Extracellular sodium regulates fibroblast growth factor 23 (FGF23) formation.

The bone-derived hormone fibroblast growth factor-23 (FGF23) has recently received much attention due to its association with chronic kidney disease and cardiovascular disease progression. Extracellular sodium concentration ([Na+]) plays a significant role in bone metabolism. Hyponatremia (lower serum [Na+]) has recently been shown to be independently associated with FGF23 levels in patients with chronic systolic heart failure. However, nothing is known about the direct impact of [Na+] on FGF23 production. Here, we show that an elevated [Na+] (+20 mM) suppressed FGF23 formation, whereas low [Na+] (-20 mM) increased FGF23 synthesis in the osteoblast-like cell lines UMR-106 and MC3T3-E1. Similar bidirectional changes in FGF23 abundance were observed when osmolality was altered by mannitol but not by urea, suggesting a role of tonicity in FGF23 formation. Moreover, these changes in FGF23 were inversely proportional to the expression of NFAT5 (nuclear factor of activated T cells-5), a transcription factor responsible for tonicity-mediated cellular adaptations. Furthermore, arginine vasopressin, which is often responsible for hyponatremia, did not affect FGF23 production. Next, we performed a comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low versus high [Na+], which revealed several novel genes involved in cellular adaptation to altered tonicity. Additional analysis of cells with Crisp-Cas9-mediated NFAT5 deletion indicated that NFAT5 controls numerous genes associated with FGF23 synthesis, thereby confirming its role in [Na+]-mediated FGF23 regulation. In line with these in vitro observations, we found that hyponatremia patients have higher FGF23 levels. Our results suggest that [Na+] is a critical regulator of FGF23 synthesis.

Characteristics and Treatment Outcomes of Transition among Patients with Inflammatory Bowel Disease.

PURPOSE: This study aimed to assess disease characteristics and outcomes of transition in patient care among adolescent patients with inflammatory bowel disease (IBD). MATERIALS AND METHODS: Data from patients younger than 18 years who were diagnosed with IBD (Crohn's disease, ulcerative colitis, or intestinal Behçet's disease) were investigated. We categorized the patients into two groups: transition IBD group (Group A, diagnosed in pediatric care followed by transfer to/attendance in adult IBD care) and non-transition group (Group B, diagnosed and followed up in pediatric care or adult IBD care without transfer). RESULTS: Data from a total of 242 patients [Group A (n=29, 12.0%), Group B (n=213, 88.0%)] were analyzed. A significantly higher number of patients was diagnosed at an earlier age in Group A than in Group B (p<0.001). Group A patients had more severe disease in terms of number of disease flare ups (p=0.011) and frequency of bowel-related complications (p<0.001). Multiple linear regression analysis showed that Group B patients had more medical non-compliance than Group A patients (ß=2.31, p=0.018). After transition, IBD-related admission frequency, emergency admission frequency, disease flare frequency, and medical non-compliance were significantly improved. CONCLUSION: The transition IBD group had more severe disease. Medical non-compliance was lower in the transition IBD group. Clinical outcomes improved after transition.

Macrophage transcription factor TonEBP promotes systemic lupus erythematosus and kidney injury via damage-induced signaling pathways.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by autoreactive B cells and dysregulation of many other types of immune cells including myeloid cells. Lupus nephritis (LN) is a common target organ manifestations of SLE. Tonicity-responsive enhancer-binding protein (TonEBP, also known as nuclear factor of activated T-cells 5 (NFAT5)), was initially identified as a central regulator of cellular responses to hypertonic stress and is a pleiotropic stress protein involved in a variety of immunometabolic diseases. To explore the role of TonEBP, we examined kidney biopsy samples from patients with LN. Kidney TonEBP expression was found to be elevated in these patients compared to control patients - in both kidney cells and infiltrating immune cells. Kidney TonEBP mRNA was elevated in LN and correlated with mRNAs encoding inflammatory cytokines and the degree of proteinuria. In a pristane-induced SLE model in mice, myeloid TonEBP deficiency blocked the development of SLE and LN. In macrophages, engagement of various toll-like receptors (TLRs) that respond to damage-associated molecular patterns induced TonEBP expression via stimulation of its promoter. Intracellular signaling downstream of the TLRs was dependent on TonEBP. Therefore, TonEBP can act as a transcriptional cofactor for NF-κB, and activated mTOR-IRF3/7 via protein-protein interactions. Additionally, TonEBP-deficient macrophages displayed elevated efferocytosis and animals with myeloid deficiency of TonEBP showed reduced Th1 and Th17 differentiation, consistent with macrophages defective in TLR signaling. Thus, our data show that myeloid TonEBP may be an attractive therapeutic target for SLE and LN.

