scholarly journals Regulation of connective tissue growth factor expression by miR-133b for the treatment of renal interstitial fibrosis in aged mice with unilateral ureteral obstruction

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dan Cao ◽  
Yuan Wang ◽  
Yingjie Zhang ◽  
Yinping Zhang ◽  
Qi Huang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Target Genes ◽  
Interstitial Fibrosis ◽  
Tissue Growth ◽  
Luciferase Reporter ◽  
Renal Interstitial Fibrosis ◽  
Mrna And Protein Expression ◽  
Hk2 Cells ◽  
Tgf Β1 ◽  
Dual Luciferase Reporter Assay

Abstract Introduction Renal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes. Methods We performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days. Results miR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70–100 times) in mouse kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO. Conclusion miR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

scholarly journals Regulation of Connective Tissue Growth Factor Expression by miR-133b for the Treatment of Renal Interstitial Fibrosis in Aged Mice with Unilateral Ureteral Obstruction

2020 ◽  
Author(s):  
Dan Cao ◽  
Yuan Wang ◽  
Yingjie Zhang ◽  
Yinping Zhang ◽  
Qi Huang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Target Genes ◽  
Interstitial Fibrosis ◽  
Tissue Growth ◽  
Luciferase Reporter ◽  
Renal Interstitial Fibrosis ◽  
Mrna And Protein Expression ◽  
Hk2 Cells ◽  
Tgf Β1 ◽  
Dual Luciferase Reporter Assay

Abstract Introduction: Renal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.Methods: We performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.Results: miR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70-100 times) in mice kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.Conclusion: miR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

scholarly journals Regulation of Connective Tissue Growth Factor Expression by miR-133b for the Treatment of Renal Interstitial Fibrosis in Aged Mice with Unilateral Ureteral Obstruction

2020 ◽  
Author(s):  
Dan Cao ◽  
Yuan Wang ◽  
Yingjie Zhang ◽  
Yinping Zhang ◽  
Qi Huang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Target Genes ◽  
Interstitial Fibrosis ◽  
Tissue Growth ◽  
Luciferase Reporter ◽  
Renal Interstitial Fibrosis ◽  
Mrna And Protein Expression ◽  
Hk2 Cells ◽  
Tgf Β1 ◽  
Dual Luciferase Reporter Assay

Abstract Introduction: Renal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.Methods: We performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.Results: miR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70-100 times) in mice kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.Conclusion: miR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

scholarly journals Regulation of CTGF Expression by miR-133b for the Treatment of Renal Interstitial Fibrosis in Old UUO Rats

2020 ◽  
Author(s):  
Dan Cao ◽  
Yuan Wang ◽  
Yingjie Zhang ◽  
Yinping Zhang ◽  
Qi Huang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Target Genes ◽  
Interstitial Fibrosis ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Renal Interstitial Fibrosis ◽  
Hk2 Cells ◽  
Tgf Β1 ◽  
Dual Luciferase Reporter Assay

Abstract Introduction: Renal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated the high expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) from old rats, which mediated the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.Methods: miR-133b expression induced during the EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and time points (0, 24, 48, and 72 h) was detected using real-time polymerase chain reaction. The target genes of miR-133b were validated using a dual-luciferase reporter assay. In vitro experiments were performed to observe mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-week-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. miR-133b、 target gene and other indexes mentioned above mRNA and protein levels and renal interstitial fibrosis were detected at 7 and 14 days.Results: miR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. Dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. miR-133b mimic transfection inhibited the TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70-100 times) in mouse kidneys after injection of the miRNA-133b overexpression complex, significantly alleviating renal interstitial fibrosis in UUO mice.Conclusion: miR-133b exerted targeted inhibitory effects on CTGF expression, consequently reducing the TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in old UUO mice.

scholarly journals Rapamycin reduces renal hypoxia, interstitial inflammation and fibrosis in a rat model of unilateral ureteral obstruction

2014 ◽  
Vol 37 (3) ◽  
pp. 142 ◽  
Author(s):  
Chun-feng Liu ◽  
Hing Liu ◽  
Yi Fang ◽  
Su-hua Jiang ◽  
Jia-ming Zhu ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Expression ◽  
Rat Model ◽  
Ureteral Obstruction ◽  
Interstitial Fibrosis ◽  
Unilateral Ureteral Obstruction ◽  
Tubulointerstitial Fibrosis ◽  
Interstitial Inflammation ◽  
Mrna And Protein Expression ◽  
Tgf Β1

