Search

Dihydromyricetin (DHM) is a flavonoid from vine tea with broad pharmacological benefits, which improve inflammation by blocking the NF-κB pathway. A growing body of research indicates that chronic kidney inflammation is vital to the pathogenesis of diabetic renal fibrosis. Sphingosine kinase-1 (SphK1) is a key regulator of diabetic renal inflammation, which triggers the NF-κB pathway. Hence, we evaluated whether DHM regulates diabetic renal inflammatory fibrosis by acting on SphK1. Here, we demonstrated that DHM effectively suppressed the synthesis of fibrotic and inflammatory adhesion factors like ICAM-1, and VCAM-1 in streptozotocin-treated high-fat diet-induced diabetic mice and HG-induced glomerular mesangial cells (GMCs). Moreover, DHM significantly suppressed NF-κB pathway activation and reduced SphK1 activity and protein expression under diabetic conditions. Mechanistically, the results of molecular docking, molecular dynamics simulation, and cellular thermal shift assay revealed that DHM stably bound to the binding pocket of SphK1, thereby reducing sphingosine-1-phosphate content and SphK1 enzymatic activity, which ultimately inhibited NF-κB DNA binding, transcriptional activity, and nuclear translocation. In conclusion, our data suggested that DHM inhibited SphK1 phosphorylation to prevent NF-κB activation thus ameliorating diabetic renal fibrosis. This supported the clinical use and further drug development of DHM as a potential candidate for treating diabetic renal fibrosis., Competing Interests: Declaration of competing interest The authors declare no potential conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Diabetic nephropathy (DN) is a complication of diabetes and is mainly characterized by renal fibrosis, which could be attributed to chronic kidney inflammation. Stimulator of interferon genes (STING), a linker between immunity and metabolism, could ameliorate various metabolic and inflammatory diseases. However, the regulatory role of STING in DN remains largely unexplored. In this study, knockdown of STING decreased extracellular matrix (ECM), pro-inflammatory, and fibrotic factors in high glucose (HG)-induced glomerular mesangial cells (GMCs), whereas overexpression of STING triggered the inflammatory fibrosis process, suggesting that STING was a potential target for DN. Polydatin (PD) is a glucoside of resveratrol and has been reported to ameliorate DN by inhibiting inflammatory responses. Nevertheless, whether PD improved DN via STING remains unclear. Here, transcriptomic profiling implied that the STING/NF-κB pathway might be an important target for PD. We further found that PD decreased the protein expression of STING, and subsequently suppressed the activation of downstream targets including TBK1 phosphorylation and NF-κB nuclear translocation, and eventually inhibited the production of ECM, pro-inflammatory and fibrotic factors in HG-induced GMCs. Notably, results of molecular docking, molecular dynamic simulations, surface plasmon resonance, cellular thermal shift assay and Co-immunoprecipitation assay indicated that PD directly bound to STING and restored the declined proteasome-mediated degradation of STING induced by HG. In diabetic mice, PD also inhibited the STING pathway and improved the pathological changes of renal inflammatory fibrosis. Our study elucidated the regulatory role of STING in DN, and the novel mechanism of PD treating DN via inhibiting STING expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Diabetic nephropathy (DN) is one of the most severe complications of diabetes mellitus. Succinate Receptor 1 (SUCNR1), a member of the G-protein-coupled receptor (GPCR) family, represents a potential target for treatment of DN. Here, utilizing multi-strategy in silico virtual screening methods containing AlphaFold2 modelling, molecular dynamics (MD) simulation, ligand-based pharmacophore screening, molecular docking and machine learning-based similarity clustering, we successfully identified a novel antagonist of SUCNR1, AK-968/12117473 (Cpd3). Through extensive in vitro experiments, including dual-luciferase reporter assay, cellular thermal shift assay, immunofluorescence, and western blotting, we substantiated that Cpd3 could specifically target SUCNR1, inhibit the activation of NF-κB pathway, and ameliorate epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in renal tubular epithelial cells (NRK-52E) under high glucose conditions. Further in silico simulations revealed the molecular basis of the SUCNR1-Cpd3 interaction, and the in vitro metabolic stability assay indicated favorable drug-like pharmacokinetic properties of Cpd3. This work not only successfully pinpointed Cpd3 as a specific antagonist of SUCNR1 to serve as a promising candidate in the realm of therapeutic interventions for DN, but also provides a paradigm of dry-wet combined discovery strategies for GPCR-based therapeutics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Renal tubulointerstitial fibrosis (RIF), featured by epithelial-to-mesenchymal-transition (EMT) of renal tubular epithelial cells and collagen deposition in the renal interstitial region, is the main pathological change of diabetic nephropathy (DN). Fraxin, the main active component of Fraxinus rhynchophylla Hance with anti-inflammatory activity, has been demonstrated to ameliorate glomerulosclerosis. However, the regulatory role of Fraxin on diabetic RIF remains unclear. In this study, we investigated the renal protective benefits of Fraxin against diabetic RIF and elucidated its mechanisms. In vitro, Fraxin inhibited the abnormal expression of EMT-related markers and proinflammatory cytokines, improved cellular morphology, and subsequently reduced the extracellular matrix (ECM) production in high glucose (HG)-induced NRK-52E cells. In vivo, Fraxin effectively ameliorated renal function, inhibited the abnormal expression of EMT-related markers and proinflammatory cytokines, and reduced ECM deposition in renal tubule interstitium in db/db mice. Notably, Fraxin could directly bind to epidermal growth factor receptor (EGFR), which contributed to the inhibition of EGFR phosphorylation and counteracted the activation of c-Src/NF-κB pathway, eventually ameliorating RIF. Thus, Fraxin may be a potential drug candidate for treating DN., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The multiple roles of TGR5 in the regulation of glucose metabolism, inflammation, and oxidative stress have drawn attention as therapeutic candidates for diabetes-related kidney disease. However, diabetes induces downregulation of renal TGR5 protein expression, and the regulatory mechanisms have not been clarified. Here, we identify that Smurf1, an E3 ubiquitin ligase, is a critical interactor of TGR5 and mediates the ubiquitination and proteasomal degradation of TGR5 under high glucose stimulation in glomerular mesangial cells. Genetic deficiency of Smurf1 restores TGR5 protein expression and attenuates renal injuries in diabetic mice. Mechanistically, Smurf1 interacts with the TGR5 ICL2 region by its HECT domain and induces K11/K48-linked polyubiquitination of TGR5 at K306 residue. Moreover, restoration of TGR5 protects db/db mice from diabetic nephropathy. These observations elucidate the critical role of Smurf1 in regulating TGR5 stability, suggesting that pharmacological targeting of the interaction between Smurf1 and TGR5 could serve as a promising therapeutic strategy against diabetic nephropathy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Renal oxidative stress (OSS) is the leading cause of diabetic nephropathy (DN). The silent information regulator 1/forkhead boxo3a (Sirt1/Foxo3a) pathway plays an essential role in regulating the antioxidant enzyme system. In this study, we aimed to investigate the mechanism of connexin32 (Cx32) on the antioxidant enzyme system in DN. Results: In this study, Cx32 overexpression significantly reduced reactive oxygen species generation and effectively inhibited the excessive production of extracellular matrix such as fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in high-glucose (HG)-induced glomerular mesangial cells. In addition, Cx32 overexpression reversed the downregulation of Sirt1, and promoted the nuclear transcription of Foxo3a, subsequently activating the antioxidant enzymes including catalase and manganese superoxide dismutase (MnSOD), however, Cx32 knockdown showed the opposite effects. A further mechanism study showed that Cx32 promoted the autoubiquitination and degradation of Smad ubiquitylation regulatory factor-1 (Smurf1), thereby reducing the ubiquitination of Sirt1 at Lys 335 and the degradation of Sirt1. Moreover, the in vivo results showed that adenovirus-mediated Cx32 overexpression activated the Sirt1/Foxo3a pathway, and inhibited OSS in the kidney tissues, eventually improving the renal function and glomerulosclerosis in diabetic mice. Innovation: This study highlighted the antioxidant role of Cx32-Sirt1-Foxo3a axis to alleviate DN, which is a new mechanism of Cx32 alleviating DN. Conclusion: Cx32 alleviated DN via activating the Sirt1/Foxo3a antioxidant pathway. The specific mechanism was that Cx32 upregulated the Sirt1 expression through reducing the ubiquitination of Lys 335 of Sirt1 by inhibiting Smurf1. Antioxid. Redox Signal. 39, 241-261.

