High glucose activates PKC-ζ and NADPH oxidase through autocrine TGF-β1 signaling in mesangial cells

2008 ◽  
Vol 295 (6) ◽  
pp. F1705-F1714 ◽  
Author(s):  
Ling Xia ◽  
Hong Wang ◽  
Snezana Munk ◽  
Janice Kwan ◽  
Howard J. Goldberg ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
High Glucose ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Mesangial Cells ◽  
Pi3 Kinase ◽  
Kinase Assay ◽  
Ros Generation ◽  
Pkc Ζ

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-β1 is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) -ζ activation. We investigated the role of TGF-β1 in this action of high glucose on primary rat mesangial cells within 1–48 h. Both high glucose and exogenous TGF-β1 stimulated PKC-ζ kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-β1 antibodies. Neutralizing TGF-β1 antibodies, or a TGF-β receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-ζ activation by high glucose. TGF-β1 also stimulated cellular membrane translocation of PKC-α, -β1, -δ, and -ε, similar to high glucose. High glucose and TGF-β1 enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-β1 antibodies, LY364947, or a specific PKC-ζ pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-ζ in normal glucose caused upregulation of p22phox, a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-β1 stimulation of PKC isozymes including PI3 kinase activation of PKC-ζ and consequent generation of ROS by NADPH oxidase.

High glucose-induced RhoA activation requires caveolae and PKCβ1-mediated ROS generation

2012 ◽  
Vol 302 (1) ◽  
pp. F159-F172 ◽  
Author(s):  
Y. Zhang ◽  
F. Peng ◽  
B. Gao ◽  
A. J. Ingram ◽  
J. C. Krepinsky
Keyword(s):  
Diabetic Nephropathy ◽  
Nadph Oxidase ◽  
High Glucose ◽  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Ros Generation ◽  
Caveolin 1 ◽  
Rhoa Activation ◽  
Rhoa Signaling ◽  
Tgf Β1

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We previously showed that RhoA activation by high glucose in mesangial cells (MC) leads to matrix upregulation (Peng F, Wu D, Gao B, Ingram AJ, Zhang B, Chorneyko K, McKenzie R, Krepinsky JC. Diabetes 57: 1683–1692, 2008). Here, we study the mechanism whereby RhoA is activated. In primary rat MC, RhoA activation required glucose entry and metabolism. Broad PKC inhibitors (PMA, bisindolylmaleimide, Gö6976), as well as specific PKCβ blockade with an inhibitor and small interfering RNA (siRNA), prevented RhoA activation by glucose. PKCβ inhibition also abrogated reactive oxygen species (ROS) generation by glucose. The ROS scavenger N-acetylcysteine (NAC) or NADPH oxidase inhibitors apocynin and DPI prevented glucose-induced RhoA activation. RhoA and some PKC isoforms localize to caveolae. Chemical disruption of these microdomains prevented RhoA and PKCβ1 activation by glucose. In caveolin-1 knockout cells, glucose did not induce RhoA and PKCβ1 activation; these responses were rescued by caveolin-1 reexpression. Furthermore, glucose-induced ROS generation was significantly attenuated by chemical disruption of caveolae and in knockout cells. Downstream of RhoA signaling, activator protein-1 (AP-1) activation was also inhibited by disrupting caveolae, was absent in caveolin-1 knockout MC and rescued by caveolin-1 reexpression. Finally, transforming growth factor (TGF)-β1 upregulation, mediated by AP-1, was prevented by RhoA signaling inhibition and by disruption or absence of caveolae. In conclusion, RhoA activation by glucose is dependent on PKCβ1-induced ROS generation, most likely through NADPH oxidase. The activation of PKCβ1 and its downstream effects, including upregulation of TGF-β1, requires caveolae. These microdomains are thus important mediators of the profibrogenic process associated with diabetic nephropathy.

