Human Neutrophil Gelatinase-associated Lipocalin Research Articles

Relationship between difference of preoperative and cardiopulmonary bypass mean arterial pressure, and acute kidney injury in cardiac surgical patients undergoing valve surgery.

Modifiable and non-modifiable factors contribute to development and progression of acute kidney injury (AKI) during cardiac surgery. We hypothesized that, the difference between preoperative mean arterial pressure (MAP) and the average mean arterial pressure maintained on cardiopulmonary bypass (CPB) would be strongly predictive of AKI. We also measured plasma Neutrophil gelatinase-associated lipocalin (NGAL), to establish its association with cardiac surgery associated-AKI (CSA-AKI). One hundred and twelve high-risk patients undergoing valve, and valve plus coronary artery bypass grafting (CABG) surgery under cardiopulmonary bypass (CPB) were included in this study. Delta mean arterial pressure (MAP) was calculated as the difference between the average of pre-operative and on-bypass MAP, and blood was sampled for NGAL levels, at baseline, and 6-h after CPB. Detailed data collection was done, tabulating most of the factors which might influence development of post-operative cardiac surgery associated-AKI (CSA-AKI). To define CSA-AKI within the first 24-h post-operatively, the Kidney Disease Improving Global Outcomes (KDIGO) classification was used. Out of 112 patients, 44 (39.3%) developed CSA-AKI postoperatively. With an ROC analysis cut-off of delta MAP of more than 25.67 mmHg, 46.4% patients developed post-operative AKI, and the average CPB flows which were 1.8 ± 0.2 were not contributory to the development of early CSA-AKI. In our study, ELISA test for human NGAL was performed on serum samples, and the estimated cut-off value of 1661ng/mL was found to be significantly associated with early CSA-AKI. Delta MAP and CPB flows are not related to early post-surgical CSA-AKI in cases with prior high-risk elements. However, baseline serum NGAL, as well as its percent change during the early post-surgical period independently predicted the development of CSA-AKI. This implies that, there may be patients with a higher pre-operative preponderance to develop this complication, which could actually be delineated by the use of serum NGAL estimations at baseline.

Evaluation of human neutrophil gelatinase-associated lipocalin level in asthma-COPD overlap syndrome of elderly patients

Aims: Human neutrophil gelatinase-associated lipocalin (NGAL) is a protein considered as a noninvasive prognostic biomarker in Asthma-COPD overlap syndrome (ACO). In this study we aimed to show the usability of NGAL as a biomarker in ACO characterized with airway inflammation in over 65 years old. Methods: Three patient groups (Group I: COPD, n: 50, Group II: Asthma, n:50, Group III: ACO, n:50) and a healhty non smoker group (Group IV, n:50) are taken into study. The serum NGAL levels of groups are compared. In Group I, II, III the level of serum NGAL is compared with the age, duration of smoking (pocket/year), number of attack per year, respiratory function values, serum C reaktive protein (CRP, mg/dL), serum IgE (IU/mL) and blood neutrophil-to-lymphocyte rate and atopy. Results: In patients having ACO when the maximum level of serum exist, it is observed that serum NGAL level is increased as the number of attacks is increased (p<0.002). On the other hand in the patients having ACO there was also positive correlation between NLR and serum NGAL level (p<0.05). Conclusion: The increase in serum NGAL levels in chronic inflammatory lung diseases suggests that serum NGAL levels can be used as a biomarker to assess inflammation severity

Slowly Relaxing Local Structure Analysis of 15N Relaxation from the Proteins p50 and Human Neutrophil Gelatinase-Associated Lipocalin: New Insights into the Dynamic Structure of β-Barrel Proteins

Nuclear magnetic resonance relaxation analysis is a powerful method for studying the internal mobility of proteins. We have developed for analysis the slowly relaxing local structure (SRLS) approach. SRLS is general in its nature in several respects, including the tensorial representation of the physical quantities comprising the dynamic model. By controlling tensor symmetry, a broad range of systems can be treated with physical relevance, typically with data-fitting techniques. In simple limits, SRLS yields the traditional model-free (MF) method. In the present context, MF simplicity means featuring the highest possible tensor symmetry. This renders MF-based data-fitting susceptible to the usage of fit parameters, yielding physically ill-defined results. A typical candidate is the Rex term, devised to represent ms-μs motions but often invoked by the fitting scheme just to improve the statistics. Here, we consider two such cases using the N-H bond as probe and the proteins p50 and human neutrophil gelatinase-associated lipocalin as paradigm systems. We illustrate the harm caused by the physically unjustified involvement of Rex in MF-based 15N relaxation analysis. Then, we show that forgoing the usage of Rex, SRLS analysis of the very same experimental data provides interesting new information.