Objective: Oxidative stress (OS) is the main cause leading to diabetic renal fibrosis. Recently, Fyn was paid much attention on OS and emerged as a pivotal player in acute kidney injury, while whether Fyn regulates oxidative stress in chronic diabetes nephropathy (DN) has not been clarified yet. The purpose of this study was to identify the role of Fyn in DN and elucidated its regulatory mechanism., Methods: The db/db mice and littermate control C57BKS/J mice were injected by tail vein with Fyn interfering adenovirus or Fyn overexpressing adenovirus to investigate the role of Fyn in vivo. Primary glomerular mesangial cells (GMCs) were used for in vitro studies., Results: Fyn was up-regulated in high glucose (HG)-induced GMCs and kidneys of diabetic mice. Additionally, Fyn knockdown reduced the level of OS in HG-induced GMCs and kidneys of diabetic mice, thereby ameliorating diabetic renal fibrosis. While overexpression of Fyn significantly increased the level of OS in GMCs and kidney tissues, resulting in renal damage. Moreover, Fyn deficiency exerted antioxidant effects by activating the Sirt1/Foxo3a pathway. Mechanistically, Fyn facilitated the combination of c-Cbl and Sirt1 by phosphorylating c-Cbl at Tyr 731 , which triggered K48-linked polyubiquitination of Sirt1 at Lys 377 and Lys 513 by c-Cbl and promoted Sirt1 degradation, impairing the antioxidant effects of Foxo3a., Conclusions: Fyn deficiency promoted Foxo3a nuclear transcription via reducing the ubiquitination of Sirt1 by c-Cbl, thereby alleviating renal oxidative damage in diabetic mice. These results identified Fyn as a potential therapeutic target against DN., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Renal chronic inflammation is an important hallmark of diabetic renal fibrosis. Casein kinase 2 interacting protein 1 (CKIP-1) performs a nephroprotective role in the pathogenesis of diabetic nephropathy (DN), which is dramatically decreased in diabetic kidneys. However, whether CKIP-1 regulates inflammation to ameliorate renal fibrosis remains unclear and it is interesting to clarify the degradation mechanism of CKIP-1. Here, we identified CKIP-1 expression was down-regulated in diabetic kidneys and knockout (KO) of CKIP-1 increased c-Jun expression and extra cellular matrix (ECM) in kidneys of normal mice, and knockout (KO) of CKIP-1 further exacerbated renal inflammatory fibrosis in diabetic mice. Moreover, the activated Src kinase interacted with CKIP-1 at Lys252 and increased K48 linked polyubiquitination and proteasome degradation of CKIP-1 in HG induced GMCs and diabetic kidneys. Mechanistically, Src facilitating the binding of c-Cbl with CKIP-1 by promoting the phosphorylation of c-Cbl, thereby increasing Cbl-mediated ubiquitination of CKIP-1 to down-regulate CKIP-1 protein expression. Thus, our study highlighted the anti-inflammation role of CKIP-1 and clarified the mechanism of CKIP-1 degradation in DN., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

