Lipocalin-2 is expressed under pernicious conditions such as intoxication, infection, inflammation and other forms of cellular stress. Experimental liver injury induces rapid and sustained LCN2 ...production by injured hepatocytes. However, the precise biological function of LCN2 in liver is still unknown. In this study, LCN2−/− mice were exposed to short term application of CCl4, lipopolysaccharide and Concanavalin A, or subjected to bile duct ligation. Subsequent injuries were assessed by liver function analysis, qRT-PCR for chemokine and cytokine expression, liver tissue Western blot, histology and TUNEL assay. Serum LCN2 levels from patients suffering from liver disease were assessed and evaluated. Acute CCl4 intoxication showed increased liver damage in LCN2−/− mice indicated by higher levels of aminotransferases, and increased expression of inflammatory cytokines and chemokines such as IL-1β, IL-6, TNF-α and MCP-1/CCL2, resulting in sustained activation of STAT1, STAT3 and JNK pathways. Hepatocytes of LCN2−/− mice showed lipid droplet accumulation and increased apoptosis. Hepatocyte apoptosis was confirmed in the Concanavalin A and lipopolysaccharide models. In chronic models (4weeks bile duct ligation or 8weeks CCl4 application), LCN2−/− mice showed slightly increased fibrosis compared to controls. Interestingly, serum LCN2 levels in diseased human livers were significantly higher compared to controls, but no differences were observed between cirrhotic and non-cirrhotic patients. Upregulation of LCN2 is a reliable indicator of liver damage and has significant hepato-protective effect in acute liver injury. LCN2 levels provide no correlation to the degree of liver fibrosis but show significant positive correlation to inflammation instead.
► Upregulation of LCN2 is a reliable indicator of liver damage. ► LCN2 has significant hepato-protective effects in acute liver injury. ► LCN2 deficient mice show increased expression of inflammatory mediators. ► LCN2 deficient mice show sustained activation of STAT1, STAT3 and JNK pathways. ►LCN2 is not correlated to hepatic fibrosis but correlates to inflammation.
Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and ...murine apolipoprotein M (apoM). Thus, isolated human ApoM⺠HDL contained S1P, whereas ApoMâ» HDL did not. Moreover, HDL in Apomâ»/â» mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-à structure of the S1P-human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM⺠HDL induced S1Pâ receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoMâ» HDL did not. Importantly, lack of S1P in the HDL fraction of Apomâ»/â» mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1Pâ receptor on endothelial cells, is a vasculoprotective constituent of HDL.
The unique viviparous Pacific Beetle cockroaches provide nutrition to their embryo by secreting milk proteins Lili-Mip, a lipid-binding glycoprotein that crystallises in-vivo. The resolved in-vivo ...crystal structure of variably glycosylated Lili-Mip shows a classical Lipocalin fold with an eight-stranded antiparallel beta-barrel enclosing a fatty acid. The availability of physiologically unaltered glycoprotein structure makes Lili-Mip a very attractive model system to investigate the role of glycans on protein structure, dynamics, and function. Towards that end, we have employed all-atom molecular dynamics simulations on various glycosylated stages of a bound and free Lili-Mip protein and characterised the impact of glycans and the bound lipid on the dynamics of this glycoconjugate. Our work provides important molecular-level mechanistic insights into the role of glycans in the nutrient storage function of the Lili-Mip protein. Our analyses show that the glycans stabilise spatially proximal residues and regulate the low amplitude opening motions of the residues at the entrance of the binding pocket. Glycans also preserve the native orientation and conformational flexibility of the ligand. However, we find that either deglycosylation or glycosylation with high-mannose and paucimannose on the core glycans, which better mimic the natural insect glycosylation state, significantly affects the conformation and dynamics. A simple but effective distance- and correlation-based network analysis of the protein also reveals the key residues regulating the barrel's architecture and ligand binding characteristics in response to glycosylation.
