RESUMO
Although accelerated cellular senescence is closely related to the progression of chronic kidney disease (CKD) and renal fibrosis, the underlying mechanisms remain largely unknown. Here, we reported that tubular aberrant expression of Brahma-related gene 1 (BRG1), an enzymatic subunit of the SWItch/Sucrose Non-Fermentable complex, is critically involved in tubular senescence and renal fibrosis. BRG1 was significantly up-regulated in the kidneys, predominantly in tubular epithelial cells, of both CKD patients and unilateral ureteral obstruction (UUO) mice. In vivo, shRNA-mediated knockdown of BRG1 significantly ameliorated renal fibrosis, improved tubular senescence, and inhibited UUO-induced activation of Wnt/ß-catenin pathway. In mouse renal tubular epithelial cells (mTECs) and primary renal tubular cells, inhibition of BRG1 diminished transforming growth factor-ß1 (TGF-ß1)-induced cellular senescence and fibrotic responses. Correspondingly, ectopic expression of BRG1 in mTECs or normal kidneys increased p16INK4a, p19ARF, and p21 expression and senescence-associated ß-galactosidase (SA-ß-gal) activity, indicating accelerated tubular senescence. Additionally, BRG1-mediated pro-fibrotic responses were largely abolished by small interfering RNA (siRNA)-mediated p16INK4a silencing in vitro or continuous senolytic treatment with ABT-263 in vivo. Moreover, BRG1 activated the Wnt/ß-catenin pathway, which further inhibited autophagy. Pharmacologic inhibition of the Wnt/ß-catenin pathway (ICG-001) or rapamycin (RAPA)-mediated activation of autophagy effectively blocked BRG1-induced tubular senescence and fibrotic responses, while bafilomycin A1 (Baf A1)-mediated inhibition of autophagy abolished the effects of ICG-001. Further, BRG1 altered the secretome of senescent tubular cells, which promoted proliferation and activation of fibroblasts. Taken together, our results indicate that BRG1 induces tubular senescence by inhibiting autophagy via the Wnt/ß-catenin pathway, which ultimately contributes to the development of renal fibrosis.
Assuntos
Autofagia , Senescência Celular , DNA Helicases/metabolismo , Células Epiteliais/metabolismo , Nefropatias/metabolismo , Túbulos Renais/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinas/metabolismo , DNA Helicases/genética , Modelos Animais de Doenças , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Células HEK293 , Humanos , Nefropatias/etiologia , Nefropatias/patologia , Túbulos Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Ratos , Fatores de Transcrição/genética , Obstrução Ureteral/complicaçõesRESUMO
Peritoneal fibrosis is a common complication of long-term peritoneal dialysis (PD) and the principal cause of ultrafiltration failure during PD. The initial and reversible step in PD-associated peritoneal fibrosis is the epithelial-mesenchymal transition (EMT). Although the mechanisms in the EMT have been the focus of many studies, only limited information is currently available concerning microRNA (miRNA) regulation in peritoneal fibrosis. In this study, we aimed to characterize the roles of microRNA-145 (miR-145) and fibroblast growth factor 10 (FGF10) in peritoneal fibrosis. After inducing EMT with transforming growth factor-ß1 (TGF-ß1) in vitro, we found that miR-145 is significantly up-regulated, whereas FGF10 is markedly down-regulated, suggesting a close link between miR-145 and FGF10 in peritoneal fibrosis, further confirmed in luciferase reporter experiments. Furthermore, in human peritoneal mesothelial cells (i.e. HMrSV5 cells), miR-145 mimics induced EMT, whereas miR-145 inhibition suppressed EMT, and we also observed that miR-145 suppressed FGF10 expression. In vivo, we found that the exogenous delivery of an miR-145 expression plasmid both blocked FGF10 and intensified the EMT, whereas miR-145 inhibition promoted the expression of FGF10 and reversed the EMT. In conclusion, miR-145 promotes the EMT during the development of peritoneal fibrosis by suppressing FGF10 activity, suggesting that miR-145 represents a potential therapeutic target for managing peritoneal fibrosis.
