Xiaogang Du

Regulatory T cells/T-helper cell 17 functional imbalance in uraemic patients on maintenance haemodialysis: A pivotal link between microinflammation and adverse cardiovascular events

Aim:  Adverse cardiovascular events resulting from accelerated atherosclerosis are the leading cause of mortality in uraemic patients on maintenance haemodialysis (MHD). Chronic inflammation due to antigen‐specific responses is an important factor in the acceleration of atherosclerosis. The balance between CD4+ CD25+ forkhead/winged helix transcription factor (Foxp3)+ regulatory T cells (Treg) and T helper (Th)17 cells has been reported to play an important role in the development of inflammatory and autoimmune diseases. The aim of the present study was to assess the Treg/Th17 pattern in uraemic patients on MHD and to explore the significance of Treg/Th17 imbalance in the development and outcome of acute cardiovascular events.

CD36 Mediated Fatty Acid-Induced Podocyte Apoptosis via Oxidative Stress.

Background Hyperlipidemia-induced apoptosis mediated by fatty acid translocase CD36 is associated with increased uptake of ox-LDL or fatty acid in macrophages, hepatocytes and proximal tubular epithelial cells, leading to atherosclerosis, liver damage and fibrosis in obese patients, and diabetic nephropathy (DN), respectively. However, the specific role of CD36 in podocyte apoptosis in DN with hyperlipidemia remains poorly investigated. Methods The expression of CD36 was measured in paraffin-embedded kidney tissue samples (Ctr = 18, DN = 20) by immunohistochemistry and immunofluorescence staining. We cultured conditionally immortalized mouse podocytes (MPC5) and treated cells with palmitic acid, and measured CD36 expression by real-time PCR, Western blot analysis and immunofluorescence; lipid uptake by Oil red O staining and BODIPY staining; apoptosis by flow cytometry assay, TUNEL assay and Western blot analysis; and ROS production by DCFH-DA fluorescence staining. All statistical analyses were performed using SPSS 21.0 statistical software. Results CD36 expression was increased in kidney tissue from DN patients with hyperlipidemia. Palmitic acid upregulated CD36 expression and promoted its translocation from cytoplasm to plasma membrane in podocytes. Furthermore, palmitic acid increased lipid uptake, ROS production and apoptosis in podocytes, Sulfo-N-succinimidyloleate (SSO), the specific inhibitor of the fatty acid binding site on CD36, decreased palmitic acid-induced fatty acid accumulation, ROS production, and apoptosis in podocytes. Antioxidant 4-hydroxy-2,2,6,6- tetramethylpiperidine -1-oxyl (tempol) inhibited the overproduction of ROS and apoptosis in podocytes induced by palmitic acid. Conclusions CD36 mediated fatty acid-induced podocyte apoptosis via oxidative stress might participate in the process of DN.

Autophagy Protects against Palmitic Acid-Induced Apoptosis in Podocytes in vitro

Autophagy is a highly conserved degradation process that is involved in the clearance of proteins and damaged organelles to maintain intracellular homeostasis and cell integrity. Type 2 diabetes is often accompanied by dyslipidemia with elevated levels of free fatty acids (FFAs). Podocytes, as an important component of the filtration barrier, are susceptible to lipid disorders. The loss of podocytes causes proteinuria, which is involved in the pathogenesis of diabetic nephropathy. In the present study, we demonstrated that palmitic acid (PA) promoted autophagy in podocytes. We further found that PA increased the production of reactive oxygen species (ROS) in podocytes and that NAC (N-acetyl-cysteine), a potent antioxidant, significantly eliminated the excessive ROS and suppressed autophagy, indicating that the increased generation of ROS was associated with the palmitic acid-induced autophagy in podocytes. Moreover, we also found that PA stimulation decreased the mitochondrial membrane potential in podocytes and induced podocyte apoptosis, while the inhibition of autophagy by chloroquine (CQ) enhanced palmitic acid-induced apoptosis accompanied by increased ROS generation, and the stimulation of autophagy by rapamycin (Rap) remarkably suppressed palmitic acid-induced ROS generation and apoptosis. Taken together, these in vitro findings suggest that PA-induced autophagy in podocytes is mediated by ROS production and that autophagy plays a protective role against PA-induced podocyte apoptosis.