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

Tonicity-responsive enhancer-binding protein promotes stemness of liver cancer and cisplatin resistance.

BACKGROUND: High recurrence and chemoresistance drive the high mortality in hepatocellular carcinoma (HCC). Although cancer stem cells are considered to be the source of recurrent and chemoresistant tumors, they remain poorly defined in HCC. Tonicity-responsive enhancer binding protein (TonEBP) is elevated in almost all HCC tumors and associated with recurrence and death. We aimed to identify function of TonEBP in stemness and chemoresistance of liver cancer. METHODS: Tumors obtained from 280 HCC patients were analyzed by immunohistochemical analyses. Stemness and chemoresistance of liver CSCs (LCSCs) were investigated using cell culture. Tumor-initiating activity was measured by implanting LCSCs into BALB/c nude mice. FINDINGS: Expression of TonEBP is higher in LCSCs in HCC cell lines and correlated with markers of LCSCs whose expression is significantly associated with poor prognosis of HCC patients. TonEBP mediates ATM-mediated activation of NF-κB, which stimulates the promoter of a key stem cell transcription factor SOX2. As expected, TonEBP is required for the tumorigenesis and self-renewal of LSCSs. Cisplatin induces the recruitment of the ERCC1/XPF dimer to the chromatin in a TonEBP-dependent manner leading to DNA repair and cisplatin resistance. The cisplatin-induced inflammation in LSCSs is also dependent on the TonEBP-ERCC1/XPF complex, and leads to enhanced stemness via the ATM-NF-κB-SOX2 pathway. In HCC patients, tumor expression of ERCC1/XPF predicts recurrence and death in a TonEBP-dependent manner. INTERPRETATION: TonEBP promotes stemness and cisplatin resistance of HCC via ATM-NF-κB. TonEBP is a key regulator of LCSCs and a promising therapeutic target for HCC and its recurrence.

Transcriptional Regulator TonEBP Mediates Oxidative Damages in Ischemic Kidney Injury.

TonEBP (tonicity-responsive enhancer binding protein) is a transcriptional regulator whose expression is elevated in response to various forms of stress including hyperglycemia, inflammation, and hypoxia. Here we investigated the role of TonEBP in acute kidney injury (AKI) using a line of TonEBP haplo-deficient mice subjected to bilateral renal ischemia followed by reperfusion (I/R). In the TonEBP haplo-deficient animals, induction of TonEBP, oxidative stress, inflammation, cell death, and functional injury in the kidney in response to I/R were all reduced. Analyses of renal transcriptome revealed that genes in several cellular pathways including peroxisome and mitochondrial inner membrane were suppressed in response to I/R, and the suppression was relieved in the TonEBP deficiency. Production of reactive oxygen species (ROS) and the cellular injury was reproduced in a renal epithelial cell line in response to hypoxia, ATP depletion, or hydrogen peroxide. The knockdown of TonEBP reduced ROS production and cellular injury in correlation with increased expression of the suppressed genes. The cellular injury was also blocked by inhibitors of necrosis. These results demonstrate that ischemic insult suppresses many genes involved in cellular metabolism leading to local oxidative stress by way of TonEBP induction. Thus, TonEBP is a promising target to prevent AKI.

TonEBP Suppresses the HO-1 Gene by Blocking Recruitment of Nrf2 to Its Promoter.

TonEBP is a key transcriptional activator in macrophages with an M1 phenotype. High expression of TonEBP is associated with many inflammatory diseases. Heme oxygenase-1 (HO-1), a stress-inducible protein, is induced by various oxidative and inflammatory signals, and its expression is regarded as an adaptive cellular response to inflammation and oxidative injury. Here, we show that TonEBP suppresses expression of HO-1 by blocking Nrf2 binding to the HO-1 promoter, thereby inducing polarization of macrophages to the M1 phenotype. Inhibition of HO-1 expression or activity significantly reduced the inhibitory responses on M1 phenotype and stimulatory effects on M2 phenotype by TonEBP knockdown. Additional experiments showed that HO-1 plays a role in the paracrine anti-inflammatory effects of TonEBP knockdown in macrophages. Identification of HO-1 as a downstream effector of TonEBP provides new possibilities for improved therapeutic approaches to inflammatory diseases.

