scholarly journals Role of MiR-155 Signal Pathway in Regulating Podocyte Injury Induced by TGF-β1

2017 ◽  
Vol 42 (4) ◽  
pp. 1469-1480 ◽  
Author(s):  
Xu Lin ◽  
Xintng Zhen ◽  
Haiting Huang ◽  
Haohao Wu ◽  
Yanwu You ◽  
...  
Keyword(s):  
Protein Expression ◽  
Antisense Oligonucleotides ◽  
Negative Control ◽  
Signal Pathway ◽  
Caspase 9 ◽  
Podocyte Injury ◽  
Apoptosis Rate ◽  
Mrna And Protein Expression ◽  
Tgf Β1

Background/Aims: Transforming growth factor beta 1 (TGF-β1) plays a critical role in the pathogenesis of glomerulosclerosis. The purpose of this study was to examine the effects of inhibition of miR-155 on podocyte injury induced by TGF-β1 and to determine further molecular mediators involved in the effects of miR-155. Methods: Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control; TGF-β1 treatment; TGF-β1 with miR-155 knockdown [using antisense oligonucleotides against miR-155 (ASO-miR-155)] and TGF-β1 with negative control antisense oligonucleotides (ASO-NC). Real time RT-PCR and Western blot analysis were employed to determine the mRNA and protein expression of nephrin, desmin and caspase-9, respectively. Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology. In addition, we examined the levels of miR-155, TGF-β1, nephrin, desmin and caspase-9 in glomerular tissues of nephropathy induced by intravenous injections of adriamycin in rats. Results: mRNA and protein expression of desmin and caspase-9 was increased in cultured TGF-β1-treated podocytes, whereas nephrin was decreased as compared with the control group. Importantly, miR-155 knockdown significantly attenuated upregulation of desmin and caspase-9, and alleviated impairment of nephrin induced by TGF-β1. Moreover, the number of apoptotic podocytes was increased after exposure to TGF-β1 and this was alleviated after miR-155 knockdown. Knocking down miR-155 also decreased an apoptosis rate of TGF-β1-treated podocytes. Note that negative control antisense oligonucleotides failed to alter an increase of the apoptosis rate in TGF-β1-treated podocytes. Consistent with in vitro results, expression of miR-155, TGF-β1, desmin and caspase-9 was increased and nephrin was decreased in glomerular tissues with nephropathy in vivo experiments. Conclusions: TGF-β1 impairs the protein expression of nephrin and amplifies the protein expression of desmin and caspase -9 via miR-155 signal pathway. Inhibition of miR-155 alleviates these changes in podocytes-treated with TGF-β1 and attenuated apoptosis of podocytes. Our data suggest that miR-155 plays a role in mediating TGF-β1-induced podocyte injury via nephrin, desmin and caspase-9. Results of the current study also indicate that blocking miR-155 signal has a protective effect on podocyte injury. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of podocyte injury observed in glomerulosclerosis.

scholarly journals Inhibition of TRPC6 Signal Pathway Alleviates Podocyte Injury Induced by TGF-β1

2017 ◽  
Vol 41 (1) ◽  
pp. 163-172 ◽  
Author(s):  
Haiting Huang ◽  
Yanwu You ◽  
Xu Lin ◽  
Chunrong Tang ◽  
Xiangjun Gu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Critical Role ◽  
Signal Pathway ◽  
Control Group ◽  
Caspase 9 ◽  
Podocyte Injury ◽  
Apoptosis Rate ◽  
Tgf Β1

