High glucose level and angiotensin II type 1 receptor stimulation synergistically amplify oxidative stress in renal mesangial cells

Abstract Oxidative stress in renal mesangial cell causes diabetic glomerular changes. High glucose levels and angiotensin II (Ang II) are known to stimulate superoxide production in renal mesangial cells. However, it has been unclear whether Ang II stimulation and pre-conditioning with high glucose affects the same pathway of superoxide production in renal mesangial cells or not. In this study, we examined the levels of oxidative stress under Ang II stimulation in renal mesangial cells preincubated for six hours at various glucose levels. Intracellular levels of reactive oxidative species (ROS) were measured using dihydroethidium or 5′,6′-chloromethyl- 2′,7′ dichlorodihydro-fluorescein diacetate, which facilitates the detection of intracellular ROS under real-time fluorescent microscope. Ang II-induced elevated intracellular ROS levels were detected only when the cells were pre-incubated with high levels of glucose (13.5 mM, 27.8 mM), but was not detected under normal glucose condition (5.5 mM). Production of Ang II-induced intracellular ROS was higher under pre-treatment with 27.8 mM glucose compared to pretreatment with 13.5 mM glucose level. This ROS production in mesangial cells was induced within several minutes of the initiation of Ang II stimulation under high glucose levels. The production of intracellular ROS was significantly reduced in the presence of angiotensin II type1-receptor (AT1R) antagonist, whereas it was augmented in the presence of angiotensin II type2-receptor antagonist. In conclusion, Ang II-induced oxidative stress was augmented by high glucose levels and ROS levels were further alleviated in the presence of AT1R antagonists

Effect of TU-100 in NASH

Background : Non-alcoholic steatohepatitis (NASH) is associated with a higher risk of hepatocellular carcinoma (HCC), and the importance of the gut–liver axis has been recognized in NASH-associated HCC. We investigated the effect of TU-100 on the intestinal microbiome and hepatocarcinogenesis in a NASH model. Methods : Seven-week-old Tsumura Suzuki obese diabetes mice, a model that shows the spontaneous onset of NASH and HCC, were used. They were divided into a TU-100 treated group and a control group. Mice were sacrificed at 24 and 48 weeks to evaluate hepatic steatosis, fibrosis, carcinogenesis, cytokine expression, and microbiome abundance. Results : At 24 weeks, the TU-100 group showed significantly lower expression of IL6, IL1B, and ACTA2 mRNA in the liver (P < 0.05). At 48 weeks, the TU-100 group showed significantly lower levels of serum alanine aminotransferase. The TU-100 group also showed a lower rate of NASH than the control group (28% vs 72% ; P = 0.1). Tumor diameter was significantly smaller in the TU-100 group compared with that in the control group (P < 0.05). Regarding the intestinal microbiome, the genera Blautia and Ruminococcus were increased in the TU-100 group (P < 0.05), whereas Dorea and Erysipelotrichaceae were decreased in the TU-100 group (P < 0.05). Conclusions : TU-100 regulates the intestinal microbiome and may suppress subsequent hepatocarcinogenesis in the NASH model

Deletion of H-ferritin in macrophages alleviates obesity and diabetes induced by high-fat diet in mice

Aims/hypothesis Iron accumulation affects obesity and diabetes, both of which are ameliorated by iron reduction. Ferritin, an iron storage protein, plays a crucial role in iron metabolism. H-ferritin exerts its cytoprotective action by reducing toxicity via its ferroxidase activity. We investigated the role of macrophage H-ferritin in obesity and diabetes. Methods Conditional macrophage-specific H-ferritin knockout (LysM-Cre FthKO) mice were used and divided into 4 groups; Wild-type (WT) and LysM-Cre FthKO mice with normal diet (ND), and WT and LysM-Cre Fth-KO mice with high-fat diet (HFD). Results Iron concentration reduced, and mRNA expression of ferroportin increased in macrophages from LysM-Cre FthKO mice. HFD-induced obesity was lower in LysM-Cre FthKO mice than in WT mice at 12 weeks (body weight (g); KO 34.6 ± 5.6 vs. WT 40.1 ± 5.2). mRNA expression of inflammatory cytokines, infiltrated macrophages, and oxidative stress increased in the adipose tissue of WT mice with HFD, but was not elevated in LysM-Cre FthKO mice with HFD. However, WT mice with HFD had elevated iron concentration in adipose tissue and spleen, which was not observed in LysM-Cre FthKO mice with HFD (adipose (μmol Fe/g protein); KO 1496 ± 479 vs. WT 2316 ± 866, spleen (μmol Fe/g protein); KO 218 ± 54 vs. WT 334 ± 83). Moreover, HFD administration impaired both glucose tolerance and insulin sensitivity in WT mice, which was ameliorated in LysM-Cre FthKO mice. In addition, energy expenditure, mRNA expression of thermogenic genes, and body temperature were higher in KO mice with HFD than WT mice with HFD. In vitro experiments showed that iron content was reduced, and LPS-induced TNF-α mRNA upregulation was inhibited in a macrophage cell line transfected with Fth siRNA. Conclusions/interpretation Deletion of macrophage H-ferritin suppresses the inflammatory response by reducing intracellular iron levels, resulting in the prevention of HFD-induced obesity and diabetes. The findings from this study highlight macrophage iron levels as a potential therapeutic target for obesity and diabetes