We previously reported that both Cx43 and CKIP-1 attenuated diabetic renal fibrosis via the activation of Nrf2 signaling pathway. However, whether CKIP-1, a scaffold protein, participates in regulating the activation of Nrf2 signaling pathway by Cx43 remains to be elucidated. In this study, the effect of adenovirus-mediated Cx43 overexpression on renal fibrosis in CKIP-1 -/- diabetic mice was investigated. We found that overexpression of Cx43 could significantly alleviate renal fibrosis by activating the Nrf2 pathway in diabetic mice, but have no obvious effect in CKIP-1 -/- diabetic mice. Cx43 overexpressed plasmid and CKIP-1 small interfering RNA were simultaneously transfected into glomerular mesangial cells and the result demonstrated that the effect of activation of Nrf2 signaling pathway by Cx43 was blocked by CKIP-1 depletion. The interaction between Cx43 and CKIP-1 was analyzed by immunofluorescence and immunoprecipitation assays. We found that Cx43 interacted with CKIP-1, and the interaction was weakened by high glucose treatment. Moreover, Cx43 regulated the expression of CKIP-1 and the interaction of CKIP-1 with Nrf2 via Cx43 carboxyl terminus (CT) domain, thereby activating Nrf2 signaling pathway. According to the results, we preliminary infer that CKIP-1 acts downstream to CX43 on the activation of Nrf2 signaling pathway to protect from renal fibrosis in diabetes, the mechanism of which might be related to the interaction of CKIP-1 with Nrf2 through Cx43 CT. Our study provides further experimental basis for targeting the Cx43-CKIP-1-Nrf2 axis to resist diabetic renal fibrosis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Hyperglycemia-induced renal epithelial-to-mesenchymal transition (EMT) is a key pathological factor in diabetic renal tubulointerstitial fibrosis (RIF). Our previous studies have shown that connexin 43 (Cx43) activation attenuated the development of diabetic renal fibrosis. However, whether Cx43 regulates the EMT of renal tubular epithelial cells (TECs) and the pathological process of RIF under the diabetic conditions remains to be elucidated. In the present study, we identified that Cx43 protein expression was down-regulated in the kidney tissues of db/db mice as well as in high glucose (HG)-induced NRK-52E cells. Overexpression of Cx43 improved renal function in db/db spontaneous diabetic model mice, increased SIRT1 levels, decreased hypoxia-inducible factor (HIF)-1α expression, and reduced production of EMT markers and extracellular matrix (ECM) components. Additionally, Cx43 overexpression inhibited the EMT process and reduced the expression of ECM components such as fibronectin (FN), Collagen I, and Collagen IV in HG-induced NRK-52E cells, whereas Cx43 deficiency had the opposite effects. Mechanistically, Cx43 in a carboxyl-terminal signal transduction-dependent manner could up-regulate SIRT1 expression and enhance SIRT1-dependent deacetylation of HIF-1α to reduce HIF-1α activity, which eventually ameliorated renal EMT and diabetic RIF. Our study indicates the essential role of Cx43 in regulating renal EMT and diabetic RIF via regulating the SIRT1-HIF-1α signaling pathway and provides an experimental basis for Cx43 as a potential target for diabetic nephropathy (DN)., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Our previous studies indicated that the G-protein-coupled bile acid receptor, Gpbar1 (TGR5), inhibits inflammation by inhibiting the NF-κB signalling pathway, eventually attenuating diabetic nephropathy (DN). Gentiopicroside (GPS), the main active secoiridoid glycoside of Gentiana manshurica Kitagawa, has been demonstrated to inhibit inflammation in various diseases via inhibiting the inflammatory signalling pathways. However, whether GPS inhibits the NF-κB signalling pathway by activating TGR5 and regulates the pathological progression of diabetic renal fibrosis requires further investigation. In this study, we found that GPS significantly reversed the downregulation of TGR5 and inhibited the overproduction of fibronectin (FN), transforming growth factor β1 (TGF-β1), intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in glomerular mesangial cells (GMCs) exposed to high glucose (HG). Additionally, GPS prevented the phosphorylation and degradation of IκBα, and subsequently inhibited the activation of the NF-κB signalling pathway. Further investigation found that GPS enhanced the stabilization of IκBα by promoting the interaction of β-arrestin2 with IκBα via TGR5 activation, which contributed to the inhibition of NF-κB signalling pathway. Importantly, the depletion of TGR5 blocked the inhibition of the NF-κB signalling pathway and reversed the downregulation of FN, ICAM-1, VCAM-1 and TGF-β1 by GPS in HG-induced GMCs. Moreover, GPS increased the TGR5 protein levels and promoted the interaction between IκBα and β-arrestin2, thereby inhibiting the reduction of IκBα and blocked NF-κB p65 nuclear translocation in the kidneys of STZ-induced diabetic mice. Collectively, these data suggested that GPS regulates the TGR5-β-arrestin2-NF-κB signalling pathway to prevent inflammation in the kidneys of diabetic mice, and ultimately ameliorates the pathological progression of diabetic renal fibrosis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background and Purpose: Nox4 is the major isoform of NADPH oxidase found in the kidney and contributes to the pathogenesis of diabetic nephropathy. However, the molecular mechanisms of increased Nox4 expression induced by hyperglycaemia remain to be elucidated. Here, the role of the connexin32-Nox4 signalling axis in diabetic nephropathy and its related mechanisms were investigated., Experimental Approach: Diabetes was induced in mice by low-dose streptozotocin (STZ) combined with a high-fat diet. Effects of connexin32 on Nox4 expression and on renal function and fibrosis in STZ-induced diabetic mice were investigated using adenovirus-overexpressing connexin32 and connexin32-deficient mice. Interactions between connexin32 and Nox4 were analysed by co-immunoprecipitation and immunofluorescence assays., Key Results: Connexin32 was down-regulated in the kidneys of STZ-induced diabetic mice. Overexpression of connexin32 reduced expression of Nox4 and improved renal function and fibrosis in diabetic mice, whereas connexin32 deficiency had opposite effects. Down-regulation of fibronectin expression by connexin32 was not dependent on gap junctional intercellular communication involving connexin32. Connexin32 interacted with Nox4 and reduced the generation of hydrogen peroxide, leading to the down-regulation of fibronectin expression. Mechanistically, connexin32 decreased Nox4 expression by promoting its K48-linked polyubiquitination. Interestingly, Smurf1 overexpression inhibited K48-linked polyubiquitination of Nox4. Furthermore, connexin32 interacted with Smurf1 and inhibited its expression., Conclusion and Implications: Connexin32 ameliorated renal fibrosis in diabetic mice by promoting K48-linked Nox4 polyubiquitination and degradation via inhibition of Smurf1 expression. Targeting the connexin32-Nox4 signalling axis may contribute to the development of novel treatments for diabetic nephropathy., (© 2019 The British Pharmacological Society.)

