TNFα-induced NLRP3 inflammasome mediates adipocyte dysfunction and activates macrophages through adipocyte-derived lipocalin 2.

BACKGROUND AND AIMS: Obesity is a state of chronic low-grade systemic inflammation. Recent studies showed that NLRP3 inflammasome initiates metabolic dysregulation in adipose tissues, primarily through activation of adipose tissue infiltrated macrophages. However, the mechanism of NLRP3 activation and its role in adipocytes remains elusive. Therefore, we aimed to examine the activation of TNFα-induced NLRP3 inflammasome in adipocytes and its role on adipocyte metabolism and crosstalk with macrophages. METHODS: The effect of TNFα on adipocyte NLRP3 inflammasome activation was measured. Caspase-1 inhibitor (Ac-YVAD-cmk) and primary adipocytes from NLRP3 and caspase-1 knockout mice were utilized to block NLRP3 inflammasome activation. Biomarkers were measured by using real-time PCR, western blotting, immunofluorescence staining, and enzyme assay kits. Conditioned media from TNFα-stimulated adipocytes was used to establish the adipocyte-macrophage crosstalk. Chromatin immunoprecipitation assay was used to identify the role of NLRP3 as a transcription factor. Mouse and human adipose tissues were collected for correlation analysis. RESULTS: TNFα treatment induced NLRP3 expression and caspase-1 activity in adipocytes, partly through autophagy dysregulation. The activated adipocyte NLRP3 inflammasome participated in mitochondrial dysfunction and insulin resistance, as evidenced by the amelioration of these effects in Ac-YVAD-cmk treated 3T3-L1 cells or primary adipocytes isolated from NLRP3 and caspase-1 knockout mice. Particularly, the adipocyte NLRP3 inflammasome was involved in glucose uptake regulation. Also, TNFα induced expression and secretion of lipocalin 2 (Lcn2) in a NLRP3-dependent manner. NLRP3 could bind to the promoter and transcriptionally regulate Lcn2 in adipocytes. Treatment with adipocyte conditioned media revealed that adipocyte-derived Lcn2 was responsible for macrophage NLRP3 inflammasome activation, working as a second signal. Adipocytes isolated from high-fat diet mice and adipose tissue from obese individuals showed a positive correlation between NLRP3 and Lcn2 gene expression. CONCLUSIONS: This study highlights the importance of adipocyte NLRP3 inflammasome activation and novel role of TNFα-NLRP3-Lcn2 axis in adipose tissue. It adds rational for the current development of NLRP3 inhibitors for treating obesity-induced metabolic diseases.

Effect of antiretroviral therapy on decreasing arterial stiffness, metabolic profile, vascular and systemic inflammatory cytokines in treatment-naïve HIV: A one-year prospective study.

INTRODUCTION: Cardiovascular disease is a major cause of death among people living with HIV (PLH). Non-treated PLH show increased levels of inflammation and biomarkers of vascular activation, and arterial stiffness as a prognostic cardiovascular disease risk factor. We investigated the effect of one year of ART on treatment-naïve HIV(+) individuals on arterial stiffness and inflammatory and vascular cytokines. METHODS: We cross-sectionally compared aortic stiffness via tonometry, inflammatory, and vascular serum cytokines on treatment-naïve (n = 20) and HIV (-) (n = 9) matched by age, sex, metabolic profile, and Framingham score. We subsequently followed young, treatment-naïve individuals after 1-year of ART and compared aortic stiffness, metabolic profile, and inflammatory and vascular serum biomarkers to baseline. Inflammatory biomarkers included: hs-CRP, D-Dimer, SAA, sCD163s, MCP-1, IL-8, IL-18, MRP8/14. Vascular cytokines included: myoglobin, NGAL, MPO, Cystatin C, ICAM-1, VCAM-1, and MMP9. RESULTS: Treatment-naïve individuals were 34.8 years old, mostly males (95%), and with high smoking prevalence (70%). Baseline T CD4+ was 512±324 cells/mcL. cfPWV was similar between HIV(-) and treatment-naïve (6.8 vs 7.3 m/s; p = 0.16) but significantly decreased after ART (-0.52 m/s; 95% CI -0.87 to -0.16; p0.006). Almost all the determined cytokines were significantly higher compared to controls, except for MCP-1, myoglobin, NGAL, cystatin C, and MMP-9. At follow-up, only total cholesterol and triglycerides increased and all inflammatory cytokines significantly decreased. Regarding vascular cytokines, MPO, ICAM-1, and VCAM-1 showed a reduction. D-Dimer tended to decrease (p = 0.06) and hs-CRP did not show a significant reduction (p = 0.17). CONCLUSION: One year of ART had a positive effect on reducing inflammatory and vascular cytokines and arterial stiffness.

