Zhiquan Chen

Connexin43 regulates high glucose-induced expression of fibronectin, ICAM-1 and TGF-β1 via Nrf2/ARE pathway in glomerular mesangial cells.

Abstract Nrf2/ARE signaling pathway is a crucial cellular defense system to cope with oxidative stress, which is adaptively activated, in diabetic condition that is not efficient enough to resist the oxidative stress provoked by hyperglycemia. We have previously demonstrated that Connexin43 (Cx43) attenuates renal fibrosis through c‐Src. However, the underlying mechanisms need to be further clarified. It has been reported that Cx43 possesses the ability of anti‐oxidative. The current study aimed to determine if Cx43 exerts protective effects on renal fibrosis in diabetes via activation of Nrf2/ARE pathway and explore the underlying molecular mechanisms. The following findings were observed: (1) Cx43 expression decreased and c‐Src activity increased in kidneys of diabetic animals; (2) Over‐expressed Cx43 in high glucose treated GMCs inhibited protein levels of FN, ICAM‐1 and TGF‐&bgr;1; (3) Nrf2/ARE signaling adaptively responded to high glucose treatment in GMCs; (4) Cx43 reduced ROS generation by boost Nrf2/ARE signaling under high glucose condition; (5) Inhibition of c‐Src activity promoted nucleus accumulation of Nrf2; (6) Over‐expressed Cx43 inhibited c‐Src activity and the interaction between c‐Src and Nrf2 in GMCs cultured in high glucose. Thus we propose that Cx43 might enhance the activation of Nrf2/ARE pathway by means of inhibiting c‐Src activity to hinder the nuclear export of Nrf2, and then reduce expression of FN, ICAM‐1 and TGF‐&bgr;1, ultimately attenuating renal fibrosis in diabetes. Graphical abstract Figure. No Caption available. HighlightsCx43 overexpression reduced ROS generation by boost Nrf2/ARE signaling under high glucose condition.Inhibition of c‐Src activity promoted nucleus accumulation of Nrf2.Over‐expressed Cx43 inhibited c‐Src activity and the interaction between c‐Src and Nrf2 in GMCs cultured in high glucose.

Polydatin promotes Nrf2-ARE anti-oxidative pathway through activating CKIP-1 to resist HG-induced up-regulation of FN and ICAM-1 in GMCs and diabetic mice kidneys

Abstract Our previous study indicated that Casein kinase 2 interacting protein‐1 (CKIP‐1) could promote the activation of the nuclear factor E2‐related factor 2 (Nrf2)/ antioxidant response element (ARE) pathway, playing a significant role in inhibiting the fibrosis of diabetic nephropathy (DN). Polydatin (PD) has been shown to possess strong resistance effects on renal fibrosis which is closely related to activating the Nrf2/ARE pathway, too. Whereas, whether PD could resist DN through regulating CKIP‐1 and consequently promoting the activation of Nrf2‐ARE pathway needs further investigation. Here, we found that PD significantly reversed the down‐regulation of CKIP‐1 and attenuated fibronectin (FN) and intercellular cell adhesion molecule‐1 (ICAM‐1) in glomerular mesangial cells (GMCs) exposed to high glucose (HG). Moreover, PD could decrease Keap1 expression and promote the nuclear content, ARE‐binding ability, and transcriptional activity of Nrf2. The activation of Nrf2‐ARE pathway by PD eventually led to the quenching of hydrogen peroxide (H2O2) and superoxide overproduction boosted by HG. Depletion of CKIP‐1 blocked the Nrf2‐ARE pathway activation and reversed FN and ICAM‐1 down‐regulation induced by PD in GMCs challenged with HG. PD increased CKIP‐1 and Nrf2 levels in the kidney tissues as well as improved the anti‐oxidative effect and renal dysfunction of diabetic mice, which eventually reversed the up‐regulation of FN and ICAM‐1. Experiments above suggested that PD could increase the CKIP‐1‐Nrf2‐ARE pathway activation to prevent the OSS‐induced insult in GMCs and diabetic mice which effectively postpone the diabetic renal fibrosis and the up‐regulation of CKIP‐1 is probably a novel mechanism in this process. Graphical abstract Figure. No Caption available. HighlightsPolydatin reversed the down‐regulation of CKIP‐1 in GMCs induced by HG.Depletion of CKIP‐1 reversed the effects of PD on Nrf2/ARE pathway.Polydatin increased the interaction of CKIP‐1 with Nrf2.Polydatin improved the antioxidant capacity and renal failure of diabetic models.