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 1h. 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 CK2α ameliorates renal inflammatory fibrosis in diabetes via NF-κB pathway. To explore potential regulatory mechanism of CK2α, the expression and activity of CK2α, 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 CK2α was upregulated in kidneys of db/db and KKAy diabetic mice; (2) Inhibition of CK2α kinase activity or knockdown of CK2α protein expression suppressed high glucose-induced expressions of FN and ICAM-1 in glomerular mesangial cells (GMCs); (3) Inhibition of CK2α kinase activity or knockdown of CK2α protein expression not only restrained IκB degradation, but also suppressed HG-induced nuclear accumulation, transcriptional activity and DNA binding activity of NF-κB in GMCs; (4) Treatment of TBB or CK2α RNAi adenovirus infection ameliorated renal fibrosis in diabetic animals; (5) Treatment of TBB or CK2α RNAi adenovirus infection suppressed IκB degradation and NF-κB nuclear accumulation in glomeruli of diabetic animals. This study indicates the essential role of CK2α in regulating the diabetic renal pathological process of inflammatory fibrosis via NF-κB pathway, and inhibition of CK2α may serve as a promising therapeutic strategy for diabetic nephropathy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 (H 2 O 2 ) 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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-β1; (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-β1, ultimately attenuating renal fibrosis in diabetes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Our previous studies have confirmed that the sphingosine kinase 1 (SphK1)-sphingosine 1-phosphate (S1P) signaling pathway in the kidney under diabetic conditions is closely correlated with the pathogenesis of diabetic nephropathy (DN). The activation of SphK1-S1P pathway by high glucose (HG) can increase the expression of fibronectin (FN), an important fibrotic component, in glomerular mesangial cells (GMCs) by promoting the DNA-binding activity of transcription factor AP-1. However, the mechanism responsible for the sustained activation of SphK1-S1P pathway remains unclear. Given the binding motifs for AP-1 within the first intron of the SphK1 gene, we speculated that the activated AP-1 in the kidney under HG condition possibly regulates SphK1 expression in a positive feedback manner, thereby promoting the sustained activation of SphK1-S1P pathway and mediating the pathological progression of DN. Here, we observed the effect of AP-1 on SphK1 expression in GMCs and explored the molecular mechanism involved in the sustained activation of SphK1-S1P pathway. We found two consensus binding motifs for AP-1 in the promoter sequences and non-coding region downstream of the transcriptional initiation of the rat SphK1 gene by chromatin immunoprecipitation assay. The treatment of GMCs with both HG and S1P significantly increased the protein expression of c-Jun and c-Fos, and obviously enhanced the phosphorylation of c-Jun at Ser63 and Ser73, and c-Fos at Ser32. Knockdown of c-Jun and c-Fos with siRNAs substantially inhibited the expression of SphK1 and FN, whereas overexpression of c-Jun and c-Fos significantly increased the expression of SphK1 and FN. Curcumin treatment greatly decreased the levels of c-Jun, c-Fos, SphK1, and FN in the kidney tissues of diabetic rats. SiRNAs targeting SphK1 and S1P2 receptor respectively inhibited the phosphorylation of c-Jun (ser63 and ser73) and c-Fos (ser32), as well as FN expression under both normal and HG conditions. Our data demonstrated that the activated SphK1-S1P signaling pathway in GMCs under diabetic conditions is closely associated with AP-1 to form a positive feedback loop. This positive feedback loop functions as an important molecular basis for the sustained activation of SphK1-S1P pathway and increased FN expression that lead to the initiation and progression of DN., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Diabetic nephropathy (DN) is the major cause of end-stage renal disease worldwide and its prevalence continues to increase. Currently, therapies for DN provide only partial renoprotection; hence new targets for therapeutic intervention need to be identified. In this review, we summarized the new target, sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway, explored its potential therapeutic role in the prevention and treatment of DN., Data Sources: Most relevant articles were mainly identified by searching PubMed in English., Study Selection: Mainly original articles and critical review articles by major pioneer investigators in this field were selected to be reviewed., Results: SphK1/S1P pathway can be activated by hyperglycemia, advanced glycation end products, and many pro-inflammatory cytokines, which leads to fibronectin, transforming growth factor-β1 up-regulation and AP-1 activation. And then it could promote glomerular mesangial cells proliferation and extracellular matrix accumulation, mediating the initiation and progression of diabetic renal fibrosis., Conclusions: SphK1/S1P pathway is closely correlated with the pathogenesis of DN. The results suggest that SphK1/S1P pathway as a new target for clinically improving DN in future is of great prospect.

