DUAL EFFECTS OF BILIRUBIN ON THE PROLIFERATION OF RAT RENAL NRK52E CELLS AND ITS ASSOCIATION WITH GAP JUNCTIONS

Objective: The effect of bilirubin on renal pathophysiology is controversial. This study aimed to observe the effects of bilirubin on the proliferation of normal rat renal tubular epithelial cell line (NRK52E) and its potential interplay with gap junction function. Methods: Cultured NRK52E cells, seeded respectively at high- or low- densities, were treated with varying concentrations of bilirubin for 24 hours. Cell injury was assessed by measuring cell viability and proliferation, and gap junction function was assessed by Parachute dye-coupling assay. Connexin 43 protein was assessed by Western blotting. Results: At doses from 17.1 to 513μmol/L, bilirubin dose-dependently enhanced cell viability and colony-formation rates when cells were seeded at either high- or low- densities (all p\u3c0.05 vs. solvent group) accompanied with enhanced intercellular fluorescence transmission and increased Cx43 protein expression in high-density cells. However, the above effects of BR were gradually reversed when its concentration increased from 684 to 1026μmol/L. In high-density cells, gap junction inhibitor 12-O-tetradecanoylphorbol 13- acetate attenuated bilirubin-induced enhancement of colony-formation and fluorescence transmission. However, in the presence of high concentration bilirubin (1026μmol/L), activation of gap junction with retinoid acid decreased colony-formation rates. Conclusion: Bilirubin can confer biphasic effects on renal NRK52E cell proliferation potentially by differentially affecting gap junction functions

Biphasic effects of bilirubin on NRK52E cells

Objective: The effect of bilirubin on renal pathophysiology is controversial. This study aimed to observe the effects of bilirubin on the proliferation of normal rat renal tubular epithelial cell line (NRK52E) and its potential interplay with gap junction function. Methods: Cultured NRK52E cells, seeded respectively at high- or low- densities, were treated with varying concentrations of bilirubin for 24 hours. Cell injury was assessed by measuring cell viability and proliferation, and gap junction function was assessed by Parachute dye-coupling assay. Connexin 43 protein was assessed by Western blotting. Results: At doses from 17.1 to 513μmol/L, bilirubin dose-dependently enhanced cell viability and colony-formation rates when cells were seeded at either high- or low- densities (all p<0.05 vs. solvent group) accompanied with enhanced intercellular fluorescence transmission and increased Cx43 protein expression in high-density cells. However, the above effects of BR were gradually reversed when its concentration increased from 684 to 1026μmol/L. In high-density cells, gap junction inhibitor 12-O-tetradecanoylphorbol 13- acetate attenuated bilirubin-induced enhancement of colony-formation and fluorescence transmission. However, in the presence of high concentration bilirubin (1026μmol/L), activation of gap junction with retinoid acid decreased colony-formation rates. Conclusion: Bilirubin can confer biphasic effects on renal NRK52E cell proliferation potentially by differentially affecting gap junction functions

Propofol Attenuates Small Intestinal Ischemia Reperfusion Injury through Inhibiting NADPH Oxidase Mediated Mast Cell Activation

Both oxidative stress and mast cell (MC) degranulation participate in the process of small intestinal ischemia reperfusion (IIR) injury, and oxidative stress induces MC degranulation. Propofol, an anesthetic with antioxidant property, can attenuate IIR injury. We postulated that propofol can protect against IIR injury by inhibiting oxidative stress subsequent from NADPH oxidase mediated MC activation. Cultured RBL-2H3 cells were pretreated with antioxidant N-acetylcysteine (NAC) or propofol and subjected to hydrogen peroxide (H2O2) stimulation without or with MC degranulator compound 48/80 (CP). H2O2 significantly increased cells degranulation, which was abolished by NAC or propofol. MC degranulation by CP further aggravated H2O2 induced cell degranulation of small intestinal epithelial cell, IEC-6 cells, stimulated by tryptase. Rats subjected to IIR showed significant increases in cellular injury and elevations of NADPH oxidase subunits p47phox and gp91phox protein expression, increases of the specific lipid peroxidation product 15-F2t-Isoprostane and interleukin-6, and reductions in superoxide dismutase activity with concomitant enhancements in tryptase and β-hexosaminidase. MC degranulation by CP further aggravated IIR injury. And all these changes were attenuated by NAC or propofol pretreatment, which also abrogated CP-mediated exacerbation of IIR injury. It is concluded that pretreatment of propofol confers protection against IIR injury by suppressing NADPH oxidase mediated MC activation

Connexin32 gap junction channels deliver miR155-3p to mediate pyroptosis in renal ischemia-reperfusion injury