Tonicity-Responsive Enhancer-Binding Protein Mediates Hyperglycemia-Induced Inflammation and Vascular and Renal Injury.

Diabetic nephropathy (DN) has become the single leading cause of ESRD in developed nations. Bearing in mind the paucity of effective treatment for DN and progressive CKD, novel targets for treatment are sorely needed. We previously reported that increased activity of tonicity-responsive enhancer-binding protein (TonEBP) in monocytes was associated with early DN in humans. We now extend these findings by testing the hypotheses that TonEBP in macrophages promotes hyperglycemia-mediated proinflammatory activation and chronic renal inflammation leading to DN and CKD, and TonEBP genetic variability in humans is associated with inflammatory, renal, and vascular function-related phenotypes. In a mouse model of DN, compared with the wild-type phenotype, TonEBP haplodeficiency associated with reduced activation of macrophages by hyperglycemia, fewer macrophages in the kidney, lower renal expression of proinflammatory genes, and attenuated DN. Furthermore, in a cohort of healthy humans, genetic variants within TonEBP associated with renal function, BP, and systemic inflammation. One of the genetic variants associated with renal function was replicated in a large population-based cohort. These findings suggest that TonEBP is a promising target for minimizing diabetes- and stress-induced inflammation and renovascular injury.

TonEBP suppresses IL-10-mediated immunomodulation.

TonEBP is a key transcriptional activator of M1 phenotype in macrophage, and its high expression is associated with many inflammatory diseases. During the progression of the inflammatory responses, the M1 to M2 phenotypic switch enables the dual role of macrophages in controlling the initiation and resolution of inflammation. Here we report that in human and mouse M1 macrophages TonEBP suppresses IL-10 expression and M2 phenotype. TonEBP knockdown promoted the transcription of the IL-10 gene by enhancing chromatin accessibility and Sp1 recruitment to its promoter. The enhanced expression of M2 genes by TonEBP knockdown was abrogated by antagonism of IL-10 by either neutralizing antibodies or siRNA-mediated silencing. In addition, pharmacological suppression of TonEBP leads to similar upregulation of IL-10 and M2 genes. Thus, TonEBP suppresses M2 phenotype via downregulation of the IL-10 in M1 macrophages.

Prognostic role of magnetic resonance imaging vs. computed tomography for hepatocellular carcinoma undergoing chemoembolization.

BACKGROUND & AIMS: Computed tomography (CT) and magnetic resonance imaging (MRI) play important roles in diagnosis and staging of hepatocellular carcinoma (HCC). However, prognostic roles of radiological characteristics are not yet determined. METHODS: Eighty-eight patients treated with chemoembolization were analysed. Radiological parameters at baseline were assessed in all patients using both dynamic CT and MRI. Treatment responses were assessed using modified RECIST 4 weeks after the first chemoembolization. RESULTS: Gross vascular invasion (GVI), bile duct invasion, irregular tumour margin (ITM), peripheral ragged enhancement (PRE) and satellite nodules on CT or MRI were associated with non-response (stable disease or progression) after chemoembolization respectively (all P ≤ 0.05). GVI, ITM and PRE on CT or MRI were also independently associated with poor overall survival (OS) respectively (all P ≤ 0.05). Using these results, a prognostic scoring system for CT and MRI were developed; 0, absence of all three features (GVI, ITM and PRE); 1, presence of one feature; 2, presence of two features; and 3, presence of three features. After adjusting tumour size, tumour number and alpha-foetoprotein level, both CT and MRI scores were independently associated with OS (both P < 0.001). Patients with CT or MRI score ≥2 had a worse OS than those with score <2 (adjusted hazard ratios, 3.837 and 2.938 respectively). MRI-specific parameters such as signal intensity on T2- or T1-weighted images, fat signal or hyperintensity on diffusion-weighted images did not have prognostic value (all P > 0.05). CONCLUSIONS: Radiological parameters by CT and MRI may be useful in biological characterization of tumours and prognostification for HCC treated with chemoembolization.