RationaleBacterial pneumonia is the most common infectious cause of death worldwide and treatment is increasingly hampered by antibiotic resistance. Mesenchymal stem cells (MSCs) have been ...demonstrated to provide protection against acute inflammatory lung injury; however, their potential therapeutic role in the setting of bacterial pneumonia has not been well studied.ObjectiveThis study focused on testing the therapeutic and mechanistic effects of MSCs in a mouse model of Gram-negative pneumonia.Methods and resultsSyngeneic MSCs from wild-type mice were isolated and administered via the intratracheal route to mice 4 h after the mice were infected with Escherichia coli. 3T3 fibroblasts and phosphate-buffered saline (PBS) were used as controls for all in vivo experiments. Survival, lung injury, bacterial counts and indices of inflammation were measured in each treatment group. Treatment with wild-type MSCs improved 48 h survival (MSC, 55%; 3T3, 8%; PBS, 0%; p<0.05 for MSC vs 3T3 and PBS groups) and lung injury compared with control mice. In addition, wild-type MSCs enhanced bacterial clearance from the alveolar space as early as 4 h after administration, an effect that was not observed with the other treatment groups. The antibacterial effect with MSCs was due, in part, to their upregulation of the antibacterial protein lipocalin 2.ConclusionsTreatment with MSCs enhanced survival and bacterial clearance in a mouse model of Gram-negative pneumonia. The bacterial clearance effect was due, in part, to the upregulation of lipocalin 2 production by MSCs.
Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarker characteristics that link the protein to the injured organ have not yet ...been described. We generated an Ngal reporter mouse by inserting a double-fusion reporter gene encoding luciferase-2 and mCherry (Luc2-mC) into the Ngal (Lcn2) locus. The Ngal-Luc2-mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real time. In the kidney, Ngal-Luc2-mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ- and cell-specific relationship with tubular stress, which correlated with the level of urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2-mC mice also revealed both the onset and the resolution of the injury, and the actions of NF-κB inhibitors and antibiotics during infection. Thus, imaging of Ngal-Luc2-mC mice and cells identified injurious and reparative agents that affect kidney damage.
BACKGROUND: Acute kidney injury (AKI) is associated with high mortality rates in dogs, which may be a consequence of late recognition using traditional diagnostic tests. Neutrophil ...gelatinase‐associated lipocalin (NGAL) is a protein‐induced during kidney injury that may identify AKI earlier than traditional tests. OBJECTIVES/HYPOTHESIS: To evaluate urinary NGAL (uNGAL) and uNGAL‐to‐urinary creatinine ratio (UNCR) as early markers of kidney injury and recovery in an AKI model in dogs. It was hypothesized that these markers would document AKI earlier than serum creatinine concentration. ANIMALS: Five purpose‐bred dogs. METHODS: Prospective study. Acute kidney injury, defined as a > 50% increase in serum creatinine concentration above baseline, was induced in dogs by gentamicin administration (8–10 mg/kg SC q8h). Blood and urine collected for biochemical analyses and uNGAL and urinary creatinine concentrations, respectively, during AKI induction and recovery. RESULTS: Acute kidney injury was diagnosed significantly earlier based on a 7‐fold increase in UNCR compared to a > 50% increase in serum creatinine concentration (day 8; range, 2–10 mg/dl vs day 16; range, 14–19 mg/dl; P = .009). During recovery, the initial decrease in UNCR preceded the decrease in serum creatinine concentration by a median of 2 days. The uNGAL changes paralleled UNCR changes, but the increase in uNGAL was triphasic; the initial peak occurred earlier than UNCR (median, day 11 versus median, day 19). CONCLUSIONS AND CLINICAL IMPORTANCE: The UNCR was early marker of gentamicin‐induced AKI and its decrease documented onset of renal recovery. Additional studies are needed to validate this marker in dogs with naturally occurring renal injury.
The present study aimed to monitor peritoneal neutrophil gelatinase-associated lipocalin (pNGAL) during peritonitis episodes and to enhance its diagnostic value by evaluating pNGAL at scheduled times ...in parallel with white blood cell (WBC) count. In addition, we investigated possible correlations between pNGAL and the etiology of peritonitis, evaluating it as a possible marker of the clinical outcome.
Twenty-two patients with peritoneal dialysis (PD)-related peritonitis were enrolled. Peritonitis was divided into Gram-positive, Gram-negative, polymicrobial, and sterile. WBC count and neutrophil gelatinase-associated lipocalin (NGAL) in PD effluent were measured at different times (days 0, 1, 5, 10, 15, and/or 20 and 10 days after antibiotic therapy discontinuation). NGAL was measured by standard quantitative laboratory-based immunoassay and by colorimetric NGAL dipstick (NGALds) (dipstick test).