Biomarkers in Primary Focal Segmental Glomerulosclerosis in Optimal Diagnostic-Therapeutic Strategy.

Focal segmental glomerulosclerosis (FSGS) involves podocyte injury. In patients with nephrotic syndrome, progression to end-stage renal disease often occurs over the course of 5 to 10 years. The diagnosis is based on a renal biopsy. It is presumed that primary FSGS is caused by an unknown plasma factor that might be responsible for the recurrence of FSGS after kidney transplantation. The nature of circulating permeability factors is not explained and particular biological molecules responsible for inducing FSGS are still unknown. Several substances have been proposed as potential circulating factors such as soluble urokinase-type plasminogen activator receptor (suPAR) and cardiolipin-like-cytokine 1 (CLC-1). Many studies have also attempted to establish which molecules are related to podocyte injury in the pathogenesis of FSGS such as plasminogen activator inhibitor type-1 (PAI-1), angiotensin II type 1 receptors (AT1R), dystroglycan(DG), microRNAs, metalloproteinases (MMPs), forkheadbox P3 (FOXP3), and poly-ADP-ribose polymerase-1 (PARP1). Some biomarkers have also been studied in the context of kidney tissue damage progression: transforming growth factor-beta (TGF-β), human neutrophil gelatinase-associated lipocalin (NGAL), malondialdehyde (MDA), and others. This paper describes molecules that could potentially be considered as circulating factors causing primary FSGS.

Assessment of Renal Function Status in Steady-State Sickle Cell Anaemic Children Using Urine Human Neutrophil Gelatinase-Associated Lipocalin and Albumin:Creatinine Ratio

Introduction: Sickle cell anaemia is characterized by defective haemoglobin synthesis and is associated with both endocrine and metabolic alterations. The effects of this clinical condition on kidney function are multifactorial and often begin early in childhood. This study aims to assess renal function in children with sickle cell anaemia using urine albumin:creatinine ratio (ACR) and urine human neutrophil gelatinase-associated lipocalin (NGAL). Methods: This case-control study was conducted on 200 children aged 5–15 years in 2 tertiary hospitals in South West Nigeria: 150 were of haemoglobin S genotype and 50 were of haemoglobin A genotype. Serum urea, creatinine, urine albumin, and NGAL were assayed by known standard methods. eGFR, urine ACR, and urine NGAL/creatinine ratio (urine NCR) were calculated. Results: The weight, height, BMI, systolic blood pressure, plasma urea, plasma creatinine, and spot urine creatinine of the HbS genotype children were significantly lower compared to that of the HbA genotype children. The eGFR, spot urine albumin, and urine ACR were significantly higher in the HbS group compared to the HbA group. There was no significant difference in the spot urine NGAL and urine NCR between the 2 groups, though both were higher in the HbS group compared to the HbA group. Conclusions: Kidney injury probably starts early in childhood in sickle cell individuals as indicated by the higher urine ACR detected in them. We infer that urine NGAL and uNCR are not sensitive markers of kidney disease especially in young sickle cell individuals possibly because of the hyperfiltration present at this age.

Role of neutrophil gelatinase-associated lipocalin in renal cell carcinoma.

Human neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein present in a wide variety of tissues and cell types. It exists as a monomer of 25 kDa, a homodimer of 45 kDa or a heterodimer of 135 kDa (disulfide bound to latent matrix metalloproteinase-9). NGAL is considered the biochemical gold standard for the early diagnosis of acute kidney injury and has attracted much attention as a diagnostic biomarker. NGAL has controversial (i.e. both beneficial and detrimental) effects on cellular processes associated with tumor development, such as cell proliferation, survival, migration, invasion and drug resistance. Therefore, the present review aimed at clarifying the role of NGAL in renal cell carcinoma (RCC). Relevant studies of NGAL and RCC were searched in PubMed and relevant information about the structure, expression, function and mechanism of NGAL in RCC were summarized. Finally, the following conclusions could be drawn from the literature: i) NGAL can be detected in cancer tissues, serum and urine of patients with RCC; ii) NGAL is not a suitable diagnostic marker for early screening of RCC; iii) NGAL expression may be used to predict the prognosis of patients with RCC; and iv) Further research on NGAL may be helpful to decrease sunitinib resistance and find new treatment strategies for RCC.