Influence of renal function and demographic data on intrarenal Doppler ultrasonography

Intrarenal Doppler ultrasonography is a non-invasive method to evaluate the renal blood flow in patients with renal arterial stenosis as well as chronic kidney diseases (CKD). Until recently, the relationship between ultrasonography findings and CKD stage has not been fully understood. Overall, 162 patients with CKD without apparent renal arterial stenosis were included in this study, and the pulsed-wave Doppler ultrasonography findings were evaluated in terms of the following parameters: peak systolic velocity (PSV), end-diastolic velocity (EDV), and resistive index (RI) at the renal arterial trunk, hilum, segmental, and interlobar regions. Age showed a significant negative correlation with the estimated glomerular filtration rate (eGFR), kidney size, and aortic PSV. Additionally, age showed a significant positive correlation with RI in all 4 regions. The eGFR showed a positive correlation with the aortic PSV and kidney size, but a negative correlation with RI. Both age and eGFR were found to be independently associated with aortic blood flow. On the intrarenal ultrasound, EDV and RI showed stronger correlations with eGFR than PSV, suggesting that the former indices would be better markers of renal function. In particular, the interlobar EDV was found to be the best index that reflects renal function. Although the RI is also a good marker of renal function, it is confounded by age; thus, its utility would be weaker than that of the EDV. In conclusion, intrarenal pulsed-wave Doppler ultrasonography is a useful tool to estimate and evaluate the renal function; the interlobar EDV may be the best index to estimate the effective perfusion and filtration of the kidneys

Granulovacuolar Degenerations Appear in Relation to Hippocampal Phosphorylated Tau Accumulation in Various Neurodegenerative Disorders

BACKGROUND: Granulovacuolar degeneration (GVD) is one of the pathological hallmarks of Alzheimer's disease (AD), and it is defined as electron-dense granules within double membrane-bound cytoplasmic vacuoles. Several lines of evidence have suggested that GVDs appear within hippocampal pyramidal neurons in AD when phosphorylated tau begins to aggregate into early-stage neurofibrillary tangles. The aim of this study is to investigate the association of GVDs with phosphorylated tau pathology to determine whether GVDs and phosphorylated tau coexist among different non-AD neurodegenerative disorders. METHODS: An autopsied series of 28 patients with a variety of neurodegenerative disorders and 9 control patients were evaluated. Standard histological stains along with immunohistochemistry using protein markers for GVD and confocal microscopy were utilized. RESULTS: The number of neurons with GVDs significantly increased with the level of phosphorylated tau accumulation in the hippocampal regions in non-AD neurodegenerative disorders. At the cellular level, diffuse staining for phosphorylated tau was detected in neurons with GVDs. CONCLUSIONS: Our data suggest that GVDs appear in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders, while the presence of phosphorylated tau in GVD-harbouring neurons in non-AD neurodegenerative disorders was indistinguishable from age-related accumulation of phosphorylated tau. Although GVDs in non-AD neurodegenerative disorders have not been studied thoroughly, our results suggest that they are not incidental findings, but rather they appear in relation to phosphorylated tau accumulation, further highlighting the role of GVD in the process of phosphorylated tau accumulation

&gamma;H2AX, a DNA Double-Strand Break Marker, Correlates with PD-L1 Expression in Smoking-Related Lung Adenocarcinoma

In recent years, the choice of immune checkpoint inhibitors (ICIs) as a treatment based on high expression of programmed death-ligand 1 (PD-L1) in lung cancers has been increasing in prevalence. The high expression of PD-L1 could be a predictor of ICI efficacy as well as high tumor mutation burden (TMB), which is determined using next-generation sequencing (NGS). However, a great deal of effort is required to perform NGS to determine TMB. The present study focused on &gamma;H2AX, a double-strand DNA break marker, and the suspected positive relation between TMB and &gamma;H2AX was investigated. We assessed the possibility of &gamma;H2AX being an alternative marker of TMB or PD-L1. One hundred formalin-fixed, paraffin-embedded specimens of lung cancer were examined. All of the patients in the study received thoracic surgery, having been diagnosed with lung adenocarcinoma or squamous cell carcinoma. The expressions of &gamma;H2AX and PD-L1 (clone: SP142) were evaluated immunohistochemically. Other immunohistochemical indicators, p53 and Ki-67, were also used to estimate the relationships of &gamma;H2AX. Positive relationships between &gamma;H2AX and PD-L1 were proven, especially in lung adenocarcinoma. Tobacco consumption was associated with higher expression of &gamma;H2AX, PD-L1, Ki-67, and p53. In conclusion, the immunoexpression of &gamma;H2AX could be a predictor for the adaptation of ICIs as well of as PD-L1 and TMB

Usefulness of rapid on-site evaluation specimens from endoscopic ultrasound-guided fine-needle aspiration for cancer gene panel testing: A retrospective study.