Silencing LCN2 suppresses oral squamous cell carcinoma progression by reducing EGFR signal activation and recycling.

BACKGROUND: EGFR is an important signal involved in tumor growth that can induce tumor metastasis and drug resistance. Exploring targets for effective EGFR regulation is an important topic in current research and drug development. Inhibiting EGFR can effectively inhibit the progression and lymph node metastasis of oral squamous cell carcinoma (OSCC) because OSCC is a type of cancer with high EGFR expression. However, the problem of EGFR drug resistance is particularly prominent, and identifying a new target for EGFR regulation could reveal an effective strategy. METHODS: We sequenced wild type or EGFR-resistant OSCC cells and samples from OSCC patients with or without lymph node metastasis to find new targets for EGFR regulation to effectively replace the strategy of directly inhibiting EGFR and exert an antitumor effect. We then investigated the effect of LCN2 on OSCC biological abilities in vitro and in vivo through protein expression regulation. Subsequently, we elucidated the regulatory mechanism of LCN2 through mass spectrometry, protein interaction, immunoblotting, and immunofluorescence analyses. As a proof of concept, a reduction-responsive nanoparticle (NP) platform was engineered for effective LCN2 siRNA (siLCN2) delivery, and a tongue orthotopic xenograft model as well as an EGFR-positive patient-derived xenograft (PDX) model were applied to investigate the curative effect of siLCN2. RESULTS: We identified lipocalin-2 (LCN2), which is upregulated in OSCC metastasis and EGFR resistance. Inhibition of LCN2 expression can effectively inhibit the proliferation and metastasis of OSCC in vitro and in vivo by inhibiting EGFR phosphorylation and downstream signal activation. Mechanistically, LCN2 binds EGFR and enhances the recycling of EGFR, thereby activating the EGFR-MEK-ERK cascade. Inhibition of LCN2 effectively inhibited the activation of EGFR. We translated this finding by systemic delivery of siLCN2 by NPs, which effectively downregulated LCN2 in the tumor tissues, thereby leading to a significant inhibition of the growth and metastasis of xenografts. CONCLUSIONS: This research indicated that targeting LCN2 could be a promising strategy for the treatment of OSCC.

Perfusate Neutrophil Gelatinase-Associated Lipocalin, Kidney Injury Molecular-1, Liver-Type Fatty Acid Binding Protein, and Interleukin-18 as Potential Biomarkers to Predict Delayed Graft Function and Long-Term Prognosis in Kidney Transplant Recipients: A Single-Center Retrospective Study.

BACKGROUND Delayed graft function (DGF) caused by ischemia-reperfusion injury is a common pathophysiological process that should be monitored by specific biomarkers in addition to serum creatinine. Thus, this single-center retrospective study aimed to investigate the association between levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecular-1 (KIM-1), liver-type fatty acid binding protein (L-FABP), and interleukin-18 (IL-18) in DGF associated with acute kidney injury in kidney transplant recipients (KTRs) and estimated glomerular filtration rate (eGFR) at 3 years post-transplant. MATERIAL AND METHODS A total of 102 KTRs [14(13.7%) of DGF and 88(86.3%) of NON-DGF] were enrolled. DGF was defined as "dialysis is needed within 1 week after kidney transplantation". NGAL, KIM-1, L-FABP, and IL-18 were obtained from perfusate samples of donation-after-cardiac-death (DCD) kidneys, and measured by ELISA. RESULTS Compared to the NON-DGF group, KTRs in the DGF group had a statistically significant increase in levels of NGAL (P<0.001) and KIM-1 (P<0.001). Multiple logistic regression analyses showed that NGAL (OR=1.204, 95% CI 1.057-1.372, P=0.005) and KIM-1 (OR=1.248, CI=1.065-1.463, P=0.006) could be regarded as independent risk factors. The accuracy of NGAL and KIM-1 was 83.3% and 82.1%, respectively, calculated using the area under the receiver operating characteristic curve. Furthermore, the eGFR at 3 years post-transplant had a moderate negative correlation with NGAL (r=-0.208, P=0.036) and KIM-1 (r=-0.260, P=0.008). CONCLUSIONS Our results support those from previous studies showing that perfusate levels of NGAL and KIM-1 are associated with DGF in KTRs and also with reduced eGFR at 3 years post-transplant.