The Role of Serum Neutrophil Gelatinase-associated Lipocalin in the Early Diagnosis of Nephropathy in Patients with Acute Alcohol Poisoning

AIM: In our study, we assessed the possibility of using the serum neutrophil gelatinase-associated lipocalin (NGAL) for the early detection of kidney damage in patients with acute alcohol poisoning (AAP).
 METHODS: The study included 89 patients and 30 healthy donors. All participants in the study were mostly represented by men (90%) aged between 20 and 40 years. The influence of alcohol poisoning severity was also taken into account in the study. The Human NGAL ELISA Kit was used for the quantitative detection of serum NGAL. We also evaluated the main laboratory indicators of kidney functions, including eGFR (calculated according to serum creatinine).
 RESULTS: We did not find a correlation between blood alcohol concentration and serum NGAL level; also, alcohol poisoning severity did not affect the NGAL values. The results of our study showed the possibility of using the serum NGAL in patients with AAP to detect the preclinical stage of reduced renal function, until the moment when it can be diagnosed with using only serum creatinine.
 CONCLUSION: We propose to consider an increase in eGFR together with an increase in serum NGAL in this group of patients as a stage, preceding nephropathy, even in the absence of clinical and laboratory signs of impaired renal function.

Early identification of tubulointerstitial renal injury in hypertensive patients: a comparative evaluation of the urine biomarkers NGAL and KIM 1

Objective. The aim of the study was a comparative assessment of urine NGAL (neutrophil gelatinase associated lipocalin) and KIM‑1 (kidney injury molecule‑1) as biomarkers of early detection of tubulointerstitial renal damage in patients with arterial hypertension (HTN) without concomitant diabetes mellitus and kidney disease.Design and methods. The study involved 52 patients of both sexes (17 man and 35 women, mean age 60,2 ± 6,54 years) hospitalized for aggravation of HTN 1–3 degrees, which were divided into 3 groups with glomerular filtration rate (GFR) > 90 ml/min/1,73 m 2 (n = 21), 60–90 ml/min/1,73 m 2 (n = 19) and < 60 ml/min/1,73 m 2 (n = 12). In a separate group, patients with albuminuria in the range 10–29 mg/g urine creatinine were isolated. The comparison group consisted of 15 normotensive individuals (6 men and 9 women, mean age 49,8 ± 9,68 years) with no obvious signs of renal, cardiovascular or other diseases. All patients and controls underwent examination, and albumin, NGAL (Human NGAL ELISA kit) and KIM‑1 (Human KIM‑1 Immunoassay ELISA) levels were assessed in the first morning urine portion.Results. In the group of patients with GFR 67,7 ± 4,92 ml/min/1,73 m 2 (mildly reduced kidney function) and in the group of patients with GFR 55,8 ± 4,44 ml/min/1,73 m 2 (renal dysfunction) urine NGAL level increased by 3,43 (р < 0,05) and 3,92 times (р < 0,05), respectively, as compared with the control group. The urine concentration of KIM‑1 increased by 2,06 times (р < 0,05) was observed only in patients with renal dysfunction. Based on the ROC analysis AUC for the urine NGAL level in patients with mildly reduced kidney function is 0,78 (95 % confidence interval, 95 %CI: 0,70–0,86; p < 0,001), specificity — 89,6 %, sensitivity — 66,7 %. Similar indicators for KIM‑1 are 0,72 (95 % CI: 0,64–0,80; p < 0,001), specificity — 85,3 %, sensitivity — 16,7 %. In the group of patients with mildly reduced kidney function the urine level of NGAL correlated with the HTN severity (r = 0,8944, р < 0,001) and its duration (r = 0,8953, р < 0,001).Conclusions. Urine NGAL as compared with KIM‑1 is a more sensitive biomarker, which indicates tubulointerstitial injury in HTN patients without diabetes mellitus and kidney diseases in the early stage of impairment renal function.

Early Diagnosis and Prognostic Value of Acute Kidney Injury in Critically Ill Patients.