Breakdown of the gut barrier in patients with multiple organ dysfunction syndrome is attenuated by continuous blood purification: effects on tight junction structural proteins.

Background Breakdown of the gut barrier increases intestinal permeability and allows movement of intraluminal contents across the mucosa, which can lead to distant organ injury and multiple organ dysfunction syndrome (MODS). Intestinal permeability is associated with alterations in cellular tight junctions involving the structural proteins occludin and zonula occludens-1 (ZO-1). The aim of this study was to investigate the effect of continuous blood purification (CBP) on gut barrier function in patients with MODS. Method Serum diamine oxidase (DAO) and endotoxin, epithelial monolayer permeability, and transepithelial electrical resistance (TER) were used as markers for the assessment of gut barrier function in 22 patients with MODS who underwent continuous venovenous hemofiltration (CVVH) for 24 hours. Blood samples were taken from patients at 0, 6, 12, and 24 hours during CVVH therapy. Serum DAO and endotoxin were determined by spectrophotography. Permeability and TER were assessed using Caco-2 cell monolayers. Occludin and ZO-1 protein levels were analyzed by immunoblotting and immunofluorescence staining. And inducible nitric oxide synthase (iNOS) mRNA levels and nitric oxide (NO) production were determined by real-time PCR and spectrophotography, respectively. Result Gut barrier dysfunction was evident in patients with MODS compared with normal controls. Serum DAO, endotoxin levels, and epithelial permeability were elevated, while TER was decreased in patients with MODS, and this change was more pronounced in nonsurvivors. Breakdown and reorganization of occludin and ZO-1 away from tight junctions was found in all MODS patients. After CBP treatment, APACHE II and MODS scores improved significantly. Serum DAO and endotoxin levels and epithelial permeability also diminished, while TER increased in all patients; CBP significantly attenuated breakdown and reorganization of tight junction proteins, and also attenuated the inflammation-induced increase in iNOS mRNA expression and NO production. Conclusion Breakdown of gut barrier function is present in patients with MODS and may be correlated with poor outcomes in the disease. CBP can not only improve the general conditions, as measured by the APACHE II score, but also improve gut barrier dysfunction by attenuating the breakdown and reorganization of occludin and ZO-1. This beneficial effect of CBP on gut barrier dysfunction is associated with down-regulation of iNOS.

Effects of 1,25-(OH)(2)D (3) on the expressions of vitamin D receptor, STAT5 and cytoskeletal rearrangement in human monocytes incubated with sera from type 2 diabetes patients and diabetic nephropathy patients with uremia.

ObjectiveTo explore the effects of 1,25-(OH)2D3 and lipopolysaccharide (LPS) plus human recombinant interleukin-15 (IL-15) on expression of vitamin D receptor (VDR) and STAT5, and cytoskeletal rearrangement in human monocytes incubated with sera from type 2 diabetes (T2DM) patients and diabetic nephropathy (DN) patients with uremia.Materials and methodsPeripheral sera were isolated from healthy volunteers (control group, T2DM patients and DN uremic non-dialysis patients). After incubation with or without 1,25(OH)2D3, THP-1 monocytes were treated with LPS plus IL-15 prior to the collection of cells and supernatants. VDR mRNA transcription was examined by RT-PCR, whilst THP-1 monocytic VDR, STAT5 and p-STAT5 expressions were investigated by Western blotting. Concentrations of IL-6 and monocyte chemoattractant protein-1 (MCP-1) in supernatants were assessed by ELISA. Immunofluorescence and a laser confocal microscopy was used to examine the expression of VDR and cytoskeletal proteins.ResultsCompared to the normal control, LPS and IL-15 down-regulate monocytic VDR expression in T2DM patients and DN uremic patients, whilst with cytoskeletal rearrangement, they up-regulate p-STAT5 expression as well as IL-6 and MCP-1 activity. Such effects could be in part blocked by 1,25-(OH)2D3.ConclusionThe above results suggest that the anti-inflammatory mechanism of 1,25-(OH)2D3 may be related to cytoskeletal proteins, VDR and STAT5 signaling pathway.