Background/Aims: Transforming growth factor beta 1 (TGF-β1) plays a critical role in the pathogenesis of glomerulosclerosis. The purpose of this study was to examine the effects of inhibition of transient receptor potential cation channel C6 (TRPC6) on podocyte injury induced by TGF-β1 via nephrin and desmin mechanisms. Methods: A rat model of nephropathy was first induced by intravenous injections of adriamycin to determine TRPC6 signal pathway engaged in glomerulosclerosis in vivo. Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control; TGF-β1 treatment; TGF-β1 with TRPC6 knockdown and TGF-β1 without TRPC6 knockdown. Real time RT-PCR and Western blot analysis were employed to determine the mRNA and protein of expression of nephrin, desmin and caspase-9, respectively. Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology. Results: In vivo experiment, adriamycin significantly upregulated the protein expression of TGF-β1, TRPC6, desmin and caspase-9, and decreased nephrin. Consistent with the latter results, in vitro experiment mRNA and protein expression of desmin and caspase-9 was increased in cultured TGF-β1-treated podocytes, whereas nephrin was declined as compared with the control group. Importantly, TRPC6 knockdown significantly attenuated the upregulated desmin and caspase-9, and alleviated impairment of nephrin induced by TGF-β1. Moreover, typical morphologic features were presented in apoptotic podocytes. The number of apoptotic podocytes was increased after exposure to TGF-β1 and this was alleviated after TRPC6 knockdown. TRPC6 knockdown also decreased an apoptosis rate of TGF-β1-treated podocytes. Note that negative TRPC6 transfection control failed to alter an increase of the apoptosis rate in TGF-β1-treated podocytes. Conclusions: TGF-β1 induced by glomerulosclerosis impairs the protein expression of nephrin and amplifies the protein expression of desmin and caspase -9 via TRPC6 signal pathway. Inhibition of TRPC6 alleviates these changes in podocytes-treated with TGF-β1 and attenuated apoptosis of podocytes. Our data suggest that TRPC6 signal plays an important role in mediating TGF-β1-induced podocyte injury via nephrin, desmin and caspase-9. Results of the current study also indicate that blocking TRPC6 signal pathway has a protective effect on podocyte injury. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of podocyte injury observed in glomerulosclerosis.

scholarly journals Eplerenone inhibits aldosterone-induced CRP generation in rat vascular smooth muscle cells by regulating the MR-ROS-ERK1/2 signal pathway

2017 ◽  
Vol 15 (3) ◽  
pp. 210-218 ◽  
Author(s):  
Xiaolu Zhang ◽  
Juntian Liu ◽  
Xiaoming Pang ◽  
Jingjing Zhao ◽  
Shouzhu Xu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Protein Expression ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Signal Pathway ◽  
Mrna And Protein Expression

Atherosclerosis is a chronic inflammatory disease in the vessel wall. As a representative inflammatory cytokine, C-reactive protein (CRP) participates in the formation and development of atherosclerosis. It is demonstrated that aldosterone induces CRP generation in vascular smooth muscle cells (VSMCs). This study explored the inhibitory effect of eplerenone on aldosterone-induced CRP expression in VSMCs and mechanism. In the in vitro experiments, rat VSMCs were cultured and aldosterone (10 nM) was used as a stimulant for CRP generation. VSMCs were pretreated with eplerenone for 1 h prior to the stimulation. Messenger RNA (mRNA) and protein expression were identified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot, respectively. The production of reactive oxygen species (ROS) was observed by a fluorescence microscope. In the in vivo experiment, a model of hyperaldosteronism was established by the subcutaneous administration of aldosterone to rats with the osmotic minipumps for 4 weeks. Serum aldosterone and CRP levels were determined with a radioimmunoassay and ELISA (enzyme-linked immunosorbent assay), respectively. The results showed that eplerenone inhibited aldosterone-induced mRNA and protein expression of CRP in VSMCs in vitro and in vivo, and decreased the circulating CRP level of hyperaldosteronism rats. Meanwhile, eplerenone reduced aldosterone-stimulated ROS generation and aldosterone-activated ERK1/2 phosphorylation in VSMCs. In summary, eplerenone inhibits aldosterone-induced CRP generation in VSMCs by regulating the MR-ROS-ERK1/2 signal pathway. These results provide new evidence for the potential anti-inflammatory effect of eplerenone.

scholarly journals Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells

2021 ◽  
Vol 30 ◽  
pp. 096368972097873
Author(s):  
Jing Li ◽  
Youming Zhu ◽  
Na Li ◽  
Tao Wu ◽  
Xianyu Zheng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Dental Pulp ◽  
Tube Formation ◽  
Endothelial Differentiation ◽  
Cell Source ◽  
Lentiviral Infection ◽  
Mrna And Protein Expression ◽  
Human Dental Pulp

The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

scholarly journals Effects of tacrolimus on autophagy protein LC3 in puromycin-damaged mouse podocytes

2020 ◽  
Vol 48 (12) ◽  
pp. 030006052097142
Author(s):  
Xiao-qing Yang ◽  
Sheng-you Yu ◽  
Li Yu ◽  
Lin Ge ◽  
Yao Zhang ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Light Chain ◽  
In Vitro Model ◽  
Group Versus ◽  
Podocyte Injury ◽  
Apoptosis Rate ◽  
Protein Levels ◽  
Vitro Model ◽  
Intercellular Connections