Background and objectives: In hospitalized children, acute kidney injury (AKI) remains to be a frequent and serious condition, associated with increased patient mortality and morbidity. Identifying early biomarkers of AKI and patient groups at the risk of developing AKI is of crucial importance in current clinical practice. Specific human protein urinary neutrophil gelatinase-associated lipocalin (uNGAL) and interleukin 18 (uIL-18) levels have been reported to peak specifically at the early stages of AKI before a rise in serum creatinine (sCr). Therefore, the aim of our study was to determine changes in uNGAL and uIL-18 levels among critically ill children and to identify the patient groups at the highest risk of developing AKI. Materials and methods: This single-center prospective observational study included 107 critically ill children aged from 1 month to 18 years, who were treated in the Pediatric Intensive Care Unit (PICU) of Lithuanian University of Health Sciences Hospital Kauno Klinikos from 1 December 2013, to 30 November 2016. The patients were divided into two groups: those who did not develop AKI (Group 1) and those who developed AKI (Group 2). Results: A total of 68 (63.6%) boys and 39 (36.4%) girls were enrolled in the study. The mean age of the patients was 101.30 ± 75.90 months. The mean length of stay in PICU and hospital was 7.91 ± 11.07 and 31.29 ± 39.09 days, respectively. A total of 32 (29.9%) children developed AKI. Of them, 29 (90.6%) cases of AKI were documented within the first three days from admission to hospital. In all cases, AKI was caused by diseases of non-renal origin. There was a significant association between the uNGAL level and AKI between Groups 1 and 2 both on day 1 (p = 0.04) and day 3 (p = 0.018). Differences in uNGAL normalized to creatinine in the urine (uCr) (uNGAL/uCr) between the groups on days 1 and 3 were also statistically significant (p = 0.007 and p = 0.015, respectively). uNGAL was found to be a good prognostic marker. No significant associations between uIL-18 or Uil-18/uCr and development of AKI were found. However, the uIL-18 level of >69.24 pg/mL during the first 24 h was associated with an eightfold greater risk of AKI progression (OR = 8.33, 95% CI = 1.39–49.87, p = 0.023). The AUC for uIL-18 was 73.4% with a sensitivity of 62.59% and a specificity of 83.3%. Age of <20 months, Pediatric Index of Mortality 2 (PIM2) score of >2.5% on admission to the PICU, multiple organ dysfunction syndrome with dysfunction of three and more organ systems, PICU length of stay more than three days, and length of mechanical ventilation of >five days were associated with a greater risk of developing AKI. Conclusions: Significant risk factors for AKI were age of <20 months, PIM2 score of >2.5% on admission to the PICU, multiple organ dysfunction syndrome with dysfunction of 3 and more organ systems, PICU length of stay of more than three days, and length of mechanical ventilation of > five days. uNGAL was identified as a good prognostic marker of AKI. On admission to PICU, uNGAL should be measured within the first three days in patients at the risk of developing AKI. The uIL-18 level on the first day was found to be as a biomarker predicting the progression of AKI.

Revisiting Neutrophil Gelatinase-Associated Lipocalin (NGAL) in Cancer: Saint or Sinner?

Human neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein present in a wide variety of tissues and cell types. NGAL exists as a 25 kDa monomer, a 46 kDa homodimer (the most abundant form in healthy subjects) and a 130 kDa disulfide-linked heterodimer bound to latent matrix metalloproteinase-9. Dysregulated expression of NGAL in human malignancies suggests its value as a clinical marker. A growing body of evidence is highlighting NGAL’s paradoxical (i.e., both beneficial and detrimental) effects on cellular processes associated with tumor development (proliferation, survival, migration, invasion, and multidrug resistance). At least two distinct cell surface receptors are identified for NGAL. This review (i) summarizes our current knowledge of NGAL’s expression profiles in solid tumors and leukemias, and (ii) critically evaluates the beneficial and detrimental activities of NGAL having been documented in a diverse range of cancer-derived cell lines. A better understanding of the causal relationships between NGAL dysregulation and tumor development will require a fine analysis of the molecular aspects and biological role(s) of NGAL both in primary tumors and at different stages of disease. Having an accurate picture of NGAL’s contribution to tumor progression is a prerequisite for attempting to modulate this protein as a putative therapeutic target.