Risk assessment of clinical outcomes in Asian patients with chronic hepatitis B using enhanced liver fibrosis test.

UNLABELLED: Serum fibrosis markers, such as the enhanced liver fibrosis (ELF) test, have been suggested as alternatives for liver biopsy (LB) in assessing liver fibrosis. We investigated the efficacy of the ELF test in predicting development of liver-related events (LREs) in patients with chronic hepatitis B (CHB). A total of 170 patients (103 men; 60.6%) with CHB who underwent LB and serological tests for determining ELFs were enrolled. All patients were followed up to monitor LRE development, defined as hepatic decompensation, hepatocellular carcinoma, and/or liver-related death. The mean age was 45.3 years. During the follow-up period (median, 41 months), 39 (22.9%) patients experienced LREs. In patients with LREs, age, proportion of male gender, ELF test results, age-spleen-platelet ratio (ASPRI), liver stiffness (LS) value, and proportion of histological cirrhosis were significantly higher than those in patients without LREs (all P < 0.05). Areas under the receiver operating characteristic curves to predict LRE development were 0.808 for the ELF test, 0.732 for LS value, 0.713 for histological fibrosis stages using Batts and Ludwig's scoring system, and 0.687 for ASPRI. On multivariate analysis, along with age, the ELF test was an independent predictor of LRE development (adjusted hazard ratio [HR], 1.438; P < 0.001). When we applied a three-tier stratification of our study population using cut-off ELF values of 8.10 and 10.40, patients with low (P = 0.002; adjusted HR: 0.045; 95% confidence interval [CI]: 0.006-0.330) and intermediate (P < 0.001; adjusted HR: 0.239; 95% CI: 0.122-0.469) ELF range were found less likely to develop LREs, compared to those with high ELF range. CONCLUSION: ELF is useful in a noninvasive prediction of LRE development. Transient elastography showed a statistically similar prognostic performance for LREs as the ELF, but other noninvasive tests were inferior.

Prediction of development of liver-related events by transient elastography in hepatitis B patients with complete virological response on antiviral therapy.

OBJECTIVES: In the era of antiviral therapy, the prognostic significance of serum hepatitis B virus (HBV) DNA level as a biological gradient substantially diminished, as most patients can achieve complete virological response (CVR). We aimed to investigate the predictive roles of liver stiffness (LS) for liver-related events (LREs) among patients with CVR. METHODS: We analyzed 192 patients with chronic HBV infection who achieved CVR (defined as HBV DNA <20 IU/ml) through entecavir therapy. LS values at CVR were measured using transient elastography. LREs were defined as any cirrhotic complication, hepatocellular carcinoma, and liver-related mortality. RESULTS: The median age of the patients was 49 years, and 134 (69.8%) were male. The median LS value at CVR was 8.8 kPa. During follow-up, LREs occurred in 25 (13.0%) patients. When the population was stratified into three groups (<8.0 kPa, 8.0-13.0 kPa, and >13.0 kPa), cumulative LRE incidences increased significantly in association with LS values (log-rank test, P=0.001). Patients with an LS value >13.0 kPa (hazard ratio (HR)=12.336, 95% confidence interval (CI) 1.335-114.010; P=0.027) and 8.0-13.0 kPa (HR=8.832, 95% CI 1.092-71.432; P=0.041) were at significantly greater risk compared with those with an LS value <8.0 kPa. On multivariate analysis, age and LS values were seen to be independent predictors (all P<0.05). When LS values were incorporated into the REACH-B scoring model instead of serum HBV DNA level, a better predictive performance was seen compared with a conventional approach (areas under the receiver operating characteristic curve, 0.814 vs. 0.629, respectively). CONCLUSIONS: LS values at CVR are useful for predicting forthcoming LRE development. Thus, in the era of potent antiviral therapy, tailored surveillance strategies might be established based upon LS values at CVR.

Prognostic value of neutrophil-to-lymphocyte ratio in patients treated with concurrent chemoradiotherapy for locally advanced oesophageal cancer.