Purpose: The purpose of this study was to explore effects of rapamycin on renal hypoxia, interstitial inflammation and fibrosis, and the expression of transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), Flk-1 and Flt-1 in a rat model of unilateral ureteral obstruction (UUO). Methods: Male Sprague-Dawley rats (n=36) were randomly divided into three groups (n=12 per group): sham surgery, UUO and UUO plus rapamycin (0.2 mg/kg/d). Serum creatinine (Scr), blood urea nitrogen, uric acid, triglycerides, cholesterol and 24-h urine protein levels were measured. The extent of interstitial fibrosis was determined by Masson's trichrome staining. ED-1 positive macrophages, type III collagen, hypoxia, TGF-1, VEGF, Flk-1, and Flt-1 mRNA and protein expressions were detected using immunohistochemical staining, real-time PCR and Western blot. Results: UUO induced an elevation in Scr, renal hypoxia, inflammation, interstitial fibrosis, TGF-β1, VEGF, Flk-1, and Flt-1 mRNA and protein expression levels (P < 0.05). Rapamycin alleviated the UUO-induced renal hypoxia, infiltration of inflammatory cells and tubulointerstitial fibrosis (at days 3 and 7). Rapamycin also down-regulated the UUO-induced elevated expression levels of TGF-β1 and Flt-1 mRNA and protein (P < 0.05). Rapamycin decreased VEGF mRNA and protein expression at day 3, and increased Flk-1 mRNA and protein expression at day 7, compared with the UUO group (P < 0.05). Conclusion: Rapamycin shows beneficial effects by reducing UUO-induced renal hypoxia, inflammation and tubulointerstitial fibrosis.

scholarly journals Transforming growth factor (TGF)-β1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis

2019 ◽  
Vol 10 (9) ◽  
Author(s):  
Peng Wei ◽  
Yan Xie ◽  
Peter W. Abel ◽  
Yapei Huang ◽  
Qin Ma ◽  
...  
Keyword(s):  
Growth Factor ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Tissue Growth ◽  
Negative Regulator ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Myofibroblast Differentiation ◽  
Dependent Manner ◽  
Tgf Β1

Abstract Transforming growth factor (TGF)-β1, a main profibrogenic cytokine in the progression of idiopathic pulmonary fibrosis (IPF), induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of collagen, leading to concomitantly loss of lung elasticity and function. Recent studies implicate the importance of microRNAs (miRNAs) in IPF but their regulation and individual pathological roles remain largely unknown. We used both RNA sequencing and quantitative RT-PCR strategies to systematically study TGF-β1-induced alternations of miRNAs in human lung fibroblasts (HFL). Our data show that miR-133a was significantly upregulated by TGF-β1 in a time- and concentration-dependent manner. Surprisingly, miR-133a inhibits TGF-β1-induced myofibroblast differentiation whereas miR-133a inhibitor enhances TGF-β1-induced myofibroblast differentiation. Interestingly, quantitative proteomics analysis indicates that miR-133a attenuates myofibroblast differentiation via targeting multiple components of TGF-β1 profibrogenic pathways. Western blot analysis confirmed that miR-133a down-regulates TGF-β1-induced expression of classic myofibroblast differentiation markers such as ɑ-smooth muscle actin (ɑ-SMA), connective tissue growth factor (CTGF) and collagens. miRNA Target Searcher analysis and luciferase reporter assays indicate that TGF-β receptor 1, CTGF and collagen type 1-alpha1 (Col1a1) are direct targets of miR-133a. More importantly, miR-133a gene transferred into lung tissues ameliorated bleomycin-induced pulmonary fibrosis in mice. Together, our study identified TGF-β1-induced miR-133a as an anti-fibrotic factor. It functions as a feed-back negative regulator of TGF-β1 profibrogenic pathways. Thus, manipulations of miR-133a expression may provide a new therapeutic strategy to halt and perhaps even partially reverse the progression of IPF.

scholarly journals Iguratimod Inhibits Renal Interstitial Fibrosis in Lupus Nephritis

2020 ◽  
Author(s):  
Leixi Xue ◽  
Jiajun Xu ◽  
Wentian Lu ◽  
Qing Wang ◽  
Chenghua Weng ◽  
...  
Keyword(s):  
Lupus Nephritis ◽  
Real Time ◽  
P38 Mapk ◽  
Real Time Pcr ◽  
Interstitial Fibrosis ◽  
Quantitative Real Time Pcr ◽  
Renal Interstitial Fibrosis ◽  
Hk2 Cells ◽  
Tgf Β1 ◽  
E Cadherin

Abstract Background: Renal interstitial fibrosis (RIF) is one of the main poor prognostic factors of lupus nephritis (LN). Here, we tested whether iguratimod could inhibit RIF in LN. Methods: MRL/lpr mice, an animal model of lupus, were treated with iguratimod or vehicle solution. Pathological changes of kidney was evaluated blindly by the same pathologist. Renal type I collagen (COL-I), IgG, E-cadherin, fibroblast-specific protein 1 (FSP-1) were detected by immunofluorescence, immunohistochemical staining or quantitative real-time PCR. After treated with transforming growth factor β1 (TGF-β1) and iguratimod, E-cadherin, fibronectin, Smad2/3, p38 MAPK, p-Smad2/3 and p-p38 MAPK in HK2 cells were measured by western blotting, quantitative real-time PCR or immunofluorescence. Results: In MRL/lpr mice, iguratimod eased the renal interstitial deposition of collagen fibers, further confirmed by decreased expression of COL-I after iguratimod treatment. Moreover, upstream pathological processes of RIF, including IgG deposition along the tubular basement membrane, infiltration of inflammatory cells into renal interstitium and tubular injury, were also prevented effectively by iguratimod treatment. Furthermore, iguratimod suppressed the expression of FSP-1 and raised the expression of E-cadherin in renal tubular epithelial cells, suggesting that iguratimod reversed the process of tubular epithelial-to-mesenchymal transition (EMT). In HK2 cells induced by TGF-β1, co-treatment with iguratimod not only reversed cellular morphologic change, but also prevented down-regulation of E-cadherin and up-regulation of fibronectin. Finally, iguratimod blocked TGF-β1-induced phosphorylation of Smad2/3 and p38 MAPK in HK2 cells. Conclusions: Our results suggest that iguratimod shows an inhibitory effect on the progress of RIF in vivo and in vitro, so iguratimod may be a potential drug for the treatment of RIF in LN.