O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

2011 ◽  
Vol 301 (4) ◽  
pp. E713-E726 ◽  
Author(s):  
Howard Goldberg ◽  
Catharine Whiteside ◽  
I. George Fantus
Keyword(s):  
Diabetic Nephropathy ◽  
P38 Mapk ◽  
High Glucose ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Mesangial Cells ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Glomerular Mesangial Cells ◽  
Matrix Accumulation

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β- N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins ( O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase ( O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr308 and Ser473 phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.

Mesangial cell NADPH oxidase upregulation in high glucose is protein kinase C dependent and required for collagen IV expression

2006 ◽  
Vol 290 (2) ◽  
pp. F345-F356 ◽  
Author(s):  
L. Xia ◽  
H. Wang ◽  
H. J. Goldberg ◽  
S. Munk ◽  
I. G. Fantus ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
High Glucose ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Fold Increase ◽  
Collagen Iv ◽  
Oxidase Inhibitor ◽  
Ros Generation ◽  
Rt Pcr ◽  
Diabetic Glomerulosclerosis

Excess collagen IV expression by mesangial cells contributes to diabetic glomerulosclerosis. We hypothesized that in high glucose reactive oxygen species (ROS) generation by NADPH oxidase is PKC dependent and required for collagen IV expression by mesangial cells. In rat mesangial cells cultured in 5 mM (NG) or 25 mM d-glucose (HG), RT-PCR and Western immunoblotting detected p22phox and p47phox mRNA and protein, respectively. Quantitative real-time RT-PCR analyzed collagen IV mRNA. With the use of confocal microscopy, ROS were detected with dichlorofluorescein and intracellular collagen IV by immunofluorescence. In HG, ROS were generated within 1 h, sustained up to 48 h, and prevented by a NADPH oxidase inhibitor, diphenylenechloride iodonium (DPI), or a conventional PKC isozyme inhibitor, Gö6976. In NG, phorbol myristate acetate stimulated ROS generation that was inhibited with DPI. In HG, expression of p22phox and p47phox was increased within 3 to 6 h and inhibited by Gö6976. In HG, Gö6976 or transfection with antisense against p22phox reversed the 1.8-fold increase in collagen IV mRNA. In HG, the antioxidants Tempol or Tiron, or transfection with antisense against p22phox or p47phox, prevented ROS generation and the 2.3-fold increase in collagen IV protein. Increased mitochondrial redox potential in HG was unaffected by transfection with antisense against p22phox. We conclude that in HG, mesangial cell ROS generation by upregulated NADPH oxidase is dependent on conventional PKC isozymes and also required for collagen IV expression.

scholarly journals Cytoprotective Effect of Liposomal Puerarin on High Glucose-Induced Injury in Rat Mesangial Cells

Antioxidants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1177
Author(s):  
Lassina Barro ◽  
Jui-Ting Hsiao ◽  
Chu-Yin Chen ◽  
Yu-Lung Chang ◽  
Ming-Fa Hsieh
Keyword(s):  
Protective Effect ◽  
Transforming Growth Factor Beta ◽  
High Glucose ◽  
High Performance ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Mesangial Cells ◽  
Diabetic Patients ◽  
Pueraria Lobata ◽  
Cytoprotective Effect

In diabetic patients, high glucose and high oxidative states activate gene expression of transforming growth factor beta (TGF-β) and further translocate Smad proteins into the nucleus of renal cells. This signal pathway is characterized as the onset of diabetic nephropathy. Puerarin is an active ingredient extracted from Pueraria lobata as an anti-hyperglycemic and anti-oxidative agent. However, the poor oral availability and aqueous solubility limit its pharmaceutical applications. The present paper reports the liposomal puerarin and its protective effect on high glucose-injured rat mesangial cells (RMCs). The purity of puerarin extracted from the root of plant Pueraria lobata was 83.4% as determined by the high-performance liquid chromatography (HPLC) method. The liposomal puerarin was fabricated by membrane hydration followed by ultrasound dispersion and membrane extrusion (pore size of 200 nm). The fabricated liposomes were examined for the loading efficiency and contents of puerarin, the particle characterizations, the radical scavenge and the protective effect in rat mesangial cells, respectively. When the liposomes were subjected to 20 times of membrane extrusion, the particle size of liposomal puerarin can be reduced to less than 200 nm. When liposomal puerarin in RMCs in high glucose concentration (33 mM) was administered, the over-expression of TGF-β and the nuclear translocation of Smad 2/3 proteins was both inhibited. Therefore, this study successfully prepared the liposomal puerarin and showed the cytoprotective effect in RMCs under high glucose condition.