Lipocalin-2 promotes acute lung inflammation and oxidative stress by enhancing macrophage iron accumulation.

Lipocalin-2 (LCN2) is an acute-phase protein that regulates inflammatory responses to bacteria or lipopolysaccharide (LPS). Although the bacteriostatic role of LCN2 is well studied, the function of LCN2 in acute lung damage remains unclear. Here, LCN2 knockout (KO) mice were used to investigate the role of LCN2 in LPS-treated mice with or without recombinant LCN2 (rLCN2). In addition, we employed patients with pneumonia. RAW264.7 cells were given LCN2 inhibition or rLCN2 with or without iron chelator deferiprone. LCN2 KO mice had a higher survival rate than wild-type (WT) mice after LPS treatment. In addition to elevated LCN2 levels in serum and bronchoalveolar lavage fluid (BALF), LPS treatment also increased LCN2 protein in alveolar macrophage lysates of BALF. LCN2 deletion attenuated neutrophil and macrophage infiltration in the lungs of LPS-treated mice as well as serum and BALF interleukin-6 (IL-6). Circulating proinflammatory cytokines and LCN2-positive macrophages were prominently increased in the BALF of pneumonia patients. In addition to increase of iron-stained macrophages in pneumonia patients, increased iron-stained macrophages and oxidative stress in LPS-treated mice were inhibited by LCN2 deletion. In contrast, rLCN2 pretreatment aggravated lung inflammation and oxidative stress in LPS-treated WT mice and then resulted in higher mortality. In RAW264.7 cells, exogenous LCN2 treatment also increased inflammation and oxidative stress, whereas LCN2 knockdown markedly diminished these effects. Furthermore, deferiprone inhibited inflammation, oxidative stress, and phagocytosis in RAW264.7 cells with high LCN2 levels, as well as LPS-induced acute lung injury in WT and LCN2 KO mice. Thus, these findings suggest that LCN2 plays a key role in inflammation and oxidative stress following acute lung injury and that LCN2 is a potential therapeutic target for pneumonia or acute lung injury.

Correlation between urine vitamin D-binding protein and early-stage renal damage in Type 2 diabetes. / å°¿ç»´ç”Ÿç´ D结合蛋白与2åž‹ç³–å°¿ç— æ‚£è€ æ—©æœŸè‚¾æŸå®³çš„ç›¸å ³æ€§.

OBJECTIVES: The excretion of urinary vitamin D-binding protein (uVDBP) is related to the occurrence and development of early-stage renal damage in patients with Type 2 diabetes (T2DM). This study aims to explore the significance of detecting uVDBP in T2DM patients and its relationship with renal tubules, and to provide a new direction for the early diagnosis of T2DM renal damage. METHODS: A total of 105 patients with T2DM, who met the inclusion criteria, were included as a patient group, and recruited 30 individuals as a normal control group. The general information and blood and urine biochemical indicators of all subjects were collected; the levels of uVDBP, and a marker of tubular injury [urine kidney injury molecule 1 (uKIM-1), urine neutrophil gelatinase-associated lipocalin (uNGAL) and urine retinol-binding protein (uRBP)] were detected by enzyme-linked immunosorbent assay. The results were corrected by urinary creatinine (Cr) to uVDBP/Cr, uKIM-1/Cr, uNGAL/Cr and uRBP/Cr. The Pearson's and Spearman's correlation tests were used to analyze the correlation between uVDBP/Cr and urine albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR) and markers of tubular injury, and multivariate linear regression and receiver operating characteristic curve were used to analyze the correlation between uVDBP/Cr and UACR or eGFR. RESULTS: Compared with the normal control group, the uVDBP/Cr level in the patient group was increased (P<0.05), and which was positively correlated with UACR (r=0.774, P<0.01), and negatively correlated with eGFR (r=-0.397, P<0.01). There were differences in the levels of uKIM-1/Cr, uNGAL/Cr, and uRBP/Cr between the 2 groups (all P<0.01). The uVDBP/Cr was positively correlated with uKIM-1/Cr (r=0.752, P<0.01), uNGAL/Cr (r=0.644, P<0.01) and uRBP/Cr (r=0.812, P<0.01). The sensitivity was 90.0% and the specificity was 82.9% (UACR>30 mg/g) for evaluation of uVDBP/Cr on T2DM patients with early-stage renal damage, while the sensitivity was 75.0% and the specificity was 72.6% for evaluation of eGFR on T2DM patients with early-stage renal damage. CONCLUSIONS: The uVDBP/Cr can be used as a biomarker in early-stage renal damage in T2DM patients.

