Neutrophil Gelatinase-associated Lipocalin Expression Research Articles

HMGB1 signaling-regulated endoplasmic reticulum stress mediates intestinal ischemia/reperfusion-induced acute renal damage

BackgroundIschemia/reperfusion of the intestine often leads to distant organ injury, but the mechanism of intestinal ischemia/reperfusion-induced renal dysfunction is still not clear. The present study aimed to investigate the mechanisms of acute renal damage after intestinal ischemia/reperfusion challenge and explore the role of released high-mobility group box-1 in this process. MethodsIntestinal ischemia/reperfusion was induced in male Sprague-Dawley rats by clamping the superior mesenteric artery for 1.5 hours. At different reperfusion time points, anti−high-mobility group box-1 neutralizing antibodies or ethyl pyruvate were administered to neutralize or inhibit circulating high-mobility group box-1, respectively. ResultsSignificant kidney injury was observed after 6 hours of intestinal reperfusion, as indicated by increased serum levels of urea nitrogen and creatinine, increased expression of neutrophil gelatinase-associated lipocalin, interleukin-6, and MIP-2, and enhanced cell apoptosis, as indicated by cleaved caspase 3 levels in renal tissues. The levels of phosphorylated eIF2ɑ, activating transcription factor 4, and C/EBP-homologous protein (CHOP) were markedly elevated, indicating the activation of endoplasmic reticulum stress in the impaired kidney. High-mobility group box-1 translocated to cytoplasm in the intestine and serum concentrations of high-mobility group box-1 increased notably during the reperfusion phase. Both anti−high-mobility group box-1 antibodies and ethyl pyruvate treatment significantly reduced serum high-mobility group box-1 concentrations, attenuated endoplasmic reticulum stress in renal tissue and inhibited the development of renal damage. Moreover, the elevated expression of receptor for advanced glycation end products in the kidneys after intestinal ischemia/reperfusion was abrogated after high-mobility group box-1 inhibition. ConclusionThese results suggested that high-mobility group box-1 signaling regulated endoplasmic reticulum stress and promoted intestinal ischemia/reperfusion-induced acute kidney injury. High-mobility group box-1 neutralization/inhibition might serve as a pharmacological intervention strategy for these pathophysiological processes.

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 < 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.

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.

Crystal structure of Leptospira leucine-rich repeat 20 reveals a novel E-cadherin binding protein to induce NGAL expression in HK2 cells.

Leptospirosis is the most common zoonotic disease caused by pathogenic Leptospira, which is classified into three groups according to virulence. Its pathogenic and intermediate species contain leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic strains. In this study, we presented the crystal structure of LSS_11580 (rLRR20) from pathogenic L. santarosai serovar Shermani. X-ray diffraction at a resolution of 1.99 Å revealed a horseshoe-shaped structure containing seven α-helices and five β-sheets. Affinity assays indicated that rLRR20 interacts with E-cadherin on the cell surface. Interestingly, its binds to the extracellular (EC) 1 domain in human epithelial (E)-cadherin, which is responsible for binding to another E-cadherin molecule in neighboring cells. Several charged residues on the concave face of LRR20 were predicted to interact with EC1 domain. In the affinity assays, these charged residues were replaced by alanine, and their affinities to E-cadherin were measured. Three vital residues and mutation variants of LRR20, namely D56A, E59A, and E123A, demonstrated significantly reduced affinity to E-cadherin compared with the control. Besides, we also demonstrated that rLRR20 induced the expression of neutrophil gelatinase-associated lipocalin (NGAL) in HK2 cells. The low ability of the three mutation variants to induce NGAL expression further demonstrates this induction. The present findings indicate that LRR20 from pathogenic Leptospira binds to E-cadherin and interacts with its EC1 domain. In addition, its induction of NGAL expression in HK2 cells is associated with acute kidney injury in human.

Assessment of neutrophil gelatinase-associated lipocalin as an early biomarker for canine renal ischemia-reperfusion injury.

