Wenyan Gong

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.

CKIP-1 ameliorates high glucose-induced expression of fibronectin and intercellular cell adhesion molecule-1 by activating the Nrf2/ARE pathway in glomerular mesangial cells

Glucose and lipid metabolism disorders as well as oxidative stress (OSS) play important roles in diabetic nephropathy (DN). Glucose and lipid metabolic dysfunctions are the basic pathological changes of chronic microvascular complications of diabetes mellitus, such as DN. OSS can lead to the accumulation of extracellular matrix and inflammatory factors which will accelerate the progress of DN. Casein kinase 2 interacting protein-1 (CKIP-1) mediates adipogenesis, cell proliferation and inflammation under many circumstances. However, whether CKIP-1 is involved in the development of DN remains unknown. Here, we show that CKIP-1 is a novel regulator of resisting the development of DN and the underlying molecular mechanism is related to activating the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) antioxidative stress pathway. The following findings were obtained: (1) The treatment of glomerular mesangial cells (GMCs) with high glucose (HG) decreased CKIP-1 levels in a time-dependent manner; (2) CKIP-1 overexpression dramatically reduced fibronectin (FN) and intercellular adhesionmolecule-1 (ICAM-1) expression. Depletion of CKIP-1 further induced the production of FN and ICAM-1; (3) CKIP-1 promoted the nuclear accumulation, DNA binding, and transcriptional activity of Nrf2. Moreover, CKIP-1 upregulated the expression of Nrf2 downstream genes, heme oxygenase (HO-1) and superoxide dismutase 1 (SOD1); and ultimately decreased the levels of reactive oxygen species (ROS). The molecular mechanisms clarify that the advantageous effect of CKIP-1 on DN are well connected with the activation of the Nrf2/ARE antioxidative stress pathway.

TGR5 activation suppressed S1P/S1P2 signaling and resisted high glucose-induced fibrosis in glomerular mesangial cells.

Glucose and lipid metabolism disorders and chronic inflammation in the kidney tissues are largely responsible for causative pathological mechanism of renal fibrosis in diabetic nephropathy (DN). As our previous findings confirmed that, sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptor 2 (S1P2) signaling activation promoted renal fibrosis in diabetes. Numerous studies have demonstrated that the G protein-coupled bile acid receptor TGR5 exhibits effective regulation of glucose and lipid metabolism and anti-inflammatory effects. TGR5 is highly expressed in kidney tissues, whether it attenuates the inflammation and renal fibrosis by inhibiting the S1P/S1P2 signaling pathway would be a new insight into the molecular mechanism of DN. Here we investigated the effects of TGR5 on diabetic renal fibrosis, and the underlying mechanism would be also discussed. We found that TGR5 activation significantly decreased the expression of intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-beta 1 (TGF-β1), as well as fibronectin (FN) induced by high glucose in glomerular mesangial cells (GMCs), which were pathological features of DN. S1P2 overexpression induced by high glucose was diminished after activation of TGR5, and AP-1 activity, including the phosphorylation of c-Jun/c-Fos and AP-1 transcription activity, was attenuated. As a G protein-coupled receptor, S1P2 interacted with TGR5 in GMCs. Furthermore, INT-777 lowered S1P2 expression and promoted S1P2 internalization. Taken together, TGR5 activation reduced ICAM-1, TGF-β1 and FN expressions induced by high glucose in GMCs, the mechanism might be through suppressing S1P/S1P2 signaling, thus ameliorating 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.

TGR5 suppresses high glucose-induced upregulation of fibronectin and transforming growth factor-β1 in rat glomerular mesangial cells by inhibiting RhoA/ROCK signaling.

RhoA/ROCK can cause renal inflammation and fibrosis in the context of diabetes by activating nuclear factor-κB (NF-κB). TGR5 is known for its role in maintaining metabolic homeostasis and anti-inflammation, which is closely related to NF-κB inhibition. Given that TGR5 is highly enriched in kidney, we aim to investigate the regulatory role of TGR5 on fibronectin (FN) and transforming growth factor-β1 (TGF-β1) in high glucose (HG)-treated rat glomerular mesangial cells (GMCs). Both the factors are closely related to renal inflammations and mediated by NF-κB. Moreover, our study determines whether such regulation is achieved by the inhibition of RhoA/ROCK and the subsequent NF-κB suppression. Polymerase chain reaction was taken to test the mRNA level of TGR5. Western blot was used to measure the protein expressions of TGR5, FN, TGF-β1, p65, IκBα, phospho-MYPT1 (Thr853), and MYPT1. Glutathione S-transferase-pull down and immunofluorescence were conducted to test the activation of RhoA, the distribution of TGR5, and p65, respectively. Electrophoretic mobility shift assay was adopted to measure the DNA binding activity of NF-κB. In GMCs, TGR5 activation or overexpression significantly suppressed FN and TGF-β1 protein expressions, NF-κB, and RhoA/ROCK activation induced by HG or transfection of constitutively active RhoA. By contrast, TGR5 RNA interference caused enhancement of FN, TGF-β1 protein expressions, increase of RhoA/ROCK activation. However, TGR5 cannot suppress RhoA/ROCK activation when a selective Protein kinase A (PKA) inhibitor was used. This study suggests that in HG-treated GMCs, TGR5 significantly suppresses the NF-κB-mediated upregulation of FN and TGF-β1, which are hallmarks of diabetic nephropathy. These functions are closely related to the suppression of RhoA/ROCK via PKA.

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.