The link between bone-derived factors osteocalcin, fibroblast growth factor 23, sclerostin, lipocalin 2 and tumor bone metastasis.

The skeleton is the third most common site of metastatic disease, which causes serious bone complications and short-term prognosis in cancer patients. Prostate and breast cancers are responsible for the majority of bone metastasis, resulting in osteolytic or osteoblastic lesions. The crosstalk between bone cells and their interactions with tumor cells are important in the development of lesions. Recently, both preclinical and clinical studies documented the clinical relevance of bone-derived factors, including osteocalcin (OC) and its undercarboxylated form (ucOC), fibroblast growth factor 23 (FGF23), sclerostin (SCL), and lipocalin 2 (LCN2) as prognostic tumor biomarkers and potential therapeutic targets in bone metastasis. Both OC and ucOC could be useful targets for the prevention of bone metastasis in breast cancer. Moreover, elevated OC level may be a metastatic marker of prostate cancer. FGF23 is particularly important for those forms of cancer that primarily affect bone and/or are characterized by bone metastasis. In other tumor entities, increased FGF23 level is enigmatic. SCL plays a significant role in the pathogenesis of both osteolytic and osteoblastic lesions, as its levels are high in metastatic breast and prostate cancers. Elevated expression levels of LCN2 have been found in aggressive subtypes of cancer. However, its role in anti-metastasis varies significantly between different cancer types. Anyway, all aforementioned bone-derived factors can be used as promising tumor biomarkers. As metastatic bone disease is generally not curable, targeting bone factors represents a new trend in the prevention of bone metastasis and patient care.

Targeting Oncolytic Adenoviruses to Cancer Cells Using a Designed Ankyrin Repeat Protein Lipocalin-2 Fusion Protein.

Oncolytic viruses are a promising technology to attack cancer cells and to recruit immune cells to the tumor site. Since the Lipocalin-2 receptor (LCN2R) is expressed on most cancer cells, we used its ligand LCN2 to target oncolytic adenoviruses (Ads) to cancer cells. Therefore, we fused a Designed Ankyrin Repeat Protein (DARPin) adapter binding the knob of Ad type 5 (knob5) to LCN2 to retarget the virus toward LCN2R with the aim of analyzing the basic characteristics of this novel targeting approach. The adapter was tested in vitro with Chinese Hamster Ovary (CHO) cells stably expressing the LCN2R and on 20 cancer cell lines (CCLs) using an Ad5 vector encoding luciferase and green fluorescent protein. Luciferase assays with the LCN2 adapter (LA) showed 10-fold higher infection compared with blocking adapter (BA) in CHO cells expressing LCN2R and in cells not expressing the LCN2R. Most CCLs showed an increased viral uptake of LA-bound virus compared with BA-bound virus and for five CCLs viral uptake was comparable to unmodified Ad5. Flow cytometry and hexon immunostainings also revealed increased uptake of LA-bound Ads compared with BA-bound Ads in most tested CCLs. Virus spread was studied in 3D cell culture models and nine CCLs showed increased and earlier fluorescence signals for LA-bound virus compared with BA-bound virus. Mechanistically, we show that the LA increases viral uptake only in the absence of its ligand Enterobactin (Ent) and independently of iron. Altogether, we characterized a novel DARPin-based system resulting in enhanced uptake demonstrating potential for future oncolytic virotherapy.

Lipocalin-2 deletion attenuates lipopolysaccharide-induced acute lung inflammation via downregulating chemotaxis-related genes.