BackgroundThe pathological mechanism of ischemia/reperfusion acute kidney injury (I/R-AKI) differs from other forms of AKI. Neutrophil gelatinase-associated lipocalin (NGAL) is a sensitive biomarker for early diagnosis of AKI, but its utility for diagnosis of canine I/R-AKI remains to be evaluated. The aims of this study were to establish an I/R-AKI model in dogs and to evaluate the diagnostic value of NGAL for canine I/R-AKI.MethodsWe randomly divided 12 beagle dogs into a sham and an I/R group. Artery and vein of the left kidneys of I/R group were cross-clamped for 60 min followed by reperfusion. The kidney samples were analyzed for histopathological lesions. Serum and urinary samples were analyzed for blood urea nitrogen (BUN), serum creatinine (sCr), serum NGAL (sNGAL), urinary creatinine (uCr), and urinary NGAL (uNGAL). Their detection sensitivities and specificities were compared using a receiver operating characteristics (ROC) method. The expression of NGAL in the renal tissues was analyzed by quantitative RT-PCR (qRT-PCR) and immunohistochemical (IHC) analysis.ResultsAfter I/R, histopathological analysis showed typical AKI lesions in the dog kidneys of the I/R group, but not in the sham group. Compared to that of the sham group, BUN and sCr of the I/R group rose to significant high levels from 24 h after I/R. Both uNGAL and sNGAL rose rapidly from 2 h, reached to the peak levels at 12 h, and then receded to the pre-operation levels by 72 h after I/R. The uNGAL/uCr ratio (uNCR) rose rapidly from 2 h and remained at variably high levels from 6 to 60 h after I/R. The ROC analysis showed that detection sensitivities of uNCR, uNGAL, and sNGAL were significantly (P<0.0001) higher than that of sCr, without significant difference in specificity. The cut-off values of sNGAL, uNGAL and uNCR were 14,642 pg/mL, 6,773 pg/mL, and 6,701 pg/mg, respectively. Both qRT-PCR and IHC analyses confirmed the dynamic expression of NGAL in the dog kidneys with ischemic acute kidney injury (I-AKI).ConclusionsThere is potential for NGAL to be used as a sensitive biomarker for early diagnosis of canine I-AKI.

Class IIa HDAC inhibitor TMP195 alleviates lipopolysaccharide-induced acute kidney injury.

Sepsis-associated acute kidney injury (SA-AKI) is associated with high mortality rates, but clinicians lack effective treatments except supportive care or renal replacement therapies. Recently, histone deacetylase (HDAC) inhibitors have been recognized as potential treatments for acute kidney injury and sepsis in animal models; however, the adverse effect generated by the use of pan inhibitors of HDACs may limit their application in people. In the present study, we explored the possible renoprotective effect of a selective class IIa HDAC inhibitor, TMP195, in a murine model of SA-AKI induced by lipopolysaccharide (LPS). Administration of TMP195 significantly reduced increased serum creatinine and blood urea nitrogen levels and renal damage induced by LPS; this was coincident with reduced expression of HDAC4, a major isoform of class IIa HDACs, and elevated histone H3 acetylation. TMP195 treatment following LPS exposure also reduced renal tubular cell apoptosis and attenuated renal expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, two biomarkers of tubular injury. Moreover, LPS exposure resulted in increased expression of BAX and cleaved caspase-3 and decreased expression of Bcl-2 and bone morphogenetic protein-7 in vivo and in vitro; TMP195 treatment reversed these responses. Finally, TMP195 inhibited LPS-induced upregulation of multiple proinflammatory cytokines/chemokines, including intercellular adhesion molecule-1, monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-1β, and accumulation of inflammatory cells in the injured kidney. Collectively, these data indicate that TMP195 has a powerful renoprotective effect in SA-AKI by mitigating renal tubular cell apoptosis and inflammation and suggest that targeting class IIa HDACs might be a novel therapeutic strategy for the treatment of SA-AKI that avoids the unintended adverse effects of a pan-HDAC inhibitor.