Assuntos
Transição Epitelial-Mesenquimal/genética , Fator 10 de Crescimento de Fibroblastos/genética , MicroRNAs/genética , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/genética , Fibrose Peritoneal/patologia , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Linhagem Celular , Fator 10 de Crescimento de Fibroblastos/deficiência , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Micheliolide (MCL), derived from parthenolide (PTL), is known for its antioxidant and anti-inflammatory effects and has multiple roles in inflammatory diseases and tumours. To investigate its effect on renal disease, we intragastrically administrated DMAMCL, a dimethylamino Michael adduct of MCL for in vivo use, in two renal fibrosis models-the unilateral ureteral occlusion (UUO) model and an ischaemia-reperfusion injury (IRI) model and used MCL in combination with transforming growth factor beta 1 (TGF-ß1) on mouse tubular epithelial cells (mTEC) in vitro. The expression of fibrotic markers (fibronectin and α-SMA) was remarkably reduced, while the expression of the epithelial marker E-cadherin was restored after DMAMCL treatment both in the UUO and IRI mice. MCL function in TGF-ß1-induced epithelial-mesenchymal transition (EMT) in mTEC was consistent with the in vivo results. Metadherin (Mtdh) was activated in the fibrotic condition, suggesting that it might be involved in fibrogenesis. Interestingly, we found that while Mtdh was upregulated in the fibrotic condition, DMAMCL/MCL could suppress its expression. The overexpression of Mtdh exerted a pro-fibrotic effect by modulating the BMP/MAPK pathway in mTECs, and MCL could specifically reverse this effect. In conclusion, DMAMCL/MCL treatment represents a novel and effective therapy for renal fibrosis by suppressing the Mtdh/BMP/MAPK pathway.
Assuntos
Nefropatias/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Substâncias Protetoras/farmacologia , Proteínas de Ligação a RNA/metabolismo , Sesquiterpenos de Guaiano/farmacologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Fibrose/metabolismo , Fibrose/patologia , Rim/citologia , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Traumatismo por Reperfusão/metabolismo , Obstrução Ureteral/metabolismoRESUMO
Diabetic kidney disease (DKD) is the principal cause of end-stage renal disease worldwide and few treatments are available. Because immunomodulators are pivotal to DKD pathophysiology, anti-inflammatory agents may be useful for treating DKD. This study was conducted to investigate the effect of micheliolide (MCL), a novel guaianolide sesquiterpene lactone with well-known anti-inflammatory effects, on DKD. Treatment with dimethylaminomicheliolide (DMAMCL), the pro-drug of MCL currently under clinical trial in oncology, protected the kidneys against proteinuria, renal failure, histopathological injury, and inflammation in db/db mice. This effect was associated with metadherin (Mtdh) downregulation. We observed aberrant upregulation of Mtdh in the kidneys of db/db mice and high-glucose (HG)-induced mouse tubular epithelial cells (mTECs). Downregulation of Mtdh obviously inhibited nuclear factor-κB signaling activation and suppressed its downstream inflammatory cytokines, such as monocyte chemotactic peptide-1, interleukin-1ß, tumor necrosis factor-α, and interleukin-6 in HG-induced mTECs, which was similar to the effect of MCL. Mtdh overexpression largely reversed the anti-inflammatory role of MCL. Moreover, MCL downregulated Mtdh by both inhibiting the transcription level and promoting ubiquitin-mediated degradation. These findings suggest that DMAMCL is a promising anti-inflammatory agent useful for preventing renal injury in DKD by inhibiting Mtdh-mediated renal inflammation.