Lipocalin-2 (LCN2) is an acute phase protein used as a biomarker for acute lung injury (ALI). Although the innate immune functions of LCN2 have been studied, how LCN2 contributes to ALI induced by lipopolysaccharide (LPS) remains unknown. In this study, we investigated the effect of LCN2 deletion on LPS-induced ALI using RNA-sequencing. LPS-treated LCN2 knockout (KO) mice had a decreased histopathological score and reduced neutrophil and macrophage infiltration in lung tissue compared with LPS-treated WT mice. RNA-sequencing analysis identified 38 differentially expressed genes (DEGs), including Cxcl5, Cxcl13, Xcl1, Saa1, and Cd14. In particular, Gene Ontology analysis of DEGs revealed a significant reduction in the inflammatory response, neutrophil chemotaxis, and chemokine-mediated signaling in LPS-treated LCN2KO mice compared with LPS-treated WT mice. Thus, these results suggest that LCN2 deletion alleviates LPS-induced ALI and that LCN2 may be involved in chemotaxis-related gene expression.

Derivation and Validation of Clinical Phenotypes of the Cardiopulmonary Bypass-Induced Inflammatory Response.

BACKGROUND: Precision medicine aims to change treatment from a "one-size-fits-all" approach to customized therapies based on the individual patient. Applying a precision medicine approach to a heterogeneous condition, such as the cardiopulmonary bypass (CPB)-induced inflammatory response, first requires identification of homogeneous subgroups that correlate with biological markers and postoperative outcomes. As a first step, we derived clinical phenotypes of the CPB-induced inflammatory response by identifying patterns in perioperative clinical variables using machine learning and simulation tools. We then evaluated whether these phenotypes were associated with biological response variables and clinical outcomes. METHODS: This single-center, retrospective cohort study used Cleveland Clinic registry data from patients undergoing cardiac surgery with CPB from January 2010 to March 2020. Biomarker data from a subgroup of patients enrolled in a clinical trial were also included. Patients undergoing emergent surgery, off-pump surgery, transplantation, descending thoracoabdominal aortic surgery, and planned ventricular assist device placement were excluded. Preoperative and intraoperative variables of patient baseline characteristics (demographics, comorbidities, and laboratory data) and perioperative data (procedural data, CPB duration, and hemodynamics) were analyzed to derive clinical phenotypes using K-means-based consensus clustering analysis. Proportion of ambiguously clustered was used to assess cluster size and optimal cluster numbers. After clusters were formed, we summarized perioperative profiles, inflammatory biomarkers (eg, interleukin [IL]-6 and IL-8), kidney biomarkers (eg, urine neutrophil gelatinase-associated lipocalin [NGAL] and IL-18), and clinical outcomes (eg, mortality and hospital length of stay). Pairwise standardized difference was reported for all summarized variables. RESULTS: Of 36,865 eligible cardiac surgery cases, 25,613 met inclusion criteria. Cluster analysis derived 3 clinical phenotypes: α, ß, and γ. Phenotype α (n = 6157 [24%]) included older patients with more comorbidities, including heart and kidney failure. Phenotype ß (n = 10,572 [41%]) patients were younger and mostly male. Phenotype γ (n = 8884 [35%]) patients were 58% female and had lower body mass index (BMI). Phenotype α patients had worse outcomes, including longer hospital length of stay (mean = 9 days for α versus 6 for both ß [absolute standardized difference {ASD} = 1.15] and γ [ASD = 1.08]), more kidney failure, and higher mortality. Inflammatory biomarkers (IL-6 and IL-8) and kidney injury biomarkers (urine NGAL and IL-18) were higher with the α phenotype compared to ß and γ immediately after surgery. CONCLUSIONS: Deriving clinical phenotypes that correlate with response biomarkers and outcomes represents an initial step toward a precision medicine approach for the management of CPB-induced inflammatory response and lays the groundwork for future investigation, including an evaluation of the heterogeneity of treatment effect.

Plasma Neutrophil Gelatinase-Associated Lipocalin Associates with New-Onset Chronic Kidney Disease in the General Population.