Rat pancreatectomy combined with isoprenaline or uninephrectomy as models of diabetic cardiomyopathy or nephropathy

Cardiovascular and renal complications are the predominant causes of morbidity and mortality amongst patients with diabetes. Development of novel treatments have been hampered by the lack of available animal models recapitulating the human disease. We hypothesized that experimental diabetes in rats combined with a cardiac or renal stressor, would mimic diabetic cardiomyopathy and nephropathy, respectively. Diabetes was surgically induced in male Sprague Dawley rats by 90% pancreatectomy (Px). Isoprenaline (Iso, 1 mg/kg, sc., 10 days) was administered 5 weeks after Px with the aim of inducing cardiomyopathy, and cardiac function and remodeling was assessed by echocardiography 10 weeks after surgery. Left ventricular (LV) fibrosis was quantified by Picro Sirius Red and gene expression analysis. Nephropathy was induced by Px combined with uninephrectomy (Px-UNx). Kidney function was assessed by measurement of glomerular filtration rate (GFR) and urine albumin excretion, and kidney injury was evaluated by histopathology and gene expression analysis. Px resulted in stable hyperglycemia, hypoinsulinemia, decreased C-peptide, and increased glycated hemoglobin (HbA1c) compared with sham-operated controls. Moreover, Px increased heart and LV weights and dimensions and caused a shift from α-myosin heavy chain (MHC) to β-MHC gene expression. Isoprenaline treatment, but not Px, decreased ejection fraction and induced LV fibrosis. There was no apparent interaction between Px and Iso treatment. The superimposition of Px and UNx increased GFR, indicating hyperfiltration. Compared with sham-operated controls, Px-UNx induced albuminuria and increased urine markers of kidney injury, including neutrophil gelatinase-associated lipocalin (NGAL) and podocalyxin, concomitant with upregulated renal gene expression of NGAL and kidney injury molecule 1 (KIM-1). Whereas Px and isoprenaline separately produced clinical endpoints related to diabetic cardiomyopathy, the combination of the two did not accentuate disease development. Conversely, Px in combination with UNx resulted in several clinical hallmarks of diabetic nephropathy indicative of early disease development.

SHP2 inhibitor PHPS1 ameliorates acute kidney injury by Erk1/2-STAT3 signaling in a combined murine hemorrhage followed by septic challenge model

BackgroundHypovolemic shock and septic challenge are two major causes of acute kidney injury (AKI) in the clinic setting. Src homology 2 domain-containing phosphatase 2 (SHP2) is one of the major protein phosphatase tyrosine phosphatase (PTPs), which play a significant role in maintaining immunological homeostasis by regulating many facets of immune cell signaling. In this study, we explored whether SHP2 signaling contributed to development of AKI sequential hemorrhage (Hem) and cecal ligation and puncture (CLP) and whether inactivation of SHP2 through administration of its selective inhibitor, phenylhydrazonopyrazolone sulfonate 1 (PHPS1), attenuated this injury.MethodsMale C57BL/6 mice were subjected to Hem (a “priming” insult) followed by CLP or sham-Hem plus sham-CLP (S/S) as controls. Samples of blood and kidney were harvested at 24 h post CLP. The expression of neutrophil gelatinase-associated lipocalin (NGAL), high mobility group box 1 (HMGB1), caspase3 as well as SHP2:phospho-SHP2, extracellular-regulated kinase (Erk1/2): phospho-Erk1/2, and signal transducer and activator of transcription 3 (STAT3):phospho-STAT3 protein in kidney tissues were detected by Western blotting. The levels of creatinine (Cre) and blood urea nitrogen (BUN) in serum were measured according to the manufacturer’s instructions. Blood inflammatory cytokine/chemokine levels were detected by ELISA.ResultsWe found that indices of kidney injury, including levels of BUN, Cre and NGAL as well as histopathologic changes, were significantly increased after Hem/CLP in comparison with that in the S/S group. Furthermore, Hem/CLP resulted in elevated serum levels of inflammatory cytokines/chemokines, and induced increased levels of HMGB1, SHP2:phospho-SHP2, Erk1/2:phospho-Erk1/2, and STAT3:phospho-STAT3 protein expression in the kidney. Treatment with PHPS1 markedly attenuated these Hem/CLP-induced changes.ConclusionsIn conclusion, our data indicate that SHP2 inhibition attenuates AKI induced by our double-hit/sequential insult model of Hem/CLP and that this protective action may be attributable to its ability to mitigate activation of the Erk1/2 and STAT3 signaling pathway. We believe this is a potentially important finding with clinical implications warranting further investigation.

