Activation of PKC-βIin glomerular mesangial cells is associated with specific NF-κB subunit translocation

2001 ◽  
Vol 281 (4) ◽  
pp. F613-F619 ◽  
Author(s):  
Anoop Kumar ◽  
Karen S. Hawkins ◽  
Meredith A. Hannan ◽  
Michael B. Ganz
Keyword(s):  
High Glucose ◽  
Nuclear Translocation ◽  
Mesangial Cells ◽  
Glomerular Mesangial Cells ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Normal Glucose ◽  
Important Pathway ◽  
Pkc Β ◽  
Early Transcription

Changes in expression and activity of protein kinase C (PKC) isoforms and early transcription factors may account for alterations in cell behavior seen in diabetes. We studied the expression of PKC-βIin rat glomerular mesangial cells (MCs) cultured in normal or high glucose and compared it with the temporal and spatial expression of dimeric transcription factor (NF-κB) p50 and p65. The results show that in unstimulated cells PKC-βIand NF-κB p50 are distributed in the cytosol and, on stimulation, their distribution is perinuclear and they are