Objective To investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury. Methods An in vitro model of puromycin-induced podocyte injury was established by dividing podocytes into three groups: controls, puromycin only (PAN group), and puromycin plus tacrolimus (FK506 group). Podocyte morphology, number, apoptosis rate and microtubule associated protein 1 light chain 3 alpha ( LC3) expression were compared. Results Puromycin caused podocyte cell body shrinkage and loose intercellular connections, but podocyte morphology in the FK506 group was similar to controls. The apoptosis rate was lower in the FK506 group versus PAN group. The low level of LC3 mRNA observed in untreated podocytes was decreased by puromycin treatment; however, levels of LC3 mRNA were higher in the FK506 group versus PAN group. Although LC3-I and LC3-II protein levels were decreased by puromycin, levels in the FK506 group were higher than the PAN group. Fewer podocyte autophagosomes were observed in the control and FK506 groups versus the PAN group. Cytoplasmic LC3-related fluorescence intensity was stronger in control and FK506 podocytes versus the PAN group. Conclusions Tacrolimus inhibited puromycin-induced mouse podocyte damage by regulating LC3 expression and enhancing autophagy.

Induction of c-jun and TGF-β1 in Fischer 344 rats during amiodarone-induced pulmonary fibrosis

2001 ◽  
Vol 281 (5) ◽  
pp. L1180-L1188 ◽  
Author(s):  
William H. Chung ◽  
Brian M. Bennett ◽  
William J. Racz ◽  
James F. Brien ◽  
Thomas E. Massey
Keyword(s):  
Pulmonary Fibrosis ◽  
Protein Expression ◽  
Pulmonary Toxicity ◽  
Transforming Growth Factor ◽  
Fischer 344 Rats ◽  
Intratracheal Administration ◽  
Fischer 344 ◽  
Fischer 344 Rat ◽  
Mrna And Protein Expression ◽  
Tgf Β1

Amiodarone (AM) is an antidysrhythmic agent with a propensity to cause pulmonary toxicity, including potentially fatal fibrosis. In the present study, the potential roles of c-Jun and transforming growth factor (TGF)-β1 in AM-induced inflammation and fibrogenesis were examined after intratracheal administration of AM (1.83 μmol/day on days 0 and 2) or an equivalent volume (0.4 ml) of distilled water to male Fischer 344 rats. Northern and immunoblot analyses demonstrated that lung TGF-β1 (mRNA and protein) expression was increased 1.5- to 1.8-fold relative to control during the early inflammation period and 1 day, 1 wk, and 2 wk post-AM treatment. Lung c-Jun protein expression was increased concomitantly with evidence of AM-induced fibrosis; at 5 wk post-AM treatment, c-Jun protein was increased 3.3-fold relative to control. The results indicate a role for induction of c- jun and TGF-β1 expression in the development of AM-induced pulmonary fibrosis in the Fischer 344 rat and provide potential targets for therapeutic intervention.

Daidzein affects steroidogenesis and oestrogen receptor expression in medium ovarian follicles of pigs

2013 ◽  
Vol 61 (1) ◽  
pp. 85-98 ◽  
Author(s):  
Anna Nynca ◽  
Dominika Słonina ◽  
Olga Jablońska ◽  
Barbara Kamińska ◽  
Renata Ciereszko
Keyword(s):  
Protein Expression ◽  
Granulosa Cells ◽  
Oestrogen Receptor ◽  
Receptor Expression ◽  
Progesterone Production ◽  
Progesterone Secretion ◽  
Mrna And Protein Expression ◽  
Induced Changes ◽  
Reproductive Processes

Daidzein, a phytoestrogen present in soybean products used in swine feed, has been demonstrated to affect both reproductive and endocrine functions. The aims of this study were to examine the in vitro effects of daidzein on (1) progesterone (P4) and oestradiol (E2) secretion by porcine luteinised granulosa cells harvested from medium follicles, and (2) the mRNA and protein expression of oestrogen receptors α and β (ERα and ERβ) in these cells. The influence of E2 on P4 secretion and ERα and ERβ expression in the granulosa cells of pigs was also investigated. It was found that daidzein inhibited progesterone secretion by luteinised granulosa cells isolated from medium follicles. In contrast, E2 did not affect progesterone production by these cells. Moreover, daidzein did not alter the granulosal secretion of E2. Both daidzein and E2 decreased mRNA expression of ERα in the cells examined. The expression of ERβ mRNA was not affected by daidzein but was inhibited by E2. ERα protein was not detected while ERβ protein was found in the nuclei of the cells. Daidzein and E2 upregulated the expression of ERβ protein in the cells. In summary, the phytoestrogen daidzein directly affected the porcine ovary by inhibiting progesterone production and increasing ERβ protein expression. Daidzein-induced changes in follicular steroidogenesis and granulosal sensitivity to oestrogens may disturb reproductive processes in pigs.

scholarly journals Hyaluronan induces monocyte chemoattractant protein-1 expression in renal tubular epithelial cells.