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.

P0512SKLB023 ATTENUATED LIPOPOLYSACCHARIDE-INDUCED ACUTE KIDNEY INJURY VIA INHIBITION OF NF-KAPPPAB AND MAPKS PATHWAYS

Abstract Background and Aims Excessive nitric oxide (NO) production by the activation of inducible nitric oxide synthase (iNOS) during sepsis was considered to contribute to acute kidney injury (AKI) and selective inhibition of iNOS activity may be a promising strategy for the treatment of sepsis-induced AKI. The novel small molecule compound (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-ylidene) methyl) phenoxy) acet-amide (SKLB023) as a selective iNOS inhibitor was designed, synthesized by our lab and exhibited therapeutic potential in arthritis and non-alcoholic steatohepatitis models with potent anti-inflammatory effects. This study aimed to evaluate whether SKLB023 as a drug candidate could offer renal protective effect against sepsis-induced AKI. Method 21 C57BL6 mice (20-22g, 6-8 weeks old) were randomly divided into three groups: control, model, SKLB023 pretreatment. AKI was induced by a single injection of 10mg/kg LPS, and SKLB023 was orally administrated at the dose of 50 mg/kg for three consecutive days and one hour before LPS injection. All mice were sacrificed 16h after LPS injection and blood and kidney tissues were collected for further evaluation. LPS-induced renal dysfunction was assessed by measurement of blood creatinine and urea nitrogen (BUN) as well as histopathological changes. Moreover, the kidney mRNA and protein expression of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1(KIM-1), newly identified kidney injury markers, were tested by polymerase chain reaction (PCR) and western blot. To explore underlying mechanisms, the renal expression of inflammatory cytokine genes IL-1β, IL-6, mcp-1 and proteins iNOS, COX-2, IL-1β, TNF-α, IL-6, HMGB1 in were measured. The total expression of nuclear factor-kappa B (NF-κB), IκBα and MAPKs proteins (ERK1/2, p38 MAPK) and their corresponding phosphoproteins (p-NF-κB, p-IκBα, p-ERK1/2, p-P38 MAPK) in mice kidneys were subsequently analyzed by western blot. Results 16hours following LPS challenge, mice developed AKI as evidenced by elevated creatinine and BUN levels and corresponding pathological changes. Additionally, kidney expression of NGAL and KIM-1 were remarkably increased after LPS injection. SKLB023 pretreatment, however, alleviated LPS-induced renal dysfunction. Moreover, the excessive production of inflammatory cytokines during sepsis was significantly suppressed by SKLB023. Further observation from PCR and immunoblot results indicated that SKLB023 attenuated the activity of NF-kB and the phosphorylation of ERK1/2 and p38 MAPK in septic AKI mice. Conclusion These data demonstrated that SKLB023 exhibited renoprotective effect in LPS-induced AKI, which was associated with its anti-inflammatory activities by inhibiting the NF-kappaB and MAPK signaling pathways

NGAL protects in nasopharyngeal carcinoma by inducing apoptosis and blocking epithelial-mesenchymal transition.