Circulating levels of neutrophil gelatinase-associated lipocalin (NGAL) have been associated with acute kidney injury and the severity and progression of chronic kidney disease (CKD). This study investigated its potential utility as a biomarker for the risk of new-onset CKD in a population-based cohort study. Individuals without CKD at baseline (n = 4660) who participated in the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) prospective population-based cohort study in the Netherlands were included. Baseline plasma NGAL concentrations were investigated for their associations with new-onset CKD, defined as a composite outcome of an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, urinary albumin excretion (UAE) > 30 mg/24-h, or both. Mean (±SD) plasma NGAL concentrations were 104.0 (±34.7) µg/L and median eGFR was 96 [IQR: 85.3-105.8] mL/min/1.73 m2. After median follow-up of 8.3 [IQR: 7.8-8.9] years, 467 participants developed new-onset CKD. Plasma NGAL concentrations were significantly associated with an increased risk of new-onset CKD (hazard ratio [HR] per doubling 1.35 [95% CI: 1.11-1.63], p = 0.002), even after adjustment for potentially confounding factors (1.37 [1.09-1.73], p = 0.007) except baseline eGFR (1.09 [0.86-1.37], p = 0.490). In secondary analyses, plasma NGAL concentrations were significantly associated with new-onset CKD as defined by eGFR < 60 mL/min/1.73 m2 alone (adjusted HR per doubling 2.54 [1.69-3.80], p < 0.001), which was abrogated after adjustment for eGFR (1.05 [0.69-1.59], p = 0.828), also when UAE > 30 mg/24-h was set as individual outcome (1.05 [0.82-1.35], p = 0.705). Higher plasma NGAL concentrations are associated with an increased risk of developing CKD in the general population. This association is dependent on renal function, and mainly driven by new-onset CKD as defined by renal function decline.

Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.

Background: The epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to <1% oxygen). We hypothesize that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of colonoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia and compare growth, differentiation, and immunological responses at 2% and 20% oxygen. Methods: Growth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data. Results: Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks. Conclusions: Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.

Assessment of Urinary Level of Neutrophil Gelatinaseassociated Lipocalin (NAGL) in Children with Renal Scar Due to Vesicoureteral Reflux.

INTRODUCTION: Renal scarring is a serious complications of urinary tract infection and vesicoureteral reflux (VUR). The dimercaptosuccinic acid (DMSA) scan is the gold standard method for diagnosing renal scars but is an expensive procedure that risks ionizing materials and is not available to everyone. Neutrophil gelatinase-associated lipocalin (NGAL) increases following inflammation, infection, and acute kidney injury in the urine. The aim of this study was to evaluate the urinary level of NGAL and determine its diagnostic value in renal scarring. METHODS: Patients aged 3 to 60 months with pyelonephritis were included in this study. Voiding cystourethrography (VCUG) was performed in the presence of hydronephrosis on ultrasonography. Children with VUR underwent DMSA scans six months after successful treatment of pyelonephritis., Patients were divided into two groups based on the result of DMSA scan: those with and those without renal scars. Levels of urinary NGAL were measured in both groups. RESULTS: Ninety-two children with VUR (grades 2 to 5) were studied, of whom 40 had renal scars and 52 did not. The urinary level of NGAL at the cutoff point of 284 ng/dL had 70% sensitivity and 100% specificity for the detection of renal scars and was higher in patients with renal scars. (P < .05). CONCLUSION: The urinary level of NGAL is considerably higher in children with renal scarring. It is not a good test for screening and early diagnosis due to its low sensitivity, although it can identify renal scars caused by VUR with high specificity.  DOI: 10.52547/ijkd.6951.

Research Progress on Lipocalin-2 in Diabetic Encephalopathy.

Diabetic encephalopathy is a central nervous complication of diabetes mellitus which is characterized by cognitive impairment and structural and neurochemical abnormalities, which is easily neglected. Lipocalin-2 (LCN2) is a 25 kDa transporter in the lipocalin family that can transport small molecules, including fatty acids, iron, steroids, and lipopolysaccharides in the circulation. Recently, LCN2 has been found to be a significant regulator of insulin resistance and glucose homeostasis. Numerous studies have shown that LCN2 is connected to central nervous system abnormalities, including neuroinflammation and neurodegeneration, while the latest researches have found that LCN2 is closely related to the development of diabetic encephalopathy. Nevertheless, its precise role in the pathogenesis of diabetic encephalopathy remains to be determined. In this paper, we review recent evidence on the role of LCN2 in diabetic encephalopathy from multiple perspectives in order to decipher the impact of LCN2 in both the aetiology and treatment of diabetic encephalopathy.

Pyruvate Kinase M2: A New Biomarker for the Early Detection of Diabetes-Induced Nephropathy.