Assuntos
Anti-Inflamatórios/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Sesquiterpenos de Guaiano/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Regulação para Baixo , Células Epiteliais/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , NF-kappa B/metabolismo , Pró-Fármacos/farmacologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sesquiterpenos de Guaiano/farmacologiaRESUMO
Pancreatic neuroendocrine tumors (PanNETs), the second most common type of primary pancreatic tumors, display notable heterogeneity in invasiveness. Current knowledge regarding genomic alterations, including DAXX/ATRX, MEN1 mutations, and copy number variations (CNVs), provides some insights into tumor invasiveness. However, the underlying reasons for the significant variation in invasiveness between insulinoma and other types of PanNETs remain unclear. To construct a comprehensive model for the stratification of prognosis, we employed analysis of both the well-established Rip1-Tag2 (RT2) mouse model of PanNETs and human PanNETs with various functional types. Firstly, by applying single-cell and bulk RNA sequencing in PanNETs from different ages and strains of RT2 mice and human PanNETs, we introduced a 2-dimensional (2D) classification system. Based on the 2-D classification system, human PanNETs were mainly classified as benign insulinomas or non-insulinomas subclusters. Non-insulinomas subtypes mainly included gastrinomas, glucagonomas, VIPomas, and NF-PanNETs, which all exhibited potential invasiveness. In addition, we discovered an enrichment of specific CNV patterns and mutations in corresponding human PanNET subclusters. Then we denoted somatic DAXX/ATRX as the 'second hit' and confounding factors for invasiveness. Finally, by combining the 2D system, DAXX/ATRX mutation status, and tumor diameter, a group of indolent PanNETs with minimal recurrence risk was identified. In conclusion, our current work constructed a comprehensive model to elucidate the heterogeneity of invasiveness in PanNETs and improve prognostic stratification.
RESUMO
INTRODUCTION: The surgical intervention approach to insulinomas in proximity to the main pancreatic duct remains controversial. Standard pancreatic resection is recommended by several guidelines; however, enucleation (EN) still attracts surgeons with less risk of late exocrine/endocrine insufficiency, despite a higher postoperative pancreatic fistula (POPF) rate. Recently, the efficacy and safety of preoperative pancreatic stent placement before the EN have been demonstrated. Thus, a multicentre open-label study is being conducted to evaluate the efficacy and safety of stent placement in improving the outcome of EN of insulinomas in proximity to the main pancreatic duct. METHODS AND ANALYSIS: This is a prospective, randomised, open-label, superiority clinical trial conducted at multiple tertiary centres in China. The major eligibility criterion is the presence of insulinoma located in the head and neck of the pancreas in proximity (≤2 mm) to the main pancreatic duct. Blocked randomisation will be performed to allocate patients into the stent EN group and the direct EN group. Patients in the stent EN group will go through stent placement by the endoscopist within 24 hours before the EN surgery, whereas other patients will receive EN surgery directly. The primary outcome is the assessment of the superiority of stent placement in reducing POPF rate measured by the International Study Group of Pancreatic Surgery standard. Both interventions will be performed in an inpatient setting and regular follow-up will be performed. The primary outcome (POPF rate) will be tested for superiority with the Χ2 test. The difference in secondary outcomes between the two groups will be analysed using appropriate tests. ETHICS AND DISSEMINATION: The study has been approved by the Peking Union Medical College Hospital Institutional Review Board (K23C0195), Ruijin Hospital Ethics Committee (2023-314), Peking University First Hospital Ethics Committee (2024033-001), Institutional Review Board of Xuanwu Hospital of Capital Medical University (2023223-002), Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University (XJTU1AF2023LSK-473), Institutional Review Board of Tongji Medical College Tongji Hospital (TJ-IRB202402059), Ethics Committee of Tongji Medical College Union Hospital (2023-0929) and Shanghai Cancer Center Institutional Review Board (2309282-16). The results of the study will be published in an international peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT05523778.