In recent years, neutrophil gelatinase-associated lipocalin (NGAL) has been considered to be a key molecule in different cancer types and its carcinogenesis may be related to the NGAL/MMP-9 complex. However, its expression pattern and role in nasopharyngeal carcinoma (NPC) has rarely been reported. In the current study, 158 tumor tissues from NPC patients were collected and immunohistochemistry was performed to determine the NGAL protein expression, to investigate the correlation between its expression and clinical and pathological parameters using Chi square analysis. Furthermore, by over-expressing NGAL in NPC cell lines, biological alteration of NPC cells with respect to cell proliferation, migration and invasion was analyzed. Results suggested that high expression of NGAL predicts better prognosis and longer survival. Overexpression of NGAL significantly reduced the proliferation and migration of NPC cells, and induced the apoptosis by activating caspase 3, 8 and 9, and blocking epithelial-mesenchymal transition by inhibiting mothers against decapentaplegic homolog 2/3 phosphorylation.

Androgen/androgen receptor affects gentamicin-induced nephrotoxicity through regulation of megalin expression

AimInvestigation whether androgen/androgen receptor (AR) might regulate megalin expression and/or functionality and thus affecting Gentamicin-induced nephrotoxicity (GIN). Main methodsMale Wistar rats were treated with gentamicin with/out AR ligands (testosterone as agonist and flutamide as antagonist). Megalin expression in the kidney tissues was determined by real-time RT-PCR and western blot. Besides, megalin functionality was assessed using immunofluorescence imaging of fluorescein isothiocyanate (FITC) conjugated bovine serum albumin (BSA) (FITC-BSA). The effects of different treatments on the kidney were assessed at the structural level by histopathological evaluation and the biochemical level by colorimetric assay of blood urea nitrogen (BUN), serum creatinine (SCr) and urinary albumin/creatinine (A/C) ratio, besides, kidney expression of neutrophil gelatinase-associated lipocalin (NGAL) by immunoblotting. Key findingsOur results revealed that treatment with testosterone either alone or combined with gentamicin increased megalin expression at mRNA and protein levels as well as at the functional level. These effects were paralleled by increased GIN as manifested by increased SCr, BUN, A/C ratio, renal expression of NGAL or histopathological changes. On the other hand, treatment with flutamide ameliorated GIN and megalin expression and functionality. Computational analysis of megalin promotor revealed the presence of multiple response elements that mediate androgen response. SignificanceAndrogen/AR regulates megalin expression at the transcriptional level and consequently GIN. This may explain the sexual dimorphism in GIN and might represent a druggable target for treatment or prevention of GIN.

CXCL14 Overexpression Attenuates Sepsis-Associated Acute Kidney Injury by Inhibiting Proinflammatory Cytokine Production.

CXCL14 is a relatively novel chemokine with a wide spectrum of biological activities. The present study was designed to investigate whether CXCL14 overexpression attenuates sepsis-associated acute kidney injury (AKI) in mice. Sepsis model has been established by cecal ligation and puncture (CLP). CLP induced AKI in mice as assessed by increased renal neutrophil gelatinase-associated lipocalin (NGAL) expression and serum creatinine levels. We found that renal CXCL14 expression in the kidney was significantly decreased at 12 hours after CLP. Correlation analysis demonstrated a negative association between renal CXCL14 expression and AKI markers including serum creatinine and renal NGAL. Moreover, CXCL14 overexpression reduced cytokine (TNF-α, IL-6, and IL-1β) production and NGAL expression in the kidney and decreased serum creatinine levels. In vivo and in vitro experiments found that CXCL14 overexpression inhibited M1 macrophage polarization but increased M2 polarization. Together, these results suggest that CXCL14 overexpression attenuates sepsis-associated AKI probably through the downregulation of macrophages-derived cytokine production. However, further studies are required to elucidate the underlying mechanism.