Both RhoA/ROCK and NF-κB signaling pathways play important roles in the pathogenesis of diabetic nephropathy (DN). However, it remains unknown whether and how RhoA/ROCK regulates NF-κB signaling in diabetic kidneys. In cultured glomerular mesangial cells (GMCs), the high glucose-activated NF-κB nuclear translocation and DNA binding activity were attenuated by ROCK inhibitor Y27632 or dominant-negative RhoA mutant, indicating that RhoA/ROCK signaling regulates high glucose-activated NF-κB pathway. Furthermore, NF-κB-regulated inflammatory factors ICAM-1 and TGF-β1 were markedly increased in high glucose-treated GMCs, leading to accumulation of fibronectin (FN), an important component of extracellular matrix (ECM), This effect was also effectively attenuated by Y27632 or dominant-negative RhoA mutant. In STZ-induced diabetic rats, treatment with ROCK inhibitor fasudil suppressed the RhoA/ROCK activation and NF-κB nuclear translocation, and significantly reduced the renal FN, ICAM-1 and TGF-β1 protein levels. Thus, the RhoA/ROCK pathway may regulate NF-κB to upregulate inflammatory genes and mediate the development of DN., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Curcumin, a major polyphenol from the golden spice Curcuma longa commonly known as turmeric, has been recently discovered to have renoprotective effects on diabetic nephropathy (DN). However, the mechanisms underlying these effects remain unclear. We previously demonstrated that the sphingosine kinase 1-sphingosine 1-phosphate (SphK1-S1P) signaling pathway plays a pivotal role in the pathogenesis of DN. This study aims to investigate whether the renoprotective effects of curcumin on DN are associated with its inhibitory effects on the SphK1-S1P signaling pathway. Our results demonstrated that the expression and activity of SphK1 and the production of S1P were significantly down-regulated by curcumin in diabetic rat kidneys and glomerular mesangial cells (GMCs) exposed to high glucose (HG). Simultaneously, SphK1-S1P-mediated fibronectin (FN) and transforming growth factor-beta 1 (TGF-β1) overproduction were inhibited. In addition, curcumin dose dependently reduced SphK1 expression and activity in GMCs transfected with SphK(WT) and significantly suppressed the increase in SphK1-mediated FN levels. Furthermore, curcumin inhibited the DNA-binding activity of activator protein 1 (AP-1), and c-Jun small interference RNA (c-Jun-siRNA) reversed the HG-induced up-regulation of SphK1. These findings suggested that down-regulation of the SphK1-S1P pathway is probably a novel mechanism by which curcumin improves the progression of DN. Inhibiting AP-1 activation is one of the therapeutic targets of curcumin to modulate the SphK1-S1P signaling pathway, thereby preventing diabetic renal fibrosis., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

A number of studies have recently demonstrated the involvement of nuclear factor-kappa B (NF-κB) activation and the subsequent coordinated inflammatory responses in the pathogenesis of diabetic nephropathy (DN). Polydatin has been shown to have the ability of anti-adhesive inflammation. However, the possible protective and beneficial effects of polydatin on DN via suppressing inflammatory damage and extracellular matrix (ECM) accumulation are not fully elucidated. We found that the polydatin could inhibit the induction and activity of NF-κB, and meanwhile ameliorating ECM accumulation in streptozotocin-diabetic rats. We aimed to investigate the effect of polydatin on fibronectin (FN) protein expression, and to elucidate its potential mechanism involving the NF-κB inflammatory signaling pathway in rat glomerular mesangial cells (GMCs) cultured under high glucose. The results revealed that polydatin significantly suppressed high glucose-induced FN production, inhibited NF-κB nuclear translocation, reduced the DNA-binding activity of NF-κB, as well as decreased the protein expression of ICAM-1 and TGF-β in GMCs. These findings suggested that polydatin significantly represses high glucose-induced FN expression in rat GMCs, which may be closely related to its inhibition of the NF-κB signaling pathway. Hence, we elucidated the potential mechanisms of the anti-inflammatory effects and ECM accumulation alleviation of polydatin in GMCs of DN in vitro., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The accumulation of glomerular extracellular matrix (ECM) is one of the critical pathological characteristics of diabetic renal fibrosis. Fibronectin (FN) is an important constituent of ECM. Our previous studies indicate that the activation of the sphingosine kinase 1 (SphK1)-sphingosine 1- phosphate (S1P) signaling pathway plays a key regulatory role in FN production in glomerular mesangial cells (GMCs) under diabetic condition. Among the five S1P receptors, the activation of S1P2 receptor is the most abundant. Berberine (BBR) treatment also effectively inhibits SphK1 activity and S1P production in the kidneys of diabetic models, thus improving renal injury. Based on these data, we further explored whether BBR could prevent FN production in GMCs under diabetic condition via the S1P2 receptor. Here, we showed that BBR significantly down-regulated the expression of S1P2 receptor in diabetic rat kidneys and GMCs exposed to high glucose (HG) and simultaneously inhibited S1P2 receptor-mediated FN overproduction. Further, BBR also obviously suppressed the activation of NF-κB induced by HG, which was accompanied by reduced S1P2 receptor and FN expression. Taken together, our findings suggest that BBR reduces FN expression by acting on the S1P2 receptor in the mesangium under diabetic condition. The role of BBR in S1P2 receptor expression regulation could closely associate with its inhibitory effect on NF-κB activation.