scholarly journals Store-operated calcium entry suppressed the TGF-β1/Smad3 signaling pathway in glomerular mesangial cells

2017 ◽  
Vol 313 (3) ◽  
pp. F729-F739 ◽  
Author(s):  
Sarika Chaudhari ◽  
Weizu Li ◽  
Yanxia Wang ◽  
Hui Jiang ◽  
Yuhong Ma ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Mesangial Cells ◽  
Underlying Mechanism ◽  
Glomerular Mesangial Cells ◽  
Critical Pathway ◽  
Smad3 Phosphorylation ◽  
Tgf Β1

Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca2+entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca2+channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La3+; 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca2+channels, significantly augmented TGF-β1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-β1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the TGF-β1/Smad3 signaling pathway.

scholarly journals Microparticles as Potential Mediators of High Glucose-Induced Renal Cell Injury

Biomolecules ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 348 ◽  
Author(s):  
Ravindran ◽  
Pasha ◽  
Agouni ◽  
Munusamy
Keyword(s):  
Er Stress ◽  
Signaling Pathways ◽  
Transforming Growth Factor Beta ◽  
High Glucose ◽  
Cell Injury ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Epithelial Mesenchymal Transition ◽  
Smooth Muscle Actin ◽  
Mesenchymal Transition

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease worldwide. Activation of signaling pathways such as the mammalian target of rapamycin (mTOR), extracellular signal-regulated kinases (ERK), endoplasmic reticulum (ER) stress, transforming growth factor-beta (TGF-β), and epithelial-mesenchymal transition (EMT), are thought to play a significant role in the etiology of DN. Microparticles (MPs), the small membrane vesicles containing bioactive signals shed by cells upon activation or during apoptosis, are elevated in diabetes and were identified as biomarkers in DN. However, their exact role in the pathophysiology of DN remains unclear. Here, we examined the effect of MPs shed from renal proximal tubular cells (RPTCs) exposed to high glucose conditions on naïve RPTCs in vitro. Our results showed significant increases in the levels of phosphorylated forms of 4E-binding protein 1 and ERK1/2 (the downstream targets of mTOR and ERK pathways), phosphorylated-eIF2α (an ER stress marker), alpha smooth muscle actin (an EMT marker), and phosphorylated-SMAD2 and nuclear translocation of SMAD4 (markers of TGF-β signaling). Together, our findings indicate that MPs activate key signaling pathways in RPTCs under high glucose conditions. Pharmacological interventions to inhibit shedding of MPs from RPTCs might serve as an effective strategy to prevent the progression of DN.

scholarly journals Gigantol has Protective Effects against High Glucose-Evoked Nephrotoxicity in Mouse Glomerulus Mesangial Cells by Suppressing ROS/MAPK/NF-κB Signaling Pathways

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 80 ◽  
Author(s):  
Mei-Fen Chen ◽  
Shorong-Shii Liou ◽  
Tang-Yao Hong ◽  
Shung-Te Kao ◽  
I-Min Liu
Keyword(s):  
Signaling Pathways ◽  
High Glucose ◽  
Cell Injury ◽  
Mesangial Cells ◽  
Therapeutic Potential ◽  
Microvascular Complications ◽  
Biologically Active ◽  
Mitogen Activated Protein Kinase ◽  
Ros Generation ◽  
Cytochrome C Release