The correlation between the human neutrophil gelatinase-associated lipocalin and the extravascular lung water index being analysised in patients with acute lung injury

Objective To study whether there is a correlation between the content of neutrophil gelatinase-asociated lipocalin(NGAL) and extravascular lung water index(EVLWI) in patients with acute lung injury(ALI) induced by septic shock . Methods We prospectively collected 110 patients with septic shock from December 2013 to June 2015, who had already completed the initial fluid resuscitation within 6 hours according to the guide. In accordance with whether combined with ALI, patients were divided into two groups: non ALI group was A group (n=61), and ALI group was B group (n=49). Baseline information, EVLWI and other indexes of hemodynamic monitored by PICCO of each group was collected prior to admission in intensive care unit. EVWI and the content of NGAL in alveolar lavage fluid, which detected by ELISA, were gathered after the initial fluid resuscitation. Results There were no significant differences at the baseline level between the two groups (P>0.05). The risk factors of EVLWI in patients with septic shock were analyzed by multivariate linear regression analysis.The risk factors contained fluid balance, NGAL, cardiac index(CI), Global end diastolic volume index(GEDI), human albumin(ALB), globulin(GLO). The results showed that EVLWI had linear regression with NGAL and GEDI. The equation of linear regression were Y(NGAL)=2.017+ 0.035X, and Y(GEDI)=-0.671+ 0.004X, respectively.Correlation analysis indicated that there was significantly positive correlation between EVLWI and GEDI(r=0.835, P=0.000) and between NGAL and GEDI(r=0.489, P=0.000). In contrast, after immobilization of GEDI, we did not find any significant correlation between EVLWI and NGAL(r=0.381, P=0.060). Conclusions In case of fluid infusion volume is relatively too much , there is a strong correlation between NGAL in alveolar lavage fluid and EVLWI levels for early fluid resuscitation in patients with sepetic shock. Key words: Intensive care unit; Sepsis; Septic shock; Acute lung injury; Bronchoalveolar lavage fluid; Extravascular lung water index; Neutrophil gelatinase-associated lipocalin; PICCO

Urinary Neutrophil Gelatinase-Associated Lipocalin and Its Association with the Histological Pattern in Idiopathic Nephrotic Syndrome

Introduction: Idiopathic nephrotic syndrome (INS) is the most common glomerular disorder of childhood. Clinical outcome of children with nephrotic syndrome depends on underlying histopathlogy and responsiveness to steroid treatment. Minimal change disease (MCD) has a favorable long-term prognosis whereas, other than minimal change nephrotic syndrome is often resistant to steroid and is more likely to progress to end-stage renal disease (ESRD). Neutrophil gelatinase-associated lipocalin (NGAL) which is a small protein belonging to the lipocalin superfamily has been demonstrated to be a powerful risk marker of chronic kidney disease progression. This study was undertaken to determine whether urinary NGAL could be used as a biomarker in differentiating minimal change disease from other glomerular histologic lesions in idiopathic nephrotic syndrome in children.&#x0D; Objectives: To evaluate the association between urinary NGAL and histological pattern in idiopathic nephrotic syndrome.&#x0D; Materials and Methods: This cross-sectional, multicenter study was conducted in the Department of Paediatric Nephrology, Dhaka Shishu Hospital and Department of Paediatric Nephrology, Bangabandhu Sheikh Mujib Medical University from June 2014 to May 2015. Fifty-one children with idiopathic nephrotic syndrome comprising 12 children with minimal change disease (MCD) and 39 with other than minimal change nephrotic syndrome were included. Urinary NGAL was measured using a commercially available HUMAN NGAL/ LIPOCALIN 2 ELISA kit which employed the quantitative sandwich enzyme immunoassay technique. Median urinary NGAL level were compared between MCD and other than MCD. Median urinary NGAL and urinary creatinine ratio also compared between two groups. The prognostic accuracy of urinary NGAL was assessed by receiver operating characteristic (ROC) curve analysis.&#x0D; Results: Median urinary NGAL (uNGAL) level of MCD group was 44.5 [IQR: 32-109.5] (ng/ml) and that of the other than MCD group was 130 [IQR:85-172] (ng/ml). This difference was statistically significant (p=0.004). Median urine NGAL and urine creatinine ratio was significant between two groups (MCD=105.5 ng/mg and other than MCD=288 ng/mg, p-value was&lt;0.001). The area under the curve (AUC) for the uNGAL as a biomarker to differentiate MCD from other than MCD was 0.78 [95% CI: 0.64-0.92] (p=0.004) and showed an optimized sensitivity of 0.82 and specificity of 0.75 with an optimal trade-off value of 72 ng/ml.&#x0D; Conclusion: Urinary NGAL was found to be a reliable biomarker to differentiate the histological pattern in idiopathic nephrotic syndrome.&#x0D; Journal of Armed Forces Medical College Bangladesh Vol.13(1) 2017: 51-55