One of the main pathological changes in diabetic nephropathy is the renal fibrosis, which includes glomerulosclerosis and tubulointerstitial fibrosis. In vivo and in vitro studies demonstrated that berberine could ameliorate renal dysfunction in diabetic rats with nephropathy and inhibit fibronectin expression in mesangial cells cultured under high glucose. However, the molecular mechanisms have not been fully elucidated. The purpose of the present study was to investigate the effects of berberine on the nuclear factor-kappa B (NF-kappaB) activation, intercellular adhesion molecule-1, transforming growth factor-beta1 and fibronectin protein expression in renal tissue from alloxan-induced diabetic mice with renal damage. The distribution of NF-kappaB p65 in glomerulus and the degradation of I kappaB-alpha in renal cortex were examined by immunohistochemistry and Western blot, respectively. The protein expression of intercellular adhesion molecule-1, transforming growth factor-beta 1 and fibronectin in renal cortex were also detected by Western blot. Our results revealed that in alloxan-induced diabetic mice, the nuclear staining of NF-kappaB p65 was increased in glomerulus, whereas renal I kappaB-alpha protein was significantly reduced. The protein levels of intercellular adhesion molecule-1, transforming growth factor-beta 1 and fibronectin were upregulated in kidney from diabetic mice. After berberine treatment, the immunostaining of NF-kappaB was decreased, and the reduced degradation of I kappaB-alpha level was partially restored. The protein levels of intercellular adhesion molecule-1, transforming growth factor-beta 1 and fibronectin were all downregulated by berberine compared with diabetic model group. In conclusion, the ameliorative effects of berberine on extracellular matrix accumulation might associate with its inhibitory function on NF-kappaB signal pathway., ((c) 2010 Elsevier B.V. All rights reserved.)

To explore the protection of emodin on renal dysfunction in the streptozotocin-induced diabetic rats with nephropathy and the role of p38 mitogen-activated protein kinase (p38 MAPK) signal transduction pathway in this protection. 30 male Spraque-Dawley rats were randomly divided into control group, model group and emodin group. The rats in the model group and emodin group were administered with streptozotocin (60 mg/kg) to induce diabetes. 40 mg/kg/day of emodin were orally given to the rats in emodin group. The rats in other groups were only given solvent. Biochemical index were analysed by oxidase and oxidase dynamical enzyme method. Glomerular area and volume were determined quantitatively by using Image Analysis System. Western blotting and immunohistochemical staining was used to detect the total p38 MAPK, phosphorylated p38 MAPK, phosphorylated cAMP response element binding protein (CREB) and fibronectin. The average kidney weight/body weight, glomerular area, glomerular volume and all biochemical indexes significantly increased in model group as compared to the control group (P<0.05), while the average body weight decreased. The expressions of phosphorylaed p38 MAPK, phosphorylated CREB and fibronectin increased by 1.98-fold, 1.94-fold and 1.96-fold respectively in model group compared with those in the control group (P<0.05). Emodin markedly decreased the average kidney weight/body weight, glomerular area, glomerular volume and all biochemical indexes (P<0.05), having a weak action on the level of blood glucose. The expressions of phosphorylated p38 MAPK, phosphorylated CREB and fibronectin also significantly downregulated in emodin group compared with those in model group (P<0.05). Emodin was efficient to ameliorate renal dysfunction in diabetic nephropathy rats probably by its inhibition of the activation of p38 MAPK pathway and downregulation of the expression of fibronectin.

Renal tubulointerstitial fibrosis (TIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells, is the typical pathological alteration in diabetic nephropathy. Gentiopicroside (GPS), a natural compound with anti-inflammatory activity, has been demonstrated to alleviate glomerulosclerosis, whereas whether GPS inhibits TIF via regulating inflammation remains unclear. In this study, diabetic db/db mice and high glucose (HG)-stimulated renal tubular epithelial cells (NRK-52E) were applied to explore the effects and mechanisms of GPS on TIF. The results in vivo showed that GPS effectively improves glycolipid metabolism disorder, renal dysfunction, and TIF. In particular, GPS treatment reversed the abnormal expressions of EMT marker proteins including elevated α-smooth muscle actin and vimentin and decreased E-cadherin in the kidney of db/db mice. Moreover, GPS treatment also inhibited protein expressions of angiotensinⅡ type 1 receptor (AT1R) and CK2α and the activation of the NF-κB pathway. Importantly, the aforementioned effects of GPS acted in vivo were further observed in vitro in HG-stimulated NRK-52E cells, which were independent of its effects on glucose and lipid-lowering activity but were reversed by AT1R over-expression. Together, our results indicate that GPS that directly inhibits the CK2/NF-κB inflammatory signaling pathway via AT1R may also contribute to the amelioration of TIF in diabetes. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is quickly becoming the largest cause of end-stage renal disease (ESRD) in diabetic patients, as well as a major source of morbidity and mortality. Our previous studies indicated that the activation of Nrf2/ARE pathway via Connexin43 (Cx43) considerably contribute to the prevention of oxidative stress in the procession of DN. Fraxin (Fr), the main active glycoside of Fraxinus rhynchophylla Hance, has been demonstrated to possess many potential pharmacological activities. Whereas, whether Fr could alleviate renal fibrosis through regulating Cx43 and consequently facilitating the activation of Nrf2/ARE pathway needs further investigation. The in vitro results showed that: 1) Fr increased the expression of antioxidant enzymes including SOD1 and HO-1 to inhibit high glucose (HG)-induced fibronectin (FN) and inflammatory cell adhesion molecule (ICAM-1) overexpression; 2) Fr exerted antioxidant effect through activating the Nrf2/ARE pathway; 3) Fr significantly up-regulated the expression of Cx43 in HG-induced glomerular mesangial cells (GMCs), while the knock down of Cx43 largely impaired the activation of Nrf2/ARE pathway induced by Fr; 4) Fr promoted the activation of Nrf2/ARE pathway via regulating the interaction between Cx43 and AKT. Moreover, in accordance with the results in vitro , elevated levels of Cx43, phosphorylated-AKT, Nrf2 and downstream antioxidant enzymes related to Nrf2 were observed in the kidneys of Fr-treated group compared with model group. Importantly, Fr significantly improved renal dysfunction pathological changes of renal fibrosis in diabetic db/db mice. Collectively, Fr could increase the Cx43-AKT-Nrf2/ARE pathway activation to postpone the diabetic renal fibrosis and the up-regulation of Cx43 is probably a novel mechanism in this process. [ABSTRACT FROM AUTHOR]