BACKGROUND: We performed a retrospective analysis of Asian patients with locally advanced oesophageal cancer to test the hypothesis that an elevated neutrophil-to-lymphocyte ratio is associated with a poor survival rate after definitive concurrent chemoradiotherapy. METHODS: In total, 138 patients diagnosed with locally advanced oesophageal cancer (TNM classification of malignant tumours stage II or III) who were treated with definitive concurrent chemoradiotherapy between January 2005 and December 2010 were retrospectively analysed. Definitive concurrent chemoradiotherapy was performed using two different chemotherapy regimens. RESULTS: The median follow-up duration was 39.5 months (range 1.1-93.4). The median progression-free survival was 14.0 months, and the median overall survival was 19.9 months. Compared with the low (<2.0) neutrophil-to-lymphocyte ratio group (n=43, 31.2%), the high (≥2.0) neutrophil-to-lymphocyte ratio group (n=95, 68.8%) exhibited significant decreases in the durations of both progression-free survival and overall survival. Using multivariate analysis, an elevated neutrophil-to-lymphocyte ratio was also significantly associated with decreased progression-free survival (HR 1.799; 95% CI, 1.050-3.083; P=0.032) and overall survival duration (HR 2.115; 95% CI, 1.193-3.749; P=0.010). CONCLUSIONS: The pretreatment neutrophil-to-lymphocyte ratio is a useful prognostic marker in patients with locally advanced oesophageal cancer treated with definitive concurrent chemoradiotherapy.

Transient elastography-based risk estimation of hepatitis B virus-related occurrence of hepatocellular carcinoma: development and validation of a predictive model.

BACKGROUND: The purpose of this study was to develop and validate a novel transient elastography-based predictive model for occurrence of hepatocellular carcinoma (HCC). METHODS: A total of 1,250 patients with chronic hepatitis B and baseline liver stiffness values were recruited between May 2005 and December 2007. The predictive model for HCC occurrence was constructed based on a Cox proportional hazards model. We estimated baseline disease-free probabilities at 3 years. Discrimination and calibration were used to validate the model. RESULTS: HCC occurred in 56 patients during a median follow-up of 30.7 months. Multivariate analysis revealed that age, male gender, and liver stiffness values were independent predictors of HCC (all P<0.05), whereas hepatitis B virus DNA ≥20,000 IU/L showed borderline statistical significance (P=0.0659). We developed a predictive model for HCC using these four variables, which showed good discrimination capability, with an area under the receiver operating characteristic curve (AUROC) of 0.806 (95% confidence interval 0.738-0.874). We used the bootstrap method to assess discrimination. The AUROC remained largely unchanged between iterations, with an average value of 0.802 (95% confidence interval 0.791-0.812). The predicted risk of occurrence of HCC calibrated well with the observed risk, with a correlation coefficient of 0.905 (P<0.001). CONCLUSION: This novel model accurately estimated the risk of HCC occurrence in patients with chronic hepatitis B.

Orthotopic liver transplantation after the combined use of locoregional therapy and sorafenib for advanced hepatocellular carcinoma.

We herein report a patient with advanced hepatitis B virus-related hepatocellular carcinoma (HCC) beyond the Milan criteria. He underwent orthotopic liver transplantation after successful HCC downstaging that satisfied the University of California, San Francisco criteria, using concurrent chemoradiation therapy with a combination of repeated hepatic arterial infusion chemotherapy (HAIC) and sorafenib. A 52-year-old male was diagnosed with advanced hepatitis B virus-related HCC beyond the Milan criteria. He underwent concurrent chemoradiation therapy (50 Gy with 20 fractions over 5 weeks with HAIC using 5-fluorouracil at a dose of 500 mg/day, which was administered during the first and fifth weeks of radiation therapy) as an initial treatment modality. This was followed by the combined use of HAIC using 5-fluorouracil (500 mg/m(2) for 5 hours on days 1-3) and cisplatin (60 mg/m(2) for 2 hours on day 2) every 4 weeks (twelve cycles) and sorafenib (from the third to the twelfth cycle of HAIC) to treat the remaining HCC. Because a remarkable decrease in the tumor burden that satisfied the University of California, San Francisco criteria was observed after these combination treatments, the patient underwent orthotopic liver transplantation with curative aim and survived for 11 months without evidence of HCC recurrence.