Molecularly Imprinted Polymer (MIP) Film with Improved Surface Area Developed by Using Metal–Organic Framework (MOF) for Sensitive Lipocalin (NGAL) Determination

Electropolymerizable functional and cross-linking monomers were used to prepare conducting molecularly imprinted polymer film with improved surface area with the help of a sacrificial metal-organic framework (MOF). Subsequent dissolution of the MOF layer resulted in a surface developed MIP film. This surface enlargement increased the analyte accessibility to imprinted molecular cavities. Application of the porous MIP film as a recognition unit of an extended-gate field effect transistor (EG-FET) chemosensor effectively enhanced analytical current signals of determination of recombinant human neutrophil gelatinase-associated lipocalin (NGAL).

Neutrophil gelatinase-associated lipocalin worsens ischemia/reperfusion damage of kidney cells by autophagy

This study aimed to explore the influence of neutrophil gelatinase-associated lipocalin on autophagy and its role in ischemia/reperfusion injury in human kidney-2 (HK-2) cells during acute kidney injury (AKI). HK-2 cells were given hypoxia/reoxygenation treatment for different times to simulate ischemia/reperfusion injury. Autophagy was evaluated by western blot and immunofluorescence of GFP-LC3. Cell viability was tested to reflect the degree of cell damage. The autophagy inhibitor 3-MA was used to inhibit autophagy and determine the role of autophagy in ischemia/reperfusion injury. HK-2 cells were hypoxia for 1 h, followed by reoxygenation treatment for 24 h. These cells were then exposed to human recombinant protein neutrophil gelatinase-associated lipocalin (NGAL) (50, 100, 200, 400, or 1000 ng/mL) with or without 3-MA. Our results showed that autophagy was induced by hypoxia treatment and was further enhanced by reoxygenation after hypoxia treatment. Cell viability was decreased with the inhibition of autophagy in the process. Autophagic flux was further induced with NGAL (>200 ng/mL), while cell viability declined in this condition. Cell viability was recovered when autophagy was inhibited. These results indicate that autophagy plays, in part, a protective role in renal ischemia/reperfusion injury. Furthermore, the data suggest that NGAL strengthens the level of autophagy in this process. Overall, a large quantity of NGAL produced by renal proximal tubular epithelial cells may induce excessive autophagy and increase renal ischemia/reperfusion injury in acute kidney injury.

Lipocalin-2 deficiency or blockade protects against aortic abdominal aneurysm development in mice.

To study the role of lipocalin-2 (Lcn2) and the effect of Lcn2 blockade via anti-Lcn2 antibody in the development of abdominal aortic aneurysm (AAA). Expression mRNA and protein levels of Lcn2 and its human orthologue neutrophil gelatinase-associated lipocalin (NGAL) in aortic wall samples from experimental mouse and human AAA samples, respectively, were analysed by real-time PCR and immunohistochemistry. Experimental AAA was induced by aortic elastase perfusion in wild-type mice (WT) and Lcn2-deficient mice (Lcn2-/-). NGAL/Lcn2 mRNA and protein levels in human and murine AAA samples were increased compared with healthy aortas. Decreased AAA incidence and reduced aortic expansion were observed in Lcn2-/- mice or mice preoperative treated with a polyclonal anti-Lcn2 antibody compared with WT mice or mice treated with control IgG, respectively, at Day 14 after elastase perfusion. Moreover, immunohistochemical analysis of AAA tissues from Lcn2-/- or anti-Lcn2-treated mice showed diminished elastin damage, reduced microvessels and polymorphonuclear neutrophil (PMN) infiltration, and enhanced preservation of vascular smooth muscle cells compared with WT aortas. Fluorescent molecular tomography revealed decreased MMP activity in AAA of Lcn2-/- mice compared with WT controls. Therapeutic administration of anti-Lcn2 antibody to WT mice 3 days after elastase perfusion decreased aortic dilatation and PMN infiltration compared with WT mice treated with control IgG. Either Lcn2 deficiency or anti-Lcn2 antibody blockade limits AAA expansion in mice by decreasing PMN infiltration in the aorta. Lcn2 modulation may therefore be a viable new therapeutic option for the treatment of AAA.

