid, platelet count, level of proteinuria). RESULTS: Of the 874 women included, 531 (61%) were obese. There were no statistically significant differences in maternal age, gestational age at delivery, BUN or creatinine levels, or amount of proteinuria between the two groups. While there was no difference in highest systolic blood pressure recorded, the non-obese cohort had a higher median maximum diastolic blood pressure [101 mm Hg (IQR 94107) v 98 mm Hg (IQR 92-104), p 0.001]. In addition, the non-obese group had a significantly increased risk of having an elevated AST (47% vs 32%, p 0.001), an elevated ALT (25% vs 18%, p 0.014), and thrombocytopenia (45% vs 30%). The non-obese women were twice as likely to develop both a platelet count of less than 100,000 and LFTs more than double of normal, consistent with the diagnosis of HELLP syndrome (6.7% v 2.8%, p 0.006). CONCLUSION: Of the women that were identified with preeclampsia, the majority were obese. However women with obesity and preeclampsia had lab abnormalities less frequently and also lower peak diastolic blood pressures. This suggests that the pathophysiology of preeclampsia may differ between obese and non-obese women. 750 Preeclampsia (PE) enhances NGAL expression at fetal-maternal interface: role of iron and hypoxia Errol Norwitz, Serkalem Tadesse, Zhonghua Tang, Seth Guller Tufts University School of Medicine, Obstetrics & Gynecology, Boston, MA, Tufts Medical Center, Obstetrics & Gynecology, Division of Maternal-Fetal Medicine, Boston, MA, Yale University School of Medicine, Obstetrics, Gynecology and Reproductive Sciences, New Haven, CT OBJECTIVE: PE is associated with both a substantial elevation in iron deposition and an aberrant maintenance of hypoxia at the maternalfetal interface. Indeed, increases in serum iron and iron transporting proteins such as neutrophil gelatinase-associated lipocalin (NGAL) have been proposed as potential antecedents of clinical symptoms in PE. This study investigates for the first time the possible role of iron, hypoxia, and NGAL in the etiology of PE using cultures of cytotrophoblasts (CTs) and decidual cells (DCs). STUDY DESIGN: To assess NGAL protein expression in PE, we analyzed placental tissues from normotensive (GA: 31 [30-32] wks) vs preeclamptic patients (GA: 31 [28-32] wks) by immunohistochemistry (IHC) and H-score analysis. CTs and DCs were isolated and purified from normal term placentas delivered by cesarean (n 8). To mimic the increased iron level and hypoxic environment in PE, cells were supplemented with iron citrate (25M) and exposed to normoxic (NMX; 20% O2) or hypoxic conditions (HPX; 2% O2 for 24h. NGAL protein and mRNA levels were measured by western blot and RTqPCR. RESULTS: (1) IHC revealed elevated NGAL protein expression in both extravillous trophoblast (EVT) and decidua in PE vs control (H-score: meanSEM): 375.175.7 vs 44.18.8 (p 0.003) and 286.352.7 vs 80.613.9 (p 0.001), respectively; (2) In CTs, iron supplementation increased NGAL expression under HPX (33.4-fold, p 0.0019), but not NMX; (3) In CTs, iron supplementation increased NGAL mRNA levels 3-fold under NMX but 175-fold under HPX (60-fold higher compared with HPX alone). Thus, HPX increased NGAL mRNA and protein expression in CTs in an iron-dependent manner. CONCLUSION: These data demonstrate that enhanced NGAL expression in EVT and decidua is a novel marker of PE. Further, our in vitro studies suggest that CT NGAL levels are elevated in PE through an iron-dependent, HPX-driven mechanism that is mediated at the level of gene transcription.

Neutrophil gelatinase-associated lipocalin as a survival factor

NGAL (human neutrophil gelatinase-associated lipocalin) and its mouse analogue 24p3 are members of the lipocalin family of small secreted proteins. These proteins are up-regulated in a number of pathological conditions, including cancers, and may function as transporters of essential factors. Although previous publications have suggested that 24p3 has pro-apoptotic functions, other data are more suggestive of a survival function. The current study was designed to determine whether NGAL is pro- or anti-apoptotic. Apoptosis induced in human adenocarcinoma A549 cells by the 5-lipoxygenase-activating-protein inhibitor MK886, or several celecoxib-derived PDK1 (phosphoinositide-dependent kinase 1) inhibitors that are devoid of cyclo-oxygenase-2 inhibitory activity, was accompanied by a dose- and time-dependent increase of NGAL mRNA levels, as was reported previously with 24p3. A similar induction of NGAL mRNA was observed in human breast cancer MCF7 cells treated with MK886, indicating this was not a cell-specific effect. Treatment of A549 cells with up to 150 mug/10(6) cells of purified recombinant NGAL protein had no effect on viability, whereas antisera against the full-length NGAL protein induced apoptosis in these cells. The stable overexpression of NGAL in A549 cells had no effect on proliferation or viability. However, the cell death induced by a PDK1 inhibitor was reduced by 50% in NGAL-overexpressing cells. Decreasing NGAL mRNA and protein expression with siRNA (small interfering RNA) in A549 cells increased the toxicity of a PDK1 inhibitor by approx. 45%. These data indicate that, although the induction of NGAL correlates with apoptosis, this induction represents a survival response. Because NGAL is a secreted protein, it may play an extracellular role in cell defence against toxicants and/or facilitate the survival of the remaining cells.