Diabetic nephropathy (DN) is a common complication of diabetes. DN progresses to end-stage renal disease, which has a high mortality rate. Current research is focused on identifying non-invasive potential biomarkers in the early stage of DN. We previously indicated that pyruvate kinase M2 (PKM2) is excreted in the urine of rats after cisplatin-induced acute kidney injury (AKI). However, it has not been reported whether PKM2 can be used as a biomarker to diagnose DN. Therefore, we try to compare whether the protein PKM2 can be detected in the urine samples from diabetic patients as shown in the results of DN models. In this study, high-fat diet (HFD)-induced Zucker diabetic fatty (ZDF) rats were used for DN phenotyping. After 19 weeks of receiving a HFD, the DN model's blood glucose, blood urea nitrogen, and serum creatinine levels were significantly increased; severe tubular and glomerular damages were also noted. The following protein-based biomarkers were increased in the urine of these models: kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and PKM2. PKM2 had the earliest detection rate. In the urine samples of patients, PKM2 protein was highly detected in the urine of diabetic patients but was not excreted in the urine of normal subjects. Therefore, PKM2 was selected as the new biomarker for the early diagnosis of DN. Our results reflect current knowledge on the role of PKM2 in DN.

Role of lipocalin 2 in stroke.

Stroke is the second leading cause of death worldwide; however, the treatment choices available to neurologists are limited in clinical practice. Lipocalin 2 (LCN2) is a secreted protein, belonging to the lipocalin superfamily, with multiple biological functions in mediating innate immune response, inflammatory response, iron-homeostasis, cell migration and differentiation, energy metabolism, and other processes in the body. LCN2 is expressed at low levels in the brain under normal physiological conditions, but its expression is significantly up-regulated in multiple acute stimulations and chronic pathologies. An up-regulation of LCN2 has been found in the blood/cerebrospinal fluid of patients with ischemic/hemorrhagic stroke, and could serve as a potential biomarker for the prediction of the severity of acute stroke. LCN2 activates reactive astrocytes and microglia, promotes neutrophil infiltration, amplifies post-stroke inflammation, promotes blood-brain barrier disruption, white matter injury, and neuronal death. Moreover, LCN2 is involved in brain injury induced by thrombin and erythrocyte lysates, as well as microvascular thrombosis after hemorrhage. In this paper, we review the role of LCN2 in the pathological processes of ischemic stroke; intracerebral hemorrhage; subarachnoid hemorrhage; and stroke-related brain diseases, such as vascular dementia and post-stroke depression, and their underlying mechanisms. We hope that this review will help elucidate the value of LCN2 as a therapeutic target in stroke.

Elevated levels of biomarkers of oxidative stress and renal injury linked to nitrogenous flame retardants exposure in e-waste dismantling site: A case study in China.

Nitrogenous flame retardants (NFRs) have aroused worldwide public concern as their nephrotoxic effect. However, knowledge regarding the pathogenesis mechanism of their exposure to induce kidney injury remains largely unknown. In this study, eight NFRs, four oxidative stress biomarkers (OSBs), and one kidney injury biomarker, namely neutrophil gelatinase-associated lipocalin (NGAL), were measured in urine specimens collected from residents living around e-waste disassembly and reference areas, representing two exposure scenarios. Significant higher concentrations of Σ8NFR (median: 70.6 vs. 33.8 µg/g Cre) and five biomarkers (124 vs. 97.4 µg/g Cre) were found in urines of populations living in e-waste site compared to those in the reference site (p < 0.05). Primary NFRs exhibited significant positive associations with OSBs and NGAL regardless of the population examined, implying that chronic NFRs exposure could induce oxidative stress and kidney damage. By using structure equation model, we found that oxidative stress, particularly DNA and RNA oxidation mediated 16.1% of the total effect of NFRs on NGAL in e-waste related people, but not on the general population. Overall, this study suggests long-term chronic exposure to NFRs can induce oxidative stress and renal injury in humans but the pathogenesis mode may be scenario-specific.

Punica granatum L. Polyphenolic Extract as an Antioxidant to Prevent Kidney Injury in Metabolic Syndrome Rats.