Effect of complement C5a on the expression of MCP-1 and NGAL in immune kidney injury of trichloroethylene sensitized mice

Objective: To explore the possible role of C5a in the pathogenesis of renal injury in TCE- sensitized mice, to analyze the impact of expression of neutrophil gelatinase-associated lipocalin (NGAL) and monocyte chemotactic protein-1 (MCP-1) in the presence or absence of C5a receptor antagonist (C5aRA) pretreatment. Methods: A total of 50 female specific pathogens free(SPF) BALB/c mice were randomly divided into blank control group (n=5) , solvent control group (n=5) , TCE group (n=20) , and TCE+C5aRA group (n= 20) . After one week for adaptive feeding, a mouse model of TCE-induced skin sensitization was established by treating with 50% TCE and 30% TCE in turn. The mice in solvent control group accept same reagents without TCE and the mice in blank control group underwent nothing. In TCE +C5aRA group, except for the TCE solution treatment, mice were intraperitoneally injected with 0.5 mg/kg C5aRA solution at the time of challenge. And the skin erythema and edema reaction were scored 24 h after the last challenge. The mice were divided into sensitization positive group and sensitization negative group according to the scoring result. The mice were aseptically sacrificed 72 h after the last challenge to obtain the kidneys. The structural damage of kidney was observed after histopathological staining. The levels of NGAL and MCP-1 mRNA and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) , respectively. Results: The sensitization rate of mice in TCE group and TCE+C5aRA group was 45.0% (9/20) and 40.0% (8/20) , respectively. No skin lesions was found in the mice of blank control group and solvent control group. The results of histopathological staining showed that the TCE sensitization positive mice showed renal tubular dilatation, vacuolar degeneration of renal tubular epithelial cells, and infiltration of interstitial cells. The pathological damage of the kidney in TCE sensitization positive group was mild, and no inflammatory cell infiltration was seen. The data of qRT-PCR showed that the expression levels of NGAL and MCP-1 mRNA in the TCE sensitization positive group were significantly increased than in solvent control group and TCE sensitization negative group (P<0.05) , while the levels of NGAL and MCP-1 mRNA in TCE+C5aRA sensitization positive group were decreased than TCE sensitization positive group (P <0.05) . The results of IHC showed that the expression levels of NGAL and MCP-1 in TCE protein sensitization positive group were significantly higher than those in solvent control group and TCE sensitization negative group (P<0.05) . After C5aRA pretreatment, the expression levels of NGAL and MCP-1 protein were decreased than the mice in TCE sensitization positive group (P<0.05) . Conclusion: The regulation of C5a on the expression of MCP-1 and NGAL may participate in TCE- induced mice kidney damage, and pharmacological inhibition of C5a seems to be an effective way to protect the kidney injury in TCE-sensitized mice.

NGAL and SMAD1 gene expression in the early detection of diabetic nephropathy by liquid biopsy

IntroductionDiabetic nephropathy (DN) is a disease that progresses with the slow and progressive decline of the glomerular filtration rate (GFR); the installation of this pathology is silent and one of...

SUN-004 Osteocrin, a bone-originated humoral factor, exerts a renoprotective role in ischemia-reperfusion injury in mice