Oxidative stress is the main factor responsible for the induction of diabetic renal fibrosis. Thus, improving the state of oxidative stress can effectively prevent the further deterioration of diabetic nephropathy (DN). Previous research has shown that formononetin (FMN), a flavonoid with significant antioxidant activity and Sirt1 activation effect, can improve diabetic renal fibrosis. However, the exact mechanisms underlying the effect of FMN on diabetic renal fibrosis have yet to be elucidated. In this study, we carried out in vivo experiments in a db/db (diabetic) mouse model and demonstrated that FMN activated the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway and improved oxidative stress by increasing levels of sirtuin-1 (Sirt1) protein level in renal tissue. We also found that this process reversed the up-regulation of fibronectin (FN) and intercellular adhesion molecule 1 (ICAM-1) and led to an improvement in renal insufficiency. In vitro results further showed that FMN significantly reversed the upregulation of FN and ICAM-1 in glomerular mesangial cells (GMCs) exposed to high glucose. FMN also promoted the expression of Nrf2 and widened its nuclear distribution. Thus, our data indicated that FMN inhibited hyperglycemia-induced superoxide overproduction by activating the Nrf2/ARE signaling pathway. We also found that FMN up-regulated the expression of Sirt1 and that Sirt1 deficiency could block the activation of the Nrf2/ARE signaling pathway in GMCs induced by high glucose. Finally, we found that Sirt1 deficiency could reverse the down-regulation of FN and ICAM-1 induced by FMN. Collectively, our data demonstrated that FMN up-regulated the expression of Sirt1 to activate the Nrf2/ARE signaling pathway, improved oxidative stress in DN to prevent the progression of renal fibrosis. Therefore, FMN probably represents an efficient therapeutic option of patients with DN. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Berberine (BBR) exerts powerful renoprotective effects on diabetic nephropathy (DN), but the underlying mechanisms remain unclear. We previously demonstrated that activation of the G protein-coupled bile acid receptor TGR5 ameliorates diabetic nephropathy by inhibiting the activation of the sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptor 2 (S1P2) signaling pathway. In this study, we explored the role of TGR5 in the BBR-induced downregulation of sphingosine 1-phosphate receptor 2 (S1P2)/mitogen-activated protein kinase (MAPK)-mediated fibrosis in glomerular mesangial cells (GMCs). Results showed that, BBR suppressed the expression of FN, ICAM-1, and TGF-β1 in high-glucose cultures of GMCs, and the phosphorylation level of c-Jun/c-Fos was downregulated. The high glucose lowered TGR5 expression in a time-dependent manner; this effect was reversed by BBR in a dose-dependent manner. The TGR5 agonist INT-777 decreased the high glucose-induced FN, ICAM-1, and TGF-β1 protein contents. In addition, TGR5 siRNA blocked S1P2 degradation by BBR. And MAPK signaling, which plays important regulatory roles in the pathological progression of DN, was activated by TGR5 siRNA. Apart from this, MAPK signaling as well as FN, ICAM-1, and TGF-β1 suppressed by BBR under high glucose conditions were limited by TGR5 depletion. Thus, BBR decreases FN, ICAM-1, and TGF-β1 levels under high glucose conditions in GMCs possibly by activating TGR5 and inhibiting S1P2/MAPK signaling. [ABSTRACT FROM AUTHOR]