Protein kinase CK2α catalytic subunit ameliorates diabetic renal inflammatory fibrosis via NF-κB signaling pathway

Graphical abstract Figure. No Caption available. ABSTRACT Activation of casein kinase 2 (CK2) is closely linked to the body disturbance of carbohydrate metabolism and inflammatory reaction. The renal chronic inflammatory reaction in the setting of diabetes is one of the important hallmarks of diabetic renal fibrosis. However, it remains unknown whether CK2 influences the process of diabetic renal fibrosis. The current study is aimed to investigate if CK2&agr; ameliorates renal inflammatory fibrosis in diabetes via NF‐&kgr;B pathway. To explore potential regulatory mechanism of CK2&agr;, the expression and activity of CK2&agr;, which were studied by plasmid transfection, selective inhibitor, small‐interfering RNA (siRNA) and adenovirus infection in vitro or in vivo, were analyzed by means of western blotting (WB), dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). The following findings were observed: (1) Expression of CK2&agr; was upregulated in kidneys of db/db and KKAy diabetic mice; (2) Inhibition of CK2&agr; kinase activity or knockdown of CK2&agr; protein expression suppressed high glucose‐induced expressions of FN and ICAM‐1 in glomerular mesangial cells (GMCs); (3) Inhibition of CK2&agr; kinase activity or knockdown of CK2&agr; protein expression not only restrained I&kgr;B degradation, but also suppressed HG‐induced nuclear accumulation, transcriptional activity and DNA binding activity of NF‐&kgr;B in GMCs; (4) Treatment of TBB or CK2&agr; RNAi adenovirus infection ameliorated renal fibrosis in diabetic animals; (5) Treatment of TBB or CK2&agr; RNAi adenovirus infection suppressed I&kgr;B degradation and NF‐&kgr;B nuclear accumulation in glomeruli of diabetic animals. This study indicates the essential role of CK2&agr; in regulating the diabetic renal pathological process of inflammatory fibrosis via NF‐&kgr;B pathway, and inhibition of CK2&agr; may serve as a promising therapeutic strategy for diabetic nephropathy.

MRTF-A mediated FN and ICAM-1 expression in AGEs-induced rat glomerular mesangial cells via activating STAT5

Advanced glycation end products (AGEs), formed at an accelerated rate under diabetes, play a role in inflammation and fibrosis in mesangial areas in diabetic nephropathy (DN). However, the transcriptional modulator that mediates the cellular response to AGEs remains largely obscure. Our goal was to determine whether myocardin-related transcription factor (MRTF)-A, a key protein involved in the transcriptional regulation of smooth muscle cell phenotype, was responsible for the glomerular mesangial cells (GMCs) injury by AGEs, and, if so, how MRTF-A promoted mesangial dysfunction initiated by AGEs. In this study, MRTF-A was activated by AGEs in terms of protein expression and nuclear translocation in rat GMCs. MRTF-A overexpression synergistically enhanced the induction of FN and ICAM-1 by AGEs. In contract, depletion of MRTF-A abrogated the pathogenic program triggered by AGEs. Then, by interfering with MRTF-A, STAT1, STAT3 and STAT5 nuclear translocation were observed and we screened out STAT5, which was decreased obviously when MRTF-A depleted. Further investigation showed that MRTF-A interacted with STAT5 and promoted its nuclear accumulation and transcriptional activity. Therefore, our present findings suggested a role of MRTF-A in AGEs-induced GMCs injury, and further revealed that the underlying molecular mechanism was related to activating the nuclear factor STAT5.

Sphingosine kinase 1 mediates diabetic renal fibrosis via NF-κB signaling pathway: involvement of CK2α

Sphingosine kinase 1 (SphK1) plays a pivotal role in regulating diabetic renal fibrotic factors such as fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1). Especially, activation of SphK1 is closely linked to the body inflammatory reaction. Casein kinase 2α subunit (CK2α), a protein kinase related to inflammatory reaction, influences diabetic renal fibrosis and expressions of FN and ICAM-1 via NF-κB pathway. However, the mechanism by which SphK1 mediates diabetic renal fibrosis has not yet fully elucidated. The current study is aimed to investigate if SphK1 mediates diabetic renal fibrotic pathological process via inflammatory pathway and activation of CK2α. The following findings were observed: (1) Expressions of SphK1 were upregulated in kidneys of diabetic mice and rats; (2) Knockdown of SphK1 expression suppressed high glucose (HG)-induced NF-κB nuclear translocation and expressions of FN and ICAM-1; (3) Compared with C57 diabetic mice, SphK1-/- diabetic mice exhibited less renal fibrotic lesions, FN accumulation and NF-κB nuclear accumulation in glomeruli of kidneys; (4) SphK1 mediated phosphorylation of CK2α, while CK2α knockdown depressed SphK1-induced activation of NF-κB pathway. This study indicates the essential role of SphK1 in regulating activation of CK2α and diabetic renal fibrotic pathological process via NF-κB.