We found strong correlations between peritoneal WBC, laboratory-based NGAL, and NGALds values, both overall and separated at each time point. On day 1, we observed no significant difference in WBC, both NGALds (p = 0.3, 0.9, and 0.2) between Gram-positive, Gram-negative, polymicrobial, and sterile peritonitis. No significant difference has been found between de novo versus relapsing peritonitis for all markers (p > 0.05). We observed a parallel decrease of WBC and both NGAL in patients with favorable outcomes. WBC count and both pNGAL resulted higher in patients with negative outcomes (defined as relapsing peritonitis, peritonitis-associated catheter removal, peritonitis-associated hemodialysis transfer, peritonitis-associated death) at day 10 (p = 0.04, p = 0.03, and p = 0.05, respectively) and day 15 (p = 0.01, p = 0.04, and tendency for p = 0.005). There was a tendency toward higher levels of WBC and NGAL in patients with a negative outcome at day 5. No significant difference in all parameters was proven at day 1 (p = 0.3, p = 0.9, p = 0.2) between groups.
This study confirms pNGAL as a valid and reliable biomarker for the diagnosis of PD-peritonitis and its monitoring. Its trend is parallel to WBC count during peritonitis episodes, in particular, patients with unfavorable outcomes.
Amyloid beta (Aβ) peptides, in particular Aβ42 and Aβ40, exert neurotoxic effects and their overproduction leads to amyloid deposits in the brain, thus constituting an important biomolecular target ...for treatments of Alzheimer's disease (AD). We describe the engineering of cognate Anticalins as a novel type of neutralizing protein reagent based on the human lipocalin scaffold. Phage display selection from a genetic random library comprising variants of the human lipocalin 2 (Lcn2) with mutations targeted at 20 exposed amino acid positions in the four loops that form the natural binding site was performed using both recombinant and synthetic target peptides and resulted in three different Anticalins. Biochemical characterization of the purified proteins produced by periplasmic secretion in Escherichia coli revealed high folding stability in a monomeric state, with Tm values ranging from 53.4°C to 74.5°C, as well as high affinities for Aβ40, between 95 pM and 563 pM, as measured by real-time surface plasmon resonance analysis. The central linear VFFAED epitope within the Aβ sequence was mapped using a synthetic peptide array on membranes and was shared by all three Anticalins, despite up to 13 mutual amino acid differences in their binding sites. All Anticalins had the ability-with varying extent-to inhibit Aβ aggregation in vitro according to the thioflavin-T fluorescence assay and, furthermore, they abolished Aβ42-mediated toxicity in neuronal cell culture. Thus, these Anticalins provide not only useful protein reagents to study the molecular pathology of AD but they also show potential as alternative drug candidates compared with antibodies.
Osteoblasts are specialized mesenchymal cells that synthesize bone matrix and coordinate the mineralization of the skeleton. These cells work in harmony with osteoclasts, which resorb bone, in a ...continuous cycle that occurs throughout life. The unique function of osteoblasts requires substantial amounts of energy production, particularly during states of new bone formation and remodelling. Over the last 15 years, studies have shown that osteoblasts secrete endocrine factors that integrate the metabolic requirements of bone formation with global energy balance through the regulation of insulin production, feeding behaviour and adipose tissue metabolism. In this article, we summarize the current understanding of three osteoblast-derived metabolic hormones (osteocalcin, lipocalin and sclerostin) and the clinical evidence that suggests the relevance of these pathways in humans, while also discussing the necessity of specific energy substrates (glucose, fatty acids and amino acids) to fuel bone formation and promote osteoblast differentiation.
In chronic kidney disease (CKD), progressive nephron loss causes glomerular sclerosis, as well as tubulointerstitial fibrosis and progressive tubular injury. In this study, we aimed to identify ...molecular changes that reflected the histopathological progression of renal tubulointerstitial fibrosis and tubular cell damage. A discovery set of renal biopsies were obtained from 48 patients with histopathologically confirmed CKD, and gene expression profiles were determined by microarray analysis. The results indicated that hepatitis A virus cellular receptor 1 (also known as Kidney Injury Molecule-1, KIM-1), lipocalin 2 (also known as neutrophil gelatinase-associated lipocalin, NGAL), SRY-box 9, WAP four-disulfide core domain 2, and NK6 homeobox 2 were differentially expressed in CKD. Their expression levels correlated with the extent of tubulointerstitial fibrosis and tubular cell injury, determined by histopathological examination. The expression of these 5 genes was also increased as kidney damage progressed in a rodent unilateral ureteral obstruction model of CKD. We calculated a molecular score using the microarray gene expression profiles of the biopsy specimens. The composite area under the receiver operating characteristics curve plotted using this molecular score showed a high accuracy for diagnosing tubulointerstitial fibrosis and tubular cell damage. The robust sensitivity of this score was confirmed in a validation set of 5 individuals with CKD. These findings identified novel molecular markers with the potential to contribute to the detection of tubular cell damage and tubulointerstitial fibrosis in the kidney.
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