scholarly journals The Cysteine-Rich Domain Protein KCP Is a Suppressor of Transforming Growth Factor β/Activin Signaling in Renal Epithelia

2006 ◽  
Vol 26 (12) ◽  
pp. 4577-4585 ◽  
Author(s):  
Jingmei Lin ◽  
Sanjeevkumar R. Patel ◽  
Min Wang ◽  
Gregory R. Dressler
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Interstitial Fibrosis ◽  
Transforming Growth Factor Β ◽  
Activin A ◽  
Bmp Signaling ◽  
Cell Junctions ◽  
Type I ◽  
Renal Interstitial Fibrosis ◽  
Tgf Β1

ABSTRACT The transforming growth factor β (TGF-β) superfamily, including the bone morphogenetic protein (BMP) and TGF-β/activin A subfamilies, is regulated by secreted proteins able to sequester or present ligands to receptors. KCP is a secreted, cysteine-rich (CR) protein with similarity to mouse Chordin and Xenopus laevis Kielin. KCP is an enhancer of BMP signaling in vertebrates and interacts with BMPs and the BMP type I receptor to promote receptor-ligand interactions. Mice homozygous for a KCP null allele are hypersensitive to developing renal interstitial fibrosis, a disease stimulated by TGF-β but inhibited by BMP7. In this report, the effects of KCP on TGF-β/activin A signaling are examined. In contrast to the enhancing effect on BMPs, KCP inhibits both activin A- and TGF-β1-mediated signaling through the Smad2/3 pathway. These inhibitory effects of KCP are mediated in a paracrine manner, suggesting that direct binding of KCP to TGF-β1 or activin A can block the interactions with prospective receptors. Consistent with this inhibitory effect, primary renal epithelial cells from KCP mutant cells are hypersensitive to TGF-β and exhibit increased apoptosis, dissociation of cadherin-based cell junctions, and expression of smooth muscle actin. Furthermore, KCP null animals show elevated levels of phosphorylated Smad2 after renal injury. The ability to enhance BMP signaling while suppressing TGF-β activation indicates a critical role for KCP in modulating the responses between these anti- and profibrotic cytokines in the initiation and progression of renal interstitial fibrosis.

Regulation of TGF-β1-induced connective tissue growth factor expression in airway smooth muscle cells

2005 ◽  
Vol 288 (1) ◽  
pp. L68-L76 ◽  
Author(s):  
Shaoping Xie ◽  
Maria B. Sukkar ◽  
Razao Issa ◽  
Ute Oltmanns ◽  
Andrew G. Nicholson ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Connective Tissue ◽  
Protein Expression ◽  
Smooth Muscle Cells ◽  
Connective Tissue Growth Factor ◽  
Tissue Growth ◽  
Airway Smooth Muscle Cells ◽  
Mrna And Protein Expression ◽  
Tgf Β1

Transforming growth factor (TGF)-β may play an important role in airway remodeling, and the fibrogenic effect of TGF-β may be mediated through connective tissue growth factor (CTGF) release. We investigated the role of MAPKs and phosphatidylinositol 3-kinase (PI3K) and the effects of inflammatory cytokines on TGF-β-induced CTGF expression in human airway smooth muscle cells (ASMC). We examined whether Smad signal was involved in the regulatory mechanisms. TGF-β1 induced a time- and concentration-dependent expression of CTGF gene and protein as analyzed by real-time RT-PCR and Western blot. Inhibition of ERK and c- jun NH2-terminal kinase (JNK), but not of p38 MAPK and PI3K, blocked the effect of TGF-β1 on CTGF mRNA and protein expression and on Smad2/3 phosphorylation. T helper lymphocyte 2-derived cytokines, IL-4 and IL-13, attenuated TGF-β1-stimulated mRNA and protein expression of CTGF and inhibited TGF-β1-stimulated ERK1/2 and Smad2/3 activation in ASMC. The proinflammatory cytokines tumor necrosis factor-α and IL-1β reduced TGF-β1-stimulated mRNA expression of CTGF but did not inhibit TGF-β-induced Smad2/3 phosphorylation. TGF-β1-stimulated CTGF expression is mediated by mechanisms involving ERK and JNK pathways and is downregulated by IL-4 and IL-13 through modulation of Smad and ERK signals.

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