Introduction: Obesity and metabolic syndrome (MetS) constitute a rapidly increasing health problem and contribute to the development of multiple comorbidities like acute and chronic kidney disease. Insulin resistance, inappropriate lipolysis, and excess of free fatty acids (FFAs) are associated with glomerulus hyperfiltration and atherosclerosis. The important component of MetS, oxidative stress, is also involved in the destabilization of kidney function and the progression of kidney injury. Natural polyphenols have the ability to reduce the harmful effect of reactive oxygen and nitrogen species (ROS/RNS). Extract derived from Punica granatum L. is rich in punicalagin that demonstrates positive effects in MetS and its associated diseases. The aim of the study was to investigate the effect of bioactive substances of pomegranate peel to kidney damage associated with the MetS. Methods: In this study, we compared biomarkers of oxidative stress in kidney tissue of adult male Zucker Diabetic Fatty (ZDF) rats with MetS and healthy controls that were treated with Punica granatum L. extract at a dose of 100 or 200 mg/kg. Additionally, we evaluated the effect of polyphenolic extract on kidney injury markers and remodeling. The concentration of ROS/RNS, oxLDL, glutathione (GSH), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), metalloproteinase 2 and 9 (MMP-2, MMP-9), and the activity of superoxide dismutase (SOD) and catalase (CAT) were measured. Results: The data showed significant differences in oxidative stress markers between treated and untreated MetS rats. ROS/RNS levels, oxLDL concentration, and SOD activity were lower, whereas CAT activity was higher in rats with MetS receiving polyphenolic extract. After administration of the extract, markers for kidney injury (NGAL, KIM-1) decreased. Conclusion: Our study confirmed the usefulness of pomegranate polyphenols in the treatment of MetS and the prevention of kidney damage. However, further, more detailed research is required to establish the mechanism of polyphenol protection.

The von Hippel-Lindau Tumor Suppressor Gene Mutations Modulate Lipocalin-2 Expression in Ferroptotic-Inflammatory Pathways.

A previous study of an animal model with tumor suppressor gene von Hippel-Lindau (VHL) conditional knockdown suggested that tissue inflammation and fibrosis play important roles in the development of clear-cell renal cell carcinoma (ccRCC), which is consistent with the epidemiological evidence linking inflammatory kidney disease and renal cancer. Ferroptosis and inflammation have been linked in a recent study, but the exact mechanism remains unclear. This study is aimed at investigating the mechanism of lipocalin-2- (LCN-2-) mediated ferroptosis and inflammation in vhl-mutated HK-2 cells and mouse primary proximal tubule cells (mRTCs) and the polarization of macrophage RAW 264.7 cells. Based on the levels of lipid reactive oxygen species (ROS) and the expression of glutathione peroxidase 4 (GPX4) in HK-2 cells, we observed that a VHL mutation increased ROS production and depressed GPX4 expression, whereas LCN-2 knockdown reversed these effects. Accordingly, VHL appears to affect ferroptosis in an LCN-2-dependent manner. We also revealed that LCN-2 sensitizes HK-2 cells to inflammation and macrophage RAW 264.7 cells to M1-like polarization. This study provides novel insights into the potential therapeutic target and strategy for attenuating the progression of ccRCC by revealing the role of VHL in regulating chronic inflammation within the LCN-2-ferroptosis pathway.

Urinary biomarkers to predict acute kidney damage and mortality in COVID-19.

INTRODUCTION: Acute kidney injury (AKI) is a frequent condition in patients hospitalized for COVID-19. There are only a few reports on the use of urinary biomarkers in COVID-19 and no data so far comparing the prognostic use of individual biomarkers in the prediction of adverse outcomes. MATERIALS AND METHODS: We performed a prospective mono-centric study on the value of urinary biomarkers in predicting the composite endpoint of a transfer to the intensive care unit, the need for renal replacement therapy, mechanical ventilation, and in-hospital mortality. 41 patients hospitalized for COVID-19 were enrolled in this study. Urine samples were obtained shortly after admission to assess neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), calprotectin, and vascular non-inflammatory molecule-1 (vanin-1). RESULTS: We identified calprotectin as a predictor of a severe course of the disease requiring intensive care treatment (AUC 0.728, p = 0.016). Positive and negative predictive values were 78.6% and 76.9%, respectively, using a cut-off concentration of 127.8 ng/mL. NGAL tended to predict COVID-19-associated AKI without reaching statistical significance (AUC 0.669, p = 0.053). The best parameter in the prediction of in-hospital mortality was NGAL as well (AUC 0.674, p = 0.077). KIM-1 and vanin-1 did not reach significance for any of the investigated endpoints. CONCLUSION: While KIM-1 and vanin-1 did not provide prognostic clinical information in the context of COVID-19, the present study shows that urinary calprotectin is moderately predictive of the need for intensive care unit (ICU) admission, and NGAL may be modestly predictive of AKI in COVID-19. Calprotectin and NGAL show promise as potential helpful adjuncts in the identification of patients at increased risk of poor outcomes or complications in COVID-19.