Continuous blood purification ameliorates RhoA-mediated endothelial permeability in severe acute pancreatitis patients with lung injury.

Background In the early phase of severe acute pancreatitis (SAP), serious pulmonary complications which are directly correlated with mortality are very common. Endothelial injury has been shown to play a key role in the pathogenesis of ALI/ARDS. Continuous blood purification (CBP) has been widely used in treating patients with multiple organ dysfunction syndrome (MODS) including ARDS. However, the impact of CBP on endothelial function has been little studied. Methods Human umbilical vein endothelial cells (HUVECs) were exposed to serum samples or replacement fluid taken from patients at specific time points during CBP, or pretreated with Y-27632 followed by treatment with serum, then, changes in cytoskeletal configuration, endothelial monolayer permeability, and RhoA activation were studied. Results Endothelial permeability, RhoA activity, and stress fiber reorganization increased in HUVECs treated with serum from patients before CBP initiation, and lessened in HUVECs treated with serum from patients after CBP initiation. Endothelial hyperpermeability and stress fiber reorganization reduced in HUVECs pretreated with Rho-kinase inhibitor, Y-27632, and in a dose-dependent fashion. Endothelial permeability and RhoA activity increased in HUVECs treated with waste replacement fluid collected 2 h after CBP initiation. Conclusions After CBP treatment, endothelial hyperpermeability induced by serum from SAP patients with lung injury was reduced. The inhibition of RhoA-mediated F-actin remodeling might be the mechanism.

Continuous blood purification ameliorates endothelial hyperpermeability in SAP patients with MODS by regulating tight junction proteins via ROCK.

Background Excessive activation of inflammatory mediator cascade during severe acute pancreatitis (SAP) is a major cause of multiple organ dysfunction and is associated with a high mortality. Recently, more and more studies have shown that continuous blood purification (CBP) could improve the prognosis of patients with multiple organ dysfunction syndrome (MODS), but the exact mechanism is still unclear. Many researchers have found that the disruption of tight junction barrier was an important factor for endothelial hyperpermeability, which played a key role in the pathogenesis of MODS. Previously, we found CBP could attenuate endothelial hyperpermeability in SAP patients with lung injury through regulating cytoskeleton reorganization mediated by RhoA/ROCK. However, the effect of CBP on the change of tight junction proteins in SAP patients with MODS was still unknown. This study aimed to investigate the role of tight junctions in endothelial hyperpermeability in SAP patients with MODS using an in vitro model, and the effect of CBP on tight junction barrier. Methods Before CBP and after CBP, blood samples were collected to observe hepatic and renal function, and arterial blood gas, while the APACHE II score was calculated to evaluate the severity of patients. To test whether RhoA/ROCK signaling pathway was involved, human umbilical vein endothelial cells (HUVECs) were exposed to serum samples taken from patients at specific time points during CBP, or preincubated with ROCK inhibitor, Y-27632, followed by treatment with serum. Then, the changes in endothelial cell permeability and the expression and distribution of tight junction proteins occludin and claudin-1 were observed. Results Compared with before CBP, the APACHE II score, serum creatinine and alanine aminotransferase decreased significantly, while PaO2/FiO2 increased significantly after CBP. Meanwhile, endothelial permeability induced by serum from patients significantly increased, while the expression of tight junction proteins occludin and claudin-1 significantly decreased, and severe disruption of occludin and claudin-1 was found in these cells. However, pretreated with Rho-kinase inhibitor, Y-27632 could lessen all of these abnormalities, and in a dose-dependent way. Endothelial hyperpermeability, the abnormal expression and distribution of occludin and claudin-1 were attenuated in HUVECs treated with serum from patients after CBP treatment. Conclusions The abnormality of tight junctions mediated by ROCK was an important mechanism for endothelial hyperpermeability induced by serum from SAP patients with MODS. CBP could ameliorate the disorganization and redistribution of tight junction proteins, hence improve the endothelial permeability.