Abstract Objectives To explore whether the gap junction (GJ) composed by connexin32(Cx32) mediated pyroptosis in renal ischemia-reperfusion(I/R) injury via transmitting miR155-3p, with aim to provide new strategies for the prevention and treatment of acute kidney injury (AKI) after renal I/R. Methods 8–10 weeks of male C57BL/ 6 wild-type mice and Cx32 knockdown mice were divided into two groups respectively: control group and renal I/R group. MCC950 (50 mg/kg. ip.) was used to inhibit NLRP3 in vivo. Human kidney tubular epithelial cells (HK - 2) and rat kidney tubular epithelial cells (NRK-52E) were divided into high-density group and low-density group, and treated with hypoxia reoxygenation (H/R) to mimic I/R. The siRNA and plasmid of Cx32, mimic and inhibitor of miR155-3p were transfected into HK - 2 cells respectively. Kidney pathological and functional injuries were measured. Western Blot and immunofluorescent staining were used to observe the expression of NLRP3, GSDMD, GSDMD-N, IL - 18, and mature IL-18. The secretion of IL-18 and IL-1β in serum, kidney tissue and cells supernatant were detected by enzyme-linked immuno sorbent assay (ELISA) kit, and the expression of NLPR3 and miR155-3p were detected by RT-qPCR and fluorescence in situ hybridization (FISH). Results Tubular pyroptosis were found to promote AKI after I/R in vivo and Cx32-GJ regulated pyroptosis by affecting the expression of miR155-3p after renal I/R injury. In vitro, H/R could lead to pyroptosis in HK-2 and NRK-52E cells. When the GJ channels were not formed, and Cx32 was inhibited or knockdown, the expression of miR155-3p was significantly reduced and the pyroptosis was obviously inhibited, leading to the reduction of injury and the increase of survival rate. Moreover, regulating the level of miR155-3p could affect survival rate and pyroptosis in vitro after H/R. Conclusions The GJ channels composed of Cx32 regulated tubular pyroptosis in renal I/R injury by transmitting miR155-3p. Inhibition of Cx32 could reduce the level of miR155-3p further to inhibit pyroptosis, leading to alleviation of renal I/R injury which provided a new strategy for preventing the occurrence of AKI. Video Abstrac

Clinical Findings in Diabetes Mellitus Patients with COVID-19

Backgrounds. Diabetes mellitus (DM) is one of the most common comorbidities in patients with coronavirus disease (COVID-19). We aim to summarize the clinical features of DM patients with COVID-19 and find out potential factors associated with severe disease. Methods. In this retrospective, single-center study, the medical records of patients with COVID-19 in Changsha, Hunan, China, from January 21, 2020, to February 19, 2020, were reviewed. Epidemiological information, clinical features, and outcomes were compared between DM patients admitted to the intensive care unit (ICU) or not. Results. A total of 241 patients confirmed with COVID-19 were enrolled, including 19 DM patients. There were more patients in DM group admitted to the ICU than non-DM group (36.8% vs. 15.8%, P=0.045). Compared with non-DM group in the ICU, there were more female patients from DM group in the ICU (85.7% vs. 31.4%, P=0.024). On admission, the mean level of glycated hemoglobin A1c (HbA1c) was higher in the ICU DM patients than that in the non-ICU DM patients (8.5% vs. 7.1%). There were more DM patients with proteinuria in the ICU group than the non-ICU group (57.1% vs. 33.3%). Twelve DM patients (63.2%) changed diabetic therapy during hospitalization, and all DM patients admitted to the ICU used insulin. As of March 14, all 19 DM patients have been discharged, and no death occurred. Conclusions. DM patients with COVID-19 are vulnerable to severe disease, especially for female patients. High levels of HbA1c and proteinuria could be potential risk factors for severe COVID-19 in DM patients. In addition to timely systemic therapy, the control of blood glucose and proper diabetic therapy is essential to improve the prognosis of severe DM patients with COVID-19

Evolution of the Raman spectrum of graphene grown on copper upon oxidation of the substrate

Significant changes in the Raman spectrum of single-layer graphene grown on a copper film were observed after the spontaneous oxidation of the underlying substrate that occurred under ambient conditions. The frequencies of the graphene G and 2D Raman modes were found to undergo red shifts, while the intensities of the two bands change by more than an order of magnitude. To understand the origin of these effects, we further characterized the samples by scanning tunneling microscopy (STM), scanning tunneling spectroscopy (STS), and atomic force microscopy (AFM). The oxidation of the substrate produced an appreciable corrugation in the substrate without disrupting the crystalline order of the graphene overlayer and/or changing the carrier doping level. We explain the red shifts of the Raman frequencies in terms of tensile strain induced by corrugation of the graphene layer. The changes in Raman intensity with oxidation arise from the influence of the thin cuprous oxide film on the efficiency of light coupling with the graphene layer in the Raman scattering process

MC stabilizers alleviated ALI 8

<p>(A) Lung histology morphology analysis after pretreatment of MC stabilizers. Lung sections were stained with H&E and visualized at 200× magnification. (B) Lung histopathology evaluation scores. (C) MC stabilization significantly decreased the W/D weight ratio of the right middle lobe lung 8 h after OALT. (D) MC stabilization improved the PaO₂ of the rats 8 h after OALT. (E) MC stabilization increased pro-SPC expression level in rats 8 h after OALT. Values are expressed as the mean ± SE. *<i>p</i><0.05 vs. saline control group, ^#<i>p</i><0.05 vs. sham group.</p

Basic data of Sprague–Dawley rats and operative variables.

<p>Note: Body weight and anhepatic time duration in different groups show no statistical significance.</p

MC stabilization downregulated pro-inflammatory cytokines 8 h after OALT.

<p>(A) Serum TNF-α level. (B) Lung tissue TNF-α level. (C) Serum IL-1β level. (D) Lung tissue IL-1β level. (E) Serum IL-6 level. (F) Lung tissue IL-6 level. Values are expressed as the mean ± SE. *<i>p</i><0.05 vs. saline control group, ^#<i>p</i><0.05 vs. sham group.</p