Natriuretic peptides (NPs; atrial, brain and C-type NPs: ANP, BNP and CNP) constitute the hormonal basis of the heart-kidney network, in which the remote organs influence each other under physiological and pathophysiological conditions. We recently reported that osteocrin (Ostn), originally identified as a secretory peptide in the bone and muscle, exerted bone elongation and cardioprotective effects through antagonizing NP clearance receptor NPR3. Of note, SNPs of the Ostn and NPPA/NPPB gene loci are reportedly related to a renal function decline risk by genome-wide association studies. However, the role of Ostn in the kidney has not been clarified yet. To investigate the role of Ostn, we generated systemic Ostn knockout (KO) mice, and mated with liver-specific SAP promoter-driven Ostn-transgenic mice (KO-Tg). Wild-type (WT), KO and KO-Tg mice were subjected to ischemia-reperfusion injury (IRI) by unilateral renal artery clamping. Samples were collected at 3 weeks after IRI. Ostn expression in the kidney was evaluated by X-gal immunostaining using Ostn-LacZ knockin mice. To explore the detailed alteration of Wnt-βcatenin pathway which is important for kidney fibrosis, we performed PCR-array analysis in the kidney between KO and KO-Tg mice. In atrophic and fibrotic kidneys after IRI of WT, Ostn mRNA was increased in parallel with NPR3 and CNP upregulation, but not in the contralateral or pre-IRI kidneys. Furthermore, CNP expression was markedly suppressed in the contralateral kidney after IRI. These changes were either enhanced or suppressed in KO or KO-Tg, respectively, suggesting that Ostn affects the regulation of CNP and NPR3 expression in the kidney. In KO kidneys, mRNA expression of neutrophil gelatinase-associated lipocalin (NGAL) and IL-1b was further enhanced. In KO-Tg, fibrosis and atrophy were significantly ameliorated compared to WT and KO, together with the decreased expression of pro-fibrotic (aSMA, TGF-b), pro-inflammatory (IL-1b, TNFa) and tubular injury maker (NGAL, KIM-1) genes. And also, PCR-array analysis revealed that the upregulation of Wnt-βcatenin pathway was suppressed in KO-Tg compared with KO. Similar results were obtained from MMP-7 and Myc which are induced in the downstream target of this pathway. Furthermore, X-gal immunostaining revealed that endogenous Ostn expression was induced by IRI in damaged-tubular epithelial cells of corticomedullary junction in the kidney. This was co-localized with the expression of MMP-7 and Wnt2, also induced by IRI. Exacerbation of IRI-induced kidney injury in Ostn-deficient mice was attenuated by ectopic overexpression of Ostn. Our data suggest a renoprotective role of Ostn, and could provide a potential therapeutic strategy against acute kidney injury.

Evaluation of Serum Level of Neutrophil Gelatinase Associated Lipocalin in Acne Vulgaris

Acne is a chronic inflammatory disease of the pilosebaceous unit resulting from androgen-induced increased sebum production, altered keratinization, inflammation, and bacterial colonization of hair follicles by Propionibacterium acne, Neutrophil gelatinase associated lipocalin expression have been reported in several normal tissues where it serves to provide protection against bacterial infection and modulate oxidative stress. The aim of the work is to evaluate the serum level of neutrophil gelatinase in patients with acne. This might have been a cross sectional instance similar study, directed in the dermatology, Venerology What's more Andrology division Furthermore clinical pathology section from claiming Benha college Hospitals, the ponder might have been led for eighty subjects ordered under two groups: aggregation i comprised for Fivty patients with skin inflammation vulgaris Furthermore assembly ii comprised for thirty sound people matched for age, sex Also figure impostor list (BMI) might have been a chance to be included Similarly as the control group, serum level from claiming neutrophil gelatinase might have been be measured utilizing Enzyme-Linked safe sorbent test. Those outcomes uncovered that there might have been Exceedingly measurable huge Contrast the middle of Assemblies Concerning illustration view NGAL for higher values Around instance assembly. Our information include novel viewpoints of the part of NGAL in the pathophysiology about skin inflammation vulgaris patients. Further investigations will require on report card those useful part of NGAL on fundamental focus receptor Previously, skin inflammation vulgaris should uncover inventive medicine methodologies.

Evaluation of serum neutrophil gelatinase-associated lipocalin in older patients with chronic kidney disease.

ObjectiveChronic kidney disease (CKD) is a major health‐care burden all over the world, and aging is an important risk factor for end‐stage renal disease (ESRD). Neutrophil gelatinase‐associated lipocalin (NGAL) has been confirmed as a novel marker for early diagnosis of acute kidney injury. Other studies have found that NGAL takes part in the mechanisms of CKD progression. The aim of this study was to evaluate the expression of serum NGAL in CKD, particularly in elderly patients who rapidly progressed to end‐stage renal failure.MethodsSerum NGAL, cystatin C, creatinine, urea, and other factors were evaluated in a cohort of 160 CKD patients (mean age 75.29 ± 12.08 years) with various etiologies.ResultsSerum NGAL was closely related to cystatin C, creatinine, urea, and estimated glomerular filtration rate (eGFR). Special correlations between NGAL and, respectively, anemia and hypoalbuminemia were also found. The cutoff value of NGAL was calculated from Stage 2 to Stage 5. Receiver–operator curve analysis showed good area under the curve (>0.8) and high sensitivity (> 70%) on the cutoff value of NGAL. The NGAL levels increased progressively with the increasing of 2‐ and 5‐year risk of ESRD using the Kidney Failure Risk Equations (KFRE).ConclusionIn elderly patients with CKD, serum NGAL reflects renal impairment and presents a strong and independent risk marker for progression of ESRD.

