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Advanced glycation end products' (AGEs) modification of extracellular matrix proteins induces crosslinking, which results in thickening of the basement membrane and activating several intracellular signaling cascades, eventually promoting the pathological progression of diabetic nephropathy (DN). We have previously confirmed that casein kinase 2α (CK2α) activates the nuclear factor of kappaB (NF-κB) signaling pathway to enhance high glucose-induced expressions of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in glomerular mesangial cells (GMCs). However, to date, the mechanism by which CK2α regulates diabetic renal fibrosis is not fully understood. In view of the regulation of inflammation and fibrosis by myocardin-related transcription factor A (MRTF-A), we are highly concerned whether CK2α promotes AGEs-induced expressions of FN and ICAM-1 in glomerular mesangial cells via activation of MRTF-A, thus affecting the pathogenesis of DN. We found that CK2α and MRTF-A proteins were overexpressed in AGEs-induced diabetic kidneys. Inhibition of CK2α kinase activity or knockdown of CK2α protein expression suppressed the upregulation of FN and ICAM-1 expressions in GMCs induced by AGEs. MRTF-A knockdown compromised the expressions of FN and ICAM-1 in GMCs induced by AGEs. Moreover, inhibition of CK2α kinase activity or knockdown of CK2α protein expression restrained the protein expression and nuclear aggregation of MRTF-A. CK2α interacted with MRTF-A. Furthermore, knockdown of MRTF-A while overexpression of CK2α blocked the upregulation effect of CK2α on the protein expressions of FN and ICAM-1. These findings suggest that CK2α promotes diabetic renal fibrosis via activation of MRTF-A and upregulation of inflammatory genes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. 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.)

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±0.12nm, 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 AUC 0-24h 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-β1) 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

We previously demonstrated that activation of sphingosine kinase 1 (SphK1)- sphingosine 1- phosphate (S1P) signaling pathway by high glucose (HG) plays a pivotal role in increasing the expression of fibronectin (FN), an important fibrotic component, by promoting the DNA-binding activity of transcription factor activator protein 1 (AP-1) in glomerular mesangial cells (GMCs) under diabetic conditions. As a multi-target anti-oxidative drug, polydatin (PD) has been shown to have renoprotective effects on experimental diabetes. However, whether PD could resist diabetic nephropathy (DN) by regulating SphK1-S1P signaling pathway needs further investigation. Here, we found that PD significantly reversed the upregulated FN and ICAM-1 expression in GMCs exposed to AGEs. Simultaneously, PD dose-dependently inhibited SphK1 levels at the protein expression and kinase activity and attenuated S1P production under AGEs treatment conditions. In addition, PD reduced SphK activity in GMCs transfected with wild-type SphK(WT) plasmid and significantly suppressed SphK1-mediated increase of FN and ICAM-1 levels under normal conditions. Furthermore, we found that the AGEs-induced upregulation of phosphorylation of c-Jun at Ser63 and Ser73 and c-Fos at Ser32, DNA-binding activity and transcriptional activity of AP-1 were blocked by PD. In comparison with db/db model group, PD treatment suppressed SphK1 levels (mRNA, protein expression, and activity) and S1P production, reversed the upregulation of FN, ICAM-1, c-Jun, and c-Fos in the kidney tissues of diabetic mice, and finally ameliorated renal injury in db/db mice. These findings suggested that the downregulation of SphK1-S1P signaling pathway is probably a novel mechanism by which PD suppressed AGEs-induced FN and ICAM-1 expression and improved renal dysfunction of diabetic models., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Ireland Ltd. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure and is characterized by excessive deposition of extracellular matrix (ECM) proteins such as fibronectin (FN), in the glomerular mesangium and tubulointerstitium. Betulinic acid (BA), a pentacyclic triterpene derived from the bark of the white birch tree, has been demonstrated to have many pharmacological activities. However, the effect of BA on DN has not been fully elucidated. To explore the possible anti-inflammatory effects of BA and their underlying mechanisms, we used streptozotocin-induced diabetic rat kidneys and high glucose-treated glomerular mesangial cells. Our study showed BA could inhibit the degradation of I κ B α and the activity of NF- κ B in diabetic rat kidneys and high glucose-induced mesangial cells, resulting in reduction of FN expression. In addition, BA suppressed the DNA binding activity and transcriptional activity of NF- κ B in high glucose-induced glomerular mesangial cells (GMCs). Furthermore, BA enhanced the interaction between I κ B α and β -arrestin2 in mesangial cells. Taken together, our data suggest BA inhibits NF- κ B activation through stabilizing NF- κ B inhibitory protein I κ B α , thereby preventing diabetic renal fibrosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]