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro

Neutrophil gelatinase-associated lipocalin (NGAL), a protein found in activated neutrophils, is expressed in kidney tubule cells in response to noxious stimuli, and is thus recognized as a marker of acute kidney injury. Recent studies have suggested that NGAL could also have pathophysiological importance in cardiovascular diseases. The aim of the present study was to examine NGAL expression in human carotid endarterectomy tissues ex vivo as well as the effects of NGAL in the main cell types involved in atherogenesis, namely in human macrophages, endothelial cells, and smooth muscle cells in vitro. NGAL protein was analyzed in human endarterectomy samples from patients with asymptomatic and symptomatic carotid stenosis by immunofluorescence, and NGAL mRNA expression was detected using RealTime-PCR. Human monocyte derived macrophages (MDM), human coronary artery smooth muscle cells (HCASMC), and human umbilical vein endothelial cells (HUVEC) were treated with recombinant human (rh) NGAL at different concentrations. Interleukin (IL)-6, IL-8, and monocyte chemo-attractant protein-1 (MCP-1) were determined by specific enzyme linked immunosorbent assays (ELISAs) in culture supernatants of such treated cells. Expression of NGAL protein was demonstrated by macrophages, smooth muscle cells, and endothelial cells in human carotid atherosclerotic tissue. NGAL mRNA expression was detected at a higher rate in atherosclerotic tissue of patients with symptomatic carotid stenosis (in 70%; n = 19) compared with asymptomatic patients (in 37%; n = 20, P < .001). Treatment of MDM, HCASMC, and HUVEC with rhNGAL led to a significant (P < .05) and concentration dependent increase of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 in all cell types analyzed. By induction of pro-inflammatory mediators in human macrophages, smooth muscle cells and endothelial cells, NGAL, which is predominantly expressed in atherosclerotic plaques of symptomatic patients, could be involved in creating the local and systemic pro-inflammatory environment characteristic for atherosclerosis.

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro

Neutrophil gelatinase-associated lipocalin (NGAL), a protein found in activated neutrophils, is expressed in kidney tubule cells in response to noxious stimuli, and is thus recognized as a marker of acute kidney injury. Recent studies have suggested that NGAL could also have pathophysiological importance in cardiovascular diseases. The aim of the present study was to examine NGAL expression in human carotid endarterectomy tissues exvivo as well as the effects of NGAL in the main cell types involved in atherogenesis, namely in human macrophages, endothelial cells, and smooth muscle cells invitro. NGAL protein was analyzed in human endarterectomy samples from patients with asymptomatic and symptomatic carotid stenosis by immunofluorescence, and NGAL mRNA expression was detected using RealTime-PCR. Human monocyte derived macrophages (MDM), human coronary artery smooth muscle cells (HCASMC), and human umbilical vein endothelial cells (HUVEC) were treated with recombinant human (rh) NGAL at different concentrations. Interleukin (IL)-6, IL-8, and monocyte chemo-attractant protein-1 (MCP-1) were determined by specific enzyme linked immunosorbent assays (ELISAs) in culture supernatants of such treated cells. Expression of NGAL protein was demonstrated by macrophages, smooth muscle cells, and endothelial cells in human carotid atherosclerotic tissue. NGAL mRNA expression was detected at a higher rate in atherosclerotic tissue of patients with symptomatic carotid stenosis (in 70%; n=19) compared with asymptomatic patients (in 37%; n=20, p<.001). Treatment of MDM, HCASMC, and HUVEC with rhNGAL led to a significant (p<0.05) and concentration dependent increase of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 in all cell types analyzed. By induction of pro-inflammatory mediators in human macrophages, smooth muscle cells and endothelial cells, NGAL, which is predominantly expressed in atherosclerotic plaques of symptomatic patients, could be involved in creating the local and systemic pro-inflammatory environment characteristic for atherosclerosis.

High-efficiency secretory expression of human neutrophil gelatinase-associated lipocalin from mammalian cell lines with human serum albumin signal peptide