Assuntos
Insulinoma , Neoplasias Pancreáticas , Humanos , Insulinoma/cirurgia , Estudos Prospectivos , China , Pâncreas , Ductos Pancreáticos/cirurgia , Fístula Pancreática/etiologia , Fístula Pancreática/prevenção & controle , Complicações Pós-Operatórias , Stents , Neoplasias Pancreáticas/cirurgia , Hospitais , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Multicêntricos como AssuntoRESUMO
Diabetic nephropathy (DN), a vascular complication of diabetes, is the leading cause of death in patients with diabetes. The contribution of aberrantly expressed circular RNAs (circRNAs) to DN in vivo is poorly understood. Integrated comparative circRNA microarray profiling was used to examine the expression of circRNAs in diabetic kidney of db/db mice. We found that circRNA_010383 expression was markedly downregulated in diabetic kidneys, mesangial cells, and tubular epithelial cells cultured in high-glucose conditions. circRNA_010383 colocalized with miRNA-135a (miR-135a) and inhibited miR-135a function by directly binding to miR-135a. In vitro, the knockdown of circRNA_010383 promoted the accumulation of extracellular matrix (ECM) proteins and downregulated the expression of transient receptor potential cation channel, subfamily C, member 1 (TRPC1), which is a target protein of miR-135a. Furthermore, circRNA_010383 overexpression effectively inhibited the high-glucose-induced accumulation of ECM and increased TRPC1 levels in vitro. More importantly, the kidney target of circRNA_010383 overexpression inhibited proteinuria and renal fibrosis in db/db mice. Mechanistically, we identified that a loss of circRNA_010383 promoted proteinuria and renal fibrosis in DN by acting as a sponge for miR-135a. This study reveals that circRNA_010383 may be a novel therapeutic target for DN in the future.
Assuntos
Nefropatias Diabéticas/metabolismo , Fibrose/metabolismo , Rim/metabolismo , MicroRNAs/metabolismo , RNA Circular/metabolismo , Animais , Proliferação de Células/fisiologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Regulação para Baixo , Fibrose/genética , Fibrose/patologia , Humanos , Rim/patologia , Masculino , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Camundongos , MicroRNAs/genética , RNA Circular/genéticaRESUMO
Diabetic nephropathy (DN), a vascular complication of diabetes mellitus, is the leading cause of death in diabetic patients. The contribution of aberrantly expressed circRNAs to diabetic nephropathy in vivo is poorly understood. Integrated comparative circRNA microarray profiling was used to examine the expression of circRNAs in diabetic kidney of db/db mice. We found that circRNA_010383 expression was markedly downregulated in diabetic kidneys, mesangial cells and tubular epithelial cells cultured in high-glucose conditions. circRNA_010383 colocalized with microRNA-135a (miR-135a) and inhibited miR-135a function by directly binding to miR-135a. In vitro, the knockdown of circRNA_010383 promoted the accumulation of extracellular matrix (ECM) proteins and downregulated the expression of transient receptor potential cation channel, subfamily C, member (TRPC1), which is a target protein of miR-135a. Furthermore, circRNA_010383 overexpression effectively inhibited the high-glucose-induced accumulation of ECM and increased TRPC1 levels in vitro More importantly, the kidney-target of circRNA_010383 overexpression inhibited proteinuria and renal fibrosis in db/db mice. Mechanistically, we identified that a loss of circRNA_010383 promoted proteinuria and renal fibrosis in DN by acting as a sponge for miRNA-135a. This study reveals that circRNA_010383 may be a novel therapeutic target for DN in the future.
RESUMO
Transforming growth factor (TGF)-ß/Smad signalling plays a central role in the pathogenesis of peritoneal fibrosis related to peritoneal dialysis (PD). Parthenolide (PTL), a naturally occurring phytochemical, is isolated from the shoots of feverfew (Tanacetum parthenium) and displays analgesia, anti-inflammation and anticancer activities. In this study, we examined the therapeutic potential of PTL on PD-related peritoneal fibrosis induced by daily intraperitoneal injection of 4.25% dextrose-containing PD fluid (PDF) in vivo and TGF-ß1-induced epithelial-mesenchymal transition (EMT) in vitro. PTL was administered daily before PDF injection or after 14â¯days of PDF injection. Both PTL treatments showed a protective effect on peritoneal fibrosis and prevented peritoneal dysfunction. Similarly, PTL suppressed the expression of fibrotic markers (fibronectin and collagen I) and restored the expression of the epithelial marker (E-cadherin) in TGF-ß1-treated HMrSV5 cells. Furthermore, PTL inhibited TGF-ß1-induced Smad2 and Smad3 phosphorylation and nuclear translocation but did not influence Smad1/5/9 phosphorylation or activate other downstream signalling pathways of TGF-ß1, including AKT, extracellular signal-regulated kinase (ERK) or p38. In conclusion, PTL treatment may represent an effective and novel therapy for PD-associated peritoneal fibrosis by suppressing the TGF-ß/Smad pathway.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/tratamento farmacológico , Sesquiterpenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Linhagem Celular , Soluções para Diálise/administração & dosagem , Soluções para Diálise/efeitos adversos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/imunologia , Feminino , Humanos , Masculino , Camundongos , Fibrose Peritoneal/etiologia , Fibrose Peritoneal/imunologia , Fibrose Peritoneal/patologia , Peritônio/citologia , Peritônio/efeitos dos fármacos , Peritônio/imunologia , Peritônio/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Sesquiterpenos/uso terapêutico , Transdução de Sinais/imunologia , Proteínas Smad/imunologia , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
OBJECTIVE: To explore the effect of telmisartan on the expression of metadherin in the kidney of mice with unilateral ureter obstruction. METHODS: Eighteen male C57 mice were randomized into sham-operated group, model group and telmisartan treatment group. In the latter two groups, renal interstitial fibrosis as the result of unilateral ureter obstruction (UUO) was induced by unilateral ureteral ligation with or without telmisartan intervention. Renal pathological changes of the mice were assessed using Masson staining, and immunohistochemistry and Western blotting were used to detect the expression of extracellular matrix proteins and metadherin in the kidney of the mice. In the in vitro experiment, cultured mouse renal tubular epithelial cells (mTECs) were stimulated with transforming growth factor-ß1 (TGF-ß1) and transfected with a siRNA targeting metadherin, and the changes in the expressions of extracellular matrix proteins and metadherin were detected using Western blotting. RESULTS: The expressions of extracellular matrix proteins and metadherin increased significantly in the kidney of mice with UUO (P < 0.05). Intervention with telmisartan significantly lowered the expressions of extracellular matrix proteins and metadherin and alleviated the pathology of renal fibrosis in mice with UUO (P < 0.05). In cultured mTECs, siRNA-mediated knockdown of metadherin obviously reversed TGF-ß1-induced increase in the expressions of extracellular matrix proteins and metadherin. CONCLUSIONS: Telmisartan can suppress the production of extracellular matrix proteins and the expression of metadhein to attenuate UUO-induced renal fibrosis in mice.
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Proteínas da Matriz Extracelular/metabolismo , Rim/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Telmisartan/farmacologia , Obstrução Ureteral/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II , Animais , Anti-Hipertensivos , Fibrose , Rim/metabolismo , Rim/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Distribuição Aleatória , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/complicaçõesRESUMO
Peritoneal fibrosis (PF) is a major cause of ultrafiltration failure in patients receiving long-term peritoneal dialysis (PD), and effective prevention and treatment strategies are urgently needed. The dimethylamino Michael adduct of a natural product-derived micheliolide (MCL), dimethylaminomicheliolide (DMAMCL), is a new lead compound with the advantages of high stability, low toxicity, and sustainable release of MCL. This study aimed to investigate the protective effect of DMAMCL against PD-related PF and the mechanisms involved. In this study, we found that DMAMCL significantly decreased PD-induced extracellular matrix (ECM) deposition in a mouse model of PD, and that delayed DMAMCL administration halted the progression of PF in an established PD model. In addition, rapamycin administration induced autophagy and significantly ameliorated PF. The protective effect of DMAMCL against PF was weakened when co-administered with DMAMCL and 3-methyladenine. Inducing autophagy by rapamycin decreased transforming growth factor-ß1-induced ECM accumulation in vitro. MCL promoted autophagy and inhibited ECM deposition. The anti-fibrotic effect of MCL was eliminated when knocking down ATG7 by siRNA. Taken together, DMAMCL might prevent against PF through activating autophagy. The anti-fibrotic effect of DMAMCL may be a new candidate for the treatment in patients with PD-related PF. KEY MESSAGES: Dimethylaminomicheliolide, the pro-drug of micheliolide, protects against peritoneal fibrosis in a mouse peritoneal dialysis model. Micheliolide inhibits TGF-ß1-induced extracellular matrix accumulation in vitro. Autophagy plays a protective role against peritoneal fibrosis. The antifibrogenic effect of dimethylaminomicheliolide may be due to the activation of autophagy.