Epidermal growth factor alleviates cerebral ischemia-induced brain injury by regulating expression of neutrophil gelatinase-associated lipocalin

Neutrophil gelatinase-associated lipocalin (NGAL) may be a biomarker candidate for brain injury and a novel therapeutic target in ischemic stroke. Epidermal growth factor (EGF) has protective effects on ischemic injury via activating EGF receptor (EGFR). Whether the protection mechanism of activating EGF-EGFR axis against brain injury is involved in regulating NGAL is still unknown. In the present study, we attempted to explore the expression of NGAL in ischemic brain and the effects of EGF on the NGAL expression in a mouse model of middle cerebral artery occlusion (MCAO). Results suggested that the NGAL expression in ischemic brain was markedly increased after cerebral ischemic damage, and specific NGAL-siRNA can attenuate ischemia-triggered infarct volume and neurological deficit. Then, we found that intracerebroventricular EGF treatment may reduce the level of NGAL in ischemic brain, accompanied by functional improvements. Meanwhile, specific JAK2/STAT3 inhibitor AG490 can reverse EGF-induced reduction of NGAL level. Therefore, the elevated NGAL level in ischemic brain may be an important participant in ischemic brain injury. EGF/EGFR activation ameliorated infarct volume of brain tissues and neurological deficit, and the underlying mechanism is involved in regulating the expression of NGAL via the activation of JAK2/STAT3 pathway.

P099 Neutrophil gelatinase-associated lipocalin (NGAL) as biomarker in collagenous colitis

Abstract Background Neutrophil gelatinase-associated lipocalin (NGAL) is upregulated in the intestinal epithelium in inflammatory bowel disease and is a biomarker with sensitivity and specificity comparable to calprotectin. Microscopic colitis (MC) is a common cause of chronic, watery diarrhoea and represents an inflammatory bowel disease with unknown aetiology and pathogenesis. Diagnosis depends on histological evaluation of colonic biopsies. There is a need for non-invasive diagnostic tools, and this study examines the potential of NGAL as a biomarker in collagenous colitis (CC) as one of the two main histological forms of MC. Methods Gene expression of colonic biopsies (n = 9/group) from active CC, budesonide treated CC in clinical remission and healthy controls (HC) was explored by RNA-sequencing analysis. An extended set of colonic biopsies (n = 17–29/group) was also examined by immunohistochemistry (IHC) and in situ hybridisation (ISH). Faecal samples from the same patient groups, in addition to samples from patients with irritable bowel disease diarrhoea (IBS-D), were assayed for NGAL and calprotectin (Calpro) by ELISA. Results The NGAL gene, LCN2, was significantly upregulated in active CC vs. HC (log2 2.694, fold change 6.471) adjusted p-value 0.005) and in pairs of active CC vs. treated CC LCN2 was significantly downregulated (log2 0.345, fold change −1.536), adjusted p-value 0.04). Both immunohistochemical staining and in situ hybridisation identified increased NGAL expression localised to the mucosal epithelial cells in active CC, compared with an almost absent and scarce expression in HC and treated CC, respectively. There were great individual differences in faecal concentrations of NGAL particularly in the active CC group, but the NGAL concentrations were significantly increased compared with HC, IBS-D and treated CC. Conclusion NGAL is upregulated and located mainly to the colonic epithelium of active CC and reduced in clinical remission after budesonide treatment. This is also reflected in the faecal concentrations. We propose NGAL as a valuable biomarker in evaluating the inflammatory activity related to CC and a potential faecal biomarker discriminating CC from IBS-D.