Effects of peripheral blood mononuclear cells morphology on vascular calcification in uremic patients on maintenance hemodialysis.

We used high‐resolution atomic force microscopy (AFM) to examine possible changes in the morphology of peripheral blood mononuclear cells (PBMCs), and to investigate their influence on vascular calcification in uremic patients on maintenance hemodialysis (MHD). 36 uremic patients had cardiovascular diseases after MHD (MHD group1) and 30 uremic patients did not (MHD group 2), and 20 healthy volunteers were the control group. The extent of coronary artery calcification was assessed with coronary artery calcification score (CACS). AFM was used to analyze PBMCs nuances. Concentrations of bone morphogenetic protein‐2 (BMP‐2) in PBMC supernatants were detected by ELISA. Protein expressions of BMP‐2 were measured by Western blot. No significant differences in PBMC morphology were observed among groups by light microscopy. AFM images revealed that uremic patients exhibited significant differences of PBMC morphology and vascular calcification when compared with healthy volunteers. The PBMCs in uremic patients were larger in volume, mean height, half‐maximum amplitude, average roughness and higher concentrations and expression of BMP‐2 and CACS (P < 0.05), with granular processes or caveolae of uneven size distributed over cell surfaces. These differences were also significant between MHD group 1 and group 2 (P < 0.05). PBMC volume, mean height, half‐maximum amplitude, and average roughness were positively correlated with BMP‐2 and CACS. Moreover, the correlation PBMC with BMP‐2 was higher than with CACS. PBMC morphology in MHD patients was related to the degree of vascular calcification. The larger mean height, half‐maximum amplitude, average roughness and cell volume were, the higher degree of vascular calcification was.

RBP4/Lp-PLA2/Netrin-1 signaling regulation of cognitive dysfunction in diabetic nephropathy complicated with silent cerebral infarction

Aim To investigate the RBP4/Lp-PLA2/Netrin-1 signal regulation of cognitive function impairment in diabetic nephropathy patients with silent cerebral infarction (SCI). Methods One-hundred patients newly diagnosed with diabetic nephropathy patients were included. The patients were divided into SCI group and NSCI group according to the radiological data. The degrees of cognitive dysfunction were evaluated. Serum RBP4 concentrations were determined by ELISA and protein expressions of Lp-PLA2 and Netrin-1 were determined by Western Blot. Results Compared with NSCI group, the cognitive function of patients in SCI group was impaired, the concentrations of RBP4 and the expressions of Lp-PLA2 and Netrin-1 increased (P<0.05). RBP4 concentrations were positively correlated with the cognitive dysfunction of SCI patients. Moreover, there existed a regression correlation between them. Conclusion RBP4 may be used as a predictive factor of diabetic nephropathy patients complicated with SCI and is positively correlated with cognitive dysfunction. RBP4/Lp-PLA2/Netrin-1 pathway activation may be one of the occurrence mechanisms in diabetic nephropathy complicated with SCI.

Characterization of Zebrafish Pax1b and Pax9 in Fin Bud Development

Both Pax1 and Pax9 belong to the important paired box gene family (PAX), which mainly participates in animal development and sclerotome differentiation. To date, the precise molecular mechanism and related signaling pathway of Pax1 remain unclear. In our study, microinjection of morpholino- (MO-) modified antisense oligonucleotides against pax1b induced pectoral fin bud defects. Furthermore, we demonstrate that the phenotypes caused by the knockdown of Pax1b in zebrafish could not be phenocopied by pax9 MO and could not be rescued by either Pax1a or Pax9 overexpression. We further find that Pax1b affects the expression of col2a1, Uncx4.1, Noggin3, and aggrecan, confirming the role of Pax1b in chondrocyte differentiation and bone maturation. Moreover, we identify an interaction between PAX1 and FOXO1 and find that the interaction was enhanced under hypoxia stress. Together, this evidence for cell death caused by pax1b knockdown provides new insight into the role of the Pax protein family in cell fate determination and tissue specification.