Sirt1 and nuclear factor-E2 related factor 2 (Nrf2)-anti-oxidant response element (ARE) anti-oxidative pathway play important regulatory roles in the pathological progression of diabetic nephropathy (DN) induced by advanced glycation-end products (AGEs). Polydatin (PD), a glucoside of resveratrol, has been shown to possess strong anti-oxidative bioactivity. Our previous study demonstrated that PD markedly resists the progression of diabetic renal fibrosis and thus, inhibits the development of DN. Whereas, whether PD could resist DN through regulating Sirt1 and consequently promoting Nrf2-ARE pathway needs further investigation. Here, we found that concomitant with decreasing RAGE (the specific receptor for AGEs) expression, PD significantly reversed the downregulation of Sirt1 in terms of protein expression and deacetylase activity and attenuated FN and TGF-β1 expression in GMCs exposed to AGEs. Under AGEs-treatment condition, PD could decrease Keap1 expression and promote the nuclear content, ARE-binding ability, and transcriptional activity of Nrf2. In addition, PD increased the protein levels of heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1), two target genes of Nrf2. The activation of Nrf2-ARE pathway by PD eventually led to the quenching of ROS overproduction sharply boosted by AGEs. Depletion of Sirt1 blocked Nrf2-ARE pathway activation and reversed FN and TGF-β1 downregulation induced by PD in GMCs challenged with AGEs. Along with reducing HO-1 and SOD1 expression, silencing of Nrf2 increased FN and TGF-β1 levels. PD treatment elevated Sirt1 and Nrf2 levels in the kidney tissues of diabetic rats, then improved the anti-oxidative capacity and renal dysfunction of diabetic models, and finally reversed the upregulation of FN and TGF-β1. Taken together, the resistance of PD on upregulated FN and TGF-β1 induced by AGEs via oxidative stress in GMCs is closely associated with its activation of Sirt1-Nrf2-ARE pathway. [ABSTRACT FROM AUTHOR]

RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose - treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose - treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs. [ABSTRACT FROM AUTHOR]

Abstract: Sphingosine kinase 1 (SphK1) pathway is critical in the pathogenesis of diabetic nephropathy. Recently, we found that berberine suppressed the activation of SphK1 pathway in diabetic kidney, protecting against diabetic nephropathy. The potential molecular mechanism, however, is still unknown. Here, we showed that berberine prevented the expression of α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) and fibronectin (FN) in mesangial cells cultured by high glucose. Furthermore, berberine suppressed the activation of SphK1 pathway via inhibition of the activity and expression of SphK1 in mesangial cells cultured by high glucose. Surprisingly, berberine blocked the increased activity and expression of SphK1 in mesangial cells transfected by wild type SphK1 under normal glucose condition. However, berberine had no inhibitory effect on the recombinant human SphK1 protein. Finally, berberine markedly attenuated the high glucose-induced activator protein-1 (AP-1) activity in mesangial cells. Altogether, these data not only demonstrate that berberine is an important agent against diabetic nephropathy through inhibition of SphK1/AP-1 pathway, but also indicate that the inhibition of SphK1/AP-1 by berberine is independent of ambient glucose concentration. [Copyright &y& Elsevier]

Abstract: Accumulation of extracellular matrix including fibronectin in mesangium is one of the major pathologic characteristics in diabetic nephropathy. In the current study, we explored role of sphingosine-1-phosphate (S1P) receptor in fibronectin expression and underlying molecular mechanism. Among five S1P receptors the mRNA level of S1P2 receptor was the most abundant in kidney of diabetic rats and mesangial cells under high glucose condition. S1P augmentation of fibronectin was significantly inhibited by S1P2 receptor antagonist JTE-013 and S1P2-siRNA. S1P-stimulated fibronectin expression was remarkably blocked by ERK1/2 inhibitor PD98059 and p38MAPK inhibitor SB203580. Phospho-ERK1/2 and phospho-p38MAPK level induced by S1P were markedly abrogated by JTE-013 and S1P2-siRNA. In conclusion, S1P2 receptor was significantly up-regulated under diabetic condition. S1P2 receptor mediated fibronectin expression through the activation of S1P-S1P2-MAPK (ERK1/2 and p38MAPK) axis in mesangial cells under high glucose condition, suggesting that it might be a potential therapeutic target for diabetic nephropathy treatment. [Copyright &y& Elsevier]

Abstract: Background: Our previous studies demonstrated that berberine could improve the renal function in rats and mice with diabetic nephropathy (DN) and inhibit extracellular matrix (ECM) component, fibronectin (FN) expression in rat mesangial cells (MCs) cultured under high glucose. However, the molecular mechanisms have not been fully elucidated. Objective: To explore the potential mechanisms of berberine in the treatment of DN, we investigated the effects of berberine on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs. Method: Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The activation of NF-κB was detected by Western blot and confocal microscopy. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot. Results: Our results revealed that berberine significantly suppressed LPS-induced cell proliferation and inhibited LPS-induced NF-κB nuclear translocation in MCs, as well as protein expression of ICAM-1, TGF-β1, iNOS and FN. Conclusion: Berberine significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of berberine on DN might be associated with this inhibition effect on NF-κB signaling pathway which was independent of its hypoglycemic effect. [ABSTRACT FROM AUTHOR]

Abstract: Berberine (BBR) was previously found to have beneficial effects on renal injury in experimental diabetic rats. However, the mechanisms underlying the effects are not fully understood. Sphingosine kinase–Sphingosine 1-phosphate (SphK–S1P) signaling pathway has been implicated in the pathogenesis of diabetic nephropathy (DN). The aim of this study was to investigate the effects of BBR on renal injury and the activation of SphK–S1P signaling pathway in alloxan-induced diabetic mice with nephropathy. Alloxan-induced diabetic mice were treated orally with BBR (300mg/kg/day) or vehicle for 12weeks. BBR inhibited the increases in fasting blood glucose, kidney/body weight ratio, blood urea nitrogen, serum creatinine and 24-h albuminuria in diabetic mice. It also prevented renal hypertrophy, TGF-β1 synthesis, FN and Col IV accumulation. Moreover, BBR down-regulated the elevated staining, activity and levels of mRNA and protein of SphK1, and S1P production as well. These findings suggest that the inhibitory effect of BBR on the activation of SphK–S1P signaling pathway in diabetic mouse kidney is a novel mechanism by which BBR partly exerts renoprotective effects on DN. [ABSTRACT FROM AUTHOR]