Gigantol is a bibenzyl compound derived from several medicinal orchids. This biologically active compound has shown promising therapeutic potential against diabetic cataracts, but whether this compound exerts beneficial effects on the other diabetic microvascular complications remains unclear. This study was carried out to examine effects of gigantol on high glucose-induced renal cell injury in cultured mouse kidney mesangial cells (MES-13). MES-13 cells were pretreated with gigantol (1, 5, 10 or 20 μmol/L) for 1 h followed by further exposure to high (33.3 mmol/L) glucose for 48 h. Gigantol concentration dependently enhanced cell viability followed by high glucose treatment in MES-13 cells. High glucose induced reactive oxygen species (ROS) generation, malondialdehyde production and glutathione deficiency were recoved in MES-13 cells pretreated with gigantol. High glucose triggered cell apoptosis via the the loss of mitochondrial membrane potential, depletion of adenosine triphosphate, upregulation of caspases 9 and 3, enhancement of cytochrome c release, and subsequent interruption of the Bax/Bcl-2 balance. These detrimental effects were ameliorated by gigantol. High glucose also induced activation of JNK, p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) in MES-13 cells, which were blocked by gigantol. The results suggest that treatment MES-13 cells with gigantol halts high glucose-induced renal dysfunction through the suppression of the ROS/MAPK/NF-κB signaling pathways. Our data are of value to the understanding the mechanism for gigantol, and would benefit the study of drug development or food supplement for diabetes and nephropathy.

Hexosamines and TGF-β1 use similar signaling pathways to mediate matrix protein synthesis in mesangial cells

2004 ◽  
Vol 286 (2) ◽  
pp. F409-F416 ◽  
Author(s):  
Lalit P. Singh ◽  
Kenneith Green ◽  
Michelle Alexander ◽  
Shira Bassly ◽  
Errol D. Crook
Keyword(s):  
High Glucose ◽  
Matrix Protein ◽  
Transforming Growth Factor ◽  
Cell Function ◽  
Mesangial Cells ◽  
Rat Kidney ◽  
Protein Accumulation ◽  
Western Blots ◽  
Matrix Production ◽  
Mes Cells

Hyperglycemia-induced alterations in mesangial (MES) cell function and extracellular matrix (ECM) protein accumulation are seen in diabetic glomerulopathy. Transforming growth factor-β1 (TGF-β1) mediates high-glucose-induced matrix production in the kidney. Recent studies demonstrated that some of the effects of high glucose on cellular metabolism are mediated by the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine (GlcN) 6-phosphate. We previously showed that the high-glucose-mediated fibronectin and laminin synthesis in MES cells is mediated by the HBP and that GlcN is more potent than glucose in inducing TGF-β1 promoter luciferase activity. In this study, we investigated the hypothesis that the effects of glucose on MES matrix production occur via hexosamine regulation of TGF-β1. Culturing simian virus (SV)-40-transformed rat kidney MES cells in 25 mM glucose (HG) for 48 h increases cellular fibronectin and laminin levels about twofold on Western blots compared with low glucose (5 mM). GlcN (1.5 mM) or TGF-β1 (2.5-5 ng/ml) for 24-48 h also increases ECM synthesis. However, the effects of HG or GlcN with TGF-β1 are not additive. The presence of anti-TGF-β1 antibodies (20 μg/ml) blocks both TGF-β1- and GlcN-induced fibronectin synthesis. TGF-β1 increased ECM levels via PKA (laminin and fibronectin) and PKC (fibronectin) pathways. Similarly, TGF-β1 and hexosamines led to nonadditive increases in phosphorylation of the cAMP responsive element binding transcription factor. These results suggest that the effects of excess glucose on MES ECM synthesis occur via HBP-mediated regulation of TGF-β1.

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