1998 ◽  
Vol 9 (12) ◽  
pp. 2283-2290
Author(s):  
B Beck-Schimmer ◽  
B Oertli ◽  
T Pasch ◽  
R P Wüthrich
Keyword(s):  
Protein Expression ◽  
Renal Injury ◽  
De Novo ◽  
Kidney Diseases ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Monocyte Chemoattractant Protein ◽  
Mrna And Protein Expression ◽  
Renal Tubular

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that accumulates in the renal interstitium in immune-mediated kidney diseases. The functional significance of such HA deposition in the kidney has not been elucidated. Several studies have suggested that HA may exhibit proinflammatory effects. Since chemokines such as monocyte chemoattractant protein-1 (MCP-1) play an important role in the recruitment of leukocytes in renal injury, this study tested whether HA and its fragments could promote MCP-1 production by renal parenchymal cells. Mouse cortical tubular cells were stimulated with fragmented HA or with high molecular weight HA (Healon) in vitro and were examined for MCP-1 expression. Fragmented HA, but not Healon, increased MCP-1 mRNA within 30 min with a peak after 2 h. In addition, a 10-fold increase of MCP-1 protein in the supernatant was found after a 6-h stimulation with fragmented HA. The enhanced MCP-1 mRNA and protein expression in response to HA was dose-dependent between 1 and 100 microg/ml. Upregulation of MCP-1 protein production could be blocked by preincubation with actinomycin D or cycloheximide, suggesting that MCP-1 mRNA and protein expression in response to HA are based on de novo synthesis. The HA-stimulated MCP-1 production was also inhibited with anti-CD44 antibodies, suggesting that MCP-1 is upregulated at least in part by signaling through CD44. In summary, fragmented HA markedly stimulates renal tubular MCP-1 production by mechanisms that involve binding to the HA receptor CD44. It is hypothesized that the accumulation of HA in immune renal injury could participate in the recruitment and activation of inflammatory cells in vivo through production of MCP-1.

scholarly journals HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation

2021 ◽  
Author(s):  
Yong-Xiang Chen ◽  
Chunhua Shi ◽  
Jingti Deng ◽  
Catherine Diao ◽  
Nadia Maarouf ◽  
...  
Keyword(s):  
Protein Expression ◽  
Immune Complex ◽  
Cholesterol Metabolism ◽  
Plaque Burden ◽  
Blood Levels ◽  
Igg Antibodies ◽  
Amyloid A ◽  
Cholesterol Levels ◽  
Mrna And Protein Expression

Objective: Elevated HSP27 (heat shock protein 27) levels predict relative freedom from cardiovascular events. Over-expression or twice daily subcutaneous injections of human HSP27 in ApoE −/− mice reduces blood and plaque cholesterol levels, as well as inflammation and atherosclerotic plaque burden. Antibodies to HSP27 are present in human blood, and the purpose of the current studies is to explore their role. Approach and Results: Blood levels of both HSP27 and anti-HSP27 IgG antibodies are elevated in healthy controls compared with patients with cardiovascular disease. ApoE −/− mice fed a high-fat diet and vaccinated with recombinant HSP25 (rHSP25, murine ortholog) show increased levels of anti-HSP25 IgG antibodies and reductions in plasma cholesterol and atherogenesis. Moreover, rHSP25 vaccination markedly lowered serum amyloid A levels as well as hepatic macrophage abundance and inflammatory cytokine expression. The effects of the HPS25 vaccination on cholesterol metabolism are divergent: increased hepatic LDLR (low-density lipoprotein receptor) mRNA and protein expression and reduced plasma PCSK9 (proprotein convertase subtilisin/kexin type 9) levels—despite no effect on PCSK9 expression. In vitro, the HSP27 immune complex upregulates hepatocyte LDLR mRNA and protein expression independent of intracellular cholesterol levels and increases LDLR promoter activity. The increase in LDLR expression by the HSP27 immune complex is dependent upon activation of the NF-κB (nuclear factor κ light chain enhancer of activated B cells) pathway. Hepatocyte PCSK9 protein levels are reduced after HSP27 immune complex treatment in vitro despite only minor transient effects on gene expression. Conclusions: HSP27 immunotherapy represents a novel means of lowering cholesterol and PCSK9 levels, primarily due to augmentation of LDLR expression and is associated with marked reductions in inflammation.

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