Human neutrophil gelatinase associated lipocalin (NGAL) is a secretory glycoprotein initially isolated from neutrophils. It is thought to be involved in the incidence and development of immunological diseases and cancers. Urinary and serum levels of NGAL have been investigated as a new biomarker of acute kidney injury (AKI), for an earlier and more accurate detection method than with creatinine level. However, expressing high-quality recombinant NGAL is difficult both in Escherichia coli and mammalian cells for the low yield. Here, we cloned and fused NGAL to the C-terminus of signal peptides of human NGAL, human interleukin-2 (IL2), gaussia luciferase (Gluc), human serum albumin preproprotein (HSA) or an hidden Markov model-generated signal sequence (HMM38) respectively for transient expression in Expi293F suspension cells to screen for their ability to improve the secretory expression of recombinant NGAL. The best results were obtained with signal peptide derived from HSA. The secretory recombinant protein could react specifically with NGAL antibody. For scaled production, we used HSA signal peptide to establish stable Chinese hamster ovary cell lines. Then we developed a convenient colony-selection system to select high-expression, stable cell lines. Moreover, we purified the NGAL with Ni-Sepharose column. The recombinant human NGAL displayed full biological activity. We provide a method to enhance the secretory expression of recombinant human NGAL by using the HSA signal peptide and produce the glycoprotein in mammalian cells.

Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications.

BackgroundNanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools. Although the routine procedure to obtain Nbs is to immunize camels with antigens, it is unavailable to immunize a camel when the antigens are highly toxic, pathogenic or nonimmunogenic. A synthetic phage display library is an alternative to generate Nbs against such targets, besides all the other ones.MethodsWe constructed a large and diverse synthetic phage display Nanobody (Nb) library based on the conserved camel single-domain antibody fragment (VHH) framework of cAbBCII10. Diversity was introduced in the complementarity-determining region 3 (CDR3) by means of randomization of synthetic oligonucleotides. Then human prealbumin (PA) and neutrophil gelatinase-associated lipocalin (NGAL) were used to select specific Nbs from this library. Furthermore, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect PA based on horseradish peroxidase (HRP)-conjugated anti-PA Nb isolated from this study and another biotinylated anti-PA Nb obtained from an immune library, in our previous study.ResultsA large and diverse synthetic phage display Nb library with CDR3 regions randomized by trinucleotide cassettes was constructed. The library size was 1.65 × 109 CFU/mL and the correct insertion ratio was nearly 100%. A Nb against human PA and against NGAL was successfully isolated from the synthetic library. The obtained anti-PA Nb was effectively used to develop a sandwich ELISA for PA detection and it demonstrated a working range from 50 to 1000 ng/mL, with a limit of detection (LOD) of 27.1 ng/mL.ConclusionThis proposed novel synthetic library was a good source for obtaining some antigen-specific Nbs. This approach could provide crucial support to an immune library and a naïve library in the acquisition of specific Nbs, potentially functioning as a great resource for medical diagnostic applications. In addition, we have successfully developed a novel sandwich ELISA to detect PA, which could provide great assistance for clinical PA detection.

Differential transcytosis and toxicity of the hNGAL receptor ligands cadmium-metallothionein and cadmium-phytochelatin in colon-like Caco-2 cells: Implications for in vivo cadmium toxicity

The environmental toxicant cadmium (Cd) enters the food chain. A substantial proportion of Cd in nutrients of plant origin is present as Cd-metallothionein (CdMT) and Cd-phytochelatin (CdPC) complexes, which may be absorbed and transcytosed intact by colonic enterocytes following bacterial fermentation and contribute to systemic Cd toxicity, e.g. in liver and kidneys. We have recently demonstrated that the receptor for human neutrophil gelatinase-associated lipocalin (hNGAL) is expressed in human colon and colon-like Caco-2 BBE cells where it mediates transcytosis of MT and PC. Here we show in colon-like Caco-2 BBE cells that hNGAL receptor (hNGAL-R) dependent toxicity is significantly higher with CdMT than with CdPC3 (2.5–50μM Cd2+ complexed to MT or PC3 for ≤24h), using MTT assay. Fluorescence-labelled A546-MT, but not A488-PC3 (both 700nM), co-localizes with the lysosomal marker cathepsin-B, as determined by confocal microscopy. In transwell experiments with confluent monolayers, transcytosis efficiency (i.e. the ratio of basal delivery to apical decrease) of A546-MT is decreased compared to A488-PC3 (both 700nM). The tubulin polymerization disruptor nocodazole (16.7μM) almost abolished CdMT and CdPC3 toxicity, reduced apical uptake of both A546-MT and A488-PC3, but increased transcytosis efficiency of A546-MT compared to that of A488-PC3 by preventing trafficking of A546-MT to lysosomes. Hence, following hNGAL-R dependent endocytosis of CdMT/CdPC3 in colonic epithelia, a nocodazole-sensitive trafficking pathway may preferentially target CdMT, but not CdPC3, to lysosomes, causing increased colonic epithelial toxicity but reduced systemic toxicity.