Pancreatic cancer (PC) is a highly lethal malignancy, with a 5-year survival rate of 6%. Cancer gene panel testing is expected to allow selection of suitable therapeutic drugs in individual patients with PC and improve their prognosis. Although somatic mutations can be identified in formalin-fixed, paraffin-embedded samples derived from surgical specimen, the rate of surgical indication among patients with PC is only 20%. To acquire genome information with a less invasive method, we used rapid on-site evaluation (ROSE) specimens from endoscopic ultrasound-guided fine-needle aspiration. The present study aimed to retrospectively evaluate the utility of comprehensive cancer gene panel testing with ROSE specimens. DNA was extracted from preserved ROSE specimens of 26 patients diagnosed with PC between 2011 and 2017. DNA sequences of oncogenes and cancer-related genes were determined using the Ion AmpliSeq Comprehensive Caner Panel. We compared KRAS mutations between cancer gene panel testing by next-generation sequencing (NGS) and KRAS mutation analysis by polymerase chain reaction. The mean yield of DNA per extraction from ROSE specimens was 171 ng (range, 34-478 ng). On cancer gene panel testing, we noted KRAS mutations (92%), TP53 mutations (50%), CDKN2A mutations (15%), and SMAD4 mutations (31%). The concordance rate of KRAS mutations between cancer gene panel testing by NGS using ROSE specimens and KRAS mutation analysis by the companion diagnostics using residual materials was 81%. Among five cases of KRAS discordance, three showed KRAS mutations in cancer gene panel testing but not in KRAS mutation analysis. Cancer gene panel testing with ROSE specimens can help stratify unresectable PC patients without additional invasive approaches, and it can be used for therapeutic drug selection

The human central nervous system discharges carbon dioxide and lactic acid into the cerebrospinal fluid

Abstract Background The central nervous system was previously thought to draw oxygen and nutrition from the arteries and discharge carbon dioxide and other metabolic wastes into the venous system. At present, the functional role of cerebrospinal fluid in brain metabolism is not fully known. Methods In this prospective observational study, we performed gas analysis on venous blood and cerebrospinal fluid simultaneously acquired from 16 consecutive preoperative patients without any known neurological disorders. Results The carbon dioxide partial pressure (pCO2) (p < 0.0001) and lactic acid level (p < 0.001) in the cerebrospinal fluid were significantly higher than those in the peripheral venous blood, suggesting that a considerable proportion of metabolic carbon dioxide and lactic acid is discharged from the central nervous system into the cerebrospinal fluid. The oxygen partial pressure (pO2) was much higher in the cerebrospinal fluid than in the venous blood, corroborating the conventional theory of cerebrospinal fluid circulatory dynamics. The pCO2 of the cerebrospinal fluid showed a strong negative correlation with age (R = − 0.65, p = 0.0065), but the other studied variables did not show significant correlation with age. Conclusion Carbon dioxide and lactic acid are discharged into the circulating cerebrospinal fluid, as well as into the venules. The level of carbon dioxide in the cerebrospinal fluid significantly decreased with age

Specific MAPK-Associated MicroRNAs in Serum Differentiate Pancreatic Cancer from Autoimmune Pancreatitis.

Pancreatic ductal adenocarcinoma (PDAC) is difficult to distinguish from autoimmune pancreatitis (AIP) because of their clinical and radiological similarities, and therefore simple and minimally invasive surrogate markers for differential diagnosis would be useful. In our previous studies, we identified four microRNAs (miRNAs)-miR-7, miR-34a, miR-181d, and miR-193b -as MAPK-associated microRNAs whose expression was altered significantly with upregulation of the MAPK signaling pathway. Recently it has been reported that these miRNAs could be used as biomarkers in serum samples for accurate diagnosis of pancreatic lesions. The aim of the present study was to evaluate whether these MAPK-associated miRNAs in serum could be used as biomarkers for differentiating PDAC from AIP. We enrolled 69 patients with PDAC, 26 with intraductal papillary mucinous neoplasm (IPMN) and 15 with AIP. The expression of MAPK-associated miRNAs in serum was measured by quantitative real-time PCR. The 2-ΔCT method was used to quantify the expression of miRNAs, and the data were normalized using spiked-in synthetic cel-miR-39. Patients with PDAC or IPMN showed significantly higher amounts of serum MAPK-associated miRNAs than those with AIP (p<0.009 for miR-7, p<0.002 for miR-34a, p<0.001 for miR-181d, p<0.002 for miR-193b). ROC curve analysis demonstrated that these miRNAs had an area under the ROC curve (AUC) of 0.723-0.882 for differentiation between PDAC or IPMN from AIP. Furthermore, serum miR-181d was significantly associated with the presence of metastasis in patients with PDA (p = 0.014). Serum MAPK-associated miRNAs could be novel noninvasive biomarkers for differentiation between PDAC or IPMN and AIP