Emodin self-emulsifying platform ameliorates the expression of FN, ICAM-1 and TGF-β1 in AGEs-induced glomerular mesangial cells by promoting absorption.

Abstract Emodin, a potential anti‐diabetic nephropathy agent, is limited by its oral use due to the poor water solubility. The present study aimed to enhance the absorption and the suppressive effects of emodin on renal fibrosis by developing a self‐microemulsifying drug delivery system (SMEDDS). Solubility studies, compatibility tests, pseudo‐ternary phase diagrams analysis and central composite design were carried out to obtain the optimized formulation. The average droplet size of emodin‐loaded SMEDDS was about 18.31 ± 0.12 nm, and the droplet size and zeta potential remained stable at different dilution ratios of water and different values of pH varying from 1.2 to 7.2. Enhanced cellular uptake in both the Caco‐2 cells and glomerular mesangial cells (GMCs) is great advantageous for the formulation. The AUC0–24 h of emodin‐loaded SMEDDS was 1.87‐fold greater than that of emodin suspension, which may be attributed to enhanced uptake in Caco‐2 cells. Moreover, emodin‐loaded SMEDDS showed better suppressive effects on the protein level of fibronectin (FN), transforming growth factor‐beta 1 (TGF‐&bgr;1) and intercellular adhesion molecule 1 (ICAM‐1) than the crude emodin in advanced glycation‐end products (AGEs)‐induced GMCs and renal tubular epithelial cells (NRK‐52E). Our study illustrated that developed SMEDDS improved the oral absorption of emodin, and attained better suppressive effects on the protein level of renal fibrosis compositions in AGEs‐induced GMCs and NRK‐52E cells.

Paeonol Ameliorates Diabetic Renal Fibrosis Through Promoting the Activation of the Nrf2/ARE Pathway via Up-Regulating Sirt1

Diabetic nephropathy (DN) is rapidly becoming the leading cause of end-stage renal disease worldwide and a major cause of morbidity and mortality in patients of diabetes. The main pathological change of DN is renal fibrosis. Paeonol (PA), a single phenolic compound extracted from the root bark of Cortex Moutan, has been demonstrated to have many potential pharmacological activities. However, the effects of PA on DN have not been fully elucidated. In this study, high glucose (HG)-treated glomerular mesangial cells (GMCs) and streptozotocin (STZ)-induced diabetic mice were analyzed in exploring the potential mechanisms of PA on DN. Results in vitro showed that: (1) PA inhibited HG-induced fibronectin (FN) and ICAM-1 overexpressions; (2) PA exerted renoprotective effect through activating the Nrf2/ARE pathway; (3) Sirt1 mediated the effects of PA on the activation of Nrf2/ARE pathway. What is more, in accordance with the in vitro results, significant elevated levels of Sirt1, Nrf2 and downstream proteins related to Nrf2 were observed in the kidneys of PA treatment group compared with model group. Taken together, our study shows that PA delays the progression of diabetic renal fibrosis, and the underlying mechanism is probably associated with regulating the Nrf2 pathway. The effect of PA on Nrf2 is at least partially dependent on Sirt1 activation.

Progestin and AdipoQ Receptor 3 Upregulates Fibronectin and Intercellular Adhesion Molecule-1 in Glomerular Mesangial Cells via Activating NF-κB Signaling Pathway Under High Glucose Conditions

Background Progestin and adipoQ receptor 3 (PAQR3), is a Golgi-anchored membrane protein containing seven transmembrane helices. It has been demonstrated that PAQR3 mediates insulin resistance, glucose and lipid metabolism, and inflammation. In addition, kidney inflammatory fibrosis is an important pathological feature of diabetic nephropathy (DN). Therefore, we aimed to investigate the role of PAQR3 in diabetic kidney fibrosis as well as inflammation in DN. Object The effect of PAQR3 on NF-κB signaling pathway, expressions of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in glomerular mesangial cells (GMCs) cultured by high glucose (HG) were examined. Method Diabetic mouse and rat models were induced by streptozotocin (STZ). GMCs were treated with HG and transfected with PAQR3 plasmids or small-interfering RNA targeting PAQR3 or NF-κB. The protein levels of FN and ICAM-1 were examined by Western blotting, and the transcriptional activity and DNA binding activity of NF-κB were measured by dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). The interaction between PAQR3 and IKKβ (inhibitor of nuclear factor κB kinase β) was analyzed by co-immunoprecipitation. Results PAQR3 was increased in both STZ-induced diabetic models and HG-treated GMCs. PAQR3 overexpression further increased HG-induced FN and ICAM-1 upregulation. In contrast, silencing of PAQR3 suppressed the expressions of FN and ICAM-1. PAQR3 overexpression promoted the nuclear accumulation, DNA binding activity, and transcriptional activity of NF-κB. Mechanically, PAQR3 directly interacted with IKKβ. The upregulation effect of PAQR3 overexpression on the expressions of FN and ICAM-1 was abolished by the treatment of NF-κB siRNA or PDTC (ammonium pyrrolidinedithiocarbamate) in HG-treated GMCs. Conclusion PAQR3 promotes the expressions of FN and ICAM-1 via activating NF-κB signaling pathway. Mechanistically, PAQR3 activates NF-κB signaling pathway to mediate kidney inflammatory fibrosis through direct interaction with IKKβ in DN.

CKIP-1 affects the polyubiquitination of Nrf2 and Keap1 via mediating Smurf1 to resist HG-induced renal fibrosis in GMCs and diabetic mice kidneys

ABSTRACT Our previous study indicated that Casein kinase 2 interacting protein‐1 (CKIP‐1) could promote the activation of the nuclear factor E2‐related factor 2 (Nrf2)/ antioxidant response element (ARE) pathway, playing a significant role in inhibiting the fibrosis of diabetic nephropathy (DN). However, the underlying mechanism is still unknown. Here, we investigated whether CKIP‐1 affects the polyubiquitination of Nrf2 and its cytosolic inhibitor kelch like ECH‐associated protein 1 (Keap1) via mediating Smad ubiquitylation regulatory factor‐1 (Smurf1) to promote the activation of the Nrf2/ARE signaling and resist high glucose (HG)‐induced renal fibrosis in glomerular mesangial cells (GMCs) and diabetic mice kidneys. Results showed that the expression of Smurf1 increased in HG‐induced GMCs, with a paramount upregulation at 1 h. Overexpression of wild‐type Smurf1 plasmid further promoted the HG‐induced the over‐production of fibronectin (FN) and intercellular adhesionmolecule‐1 (ICAM‐1), and depletion of Smurf1 dramatically reduced the expression of FN and ICAM‐1. Overexpression of CKIP‐1 decreased the K48‐linked polyubiquitination and increased the K63‐linked polyubiquitination of Nrf2 as well as enhanced the K48‐linked polyubiquitination and reduced K63‐linked polyubiquitination of Keap1, promoting the activation of the Nrf2/ARE pathway. Overexpression of Smurf1 increased the K48‐linked polyubiquitination and decreased the K63‐linked polyubiquitination of Nrf2, and down‐regulated the K48‐linked polyubiquitination and up‐regulated the K63‐linked polyubiquitination of Keap1, inhibiting the activation of the Nrf2/ARE pathway. CKIP‐1 promoted the degradation of Smurf1 by increasing the ubiquitination of Smurf1. Treatment of CKIP‐1 adenovirus infection reduced the Smurf1 levels, promoted the activation of the Nrf2/ARE pathway as well as suppressed the production of reactive oxygen species (ROS), and then improved the failure of renal function of diabetic mice. Experiments above suggested that CKIP‐1 affects the polyubiquitination of Nrf2 and Keap1 and promotes the Nrf2‐ARE pathway through down‐regulating Smurf1 to resist HG‐induced up‐regulation of FN and ICAM‐1 in GMCs and diabetic mice kidneys. HighlightsSmurf1 involves in the progress of DN because of its ubiquitin ligase activity.Both Nrf2 and Keap1 can be polyubiquitinated.CKIP‐1 affects the polyubiquitination of Nrf2 and Keap1 via Smurf1.CKIP‐1 adenovirus improved antioxidant ability and renal failure of diabetic mice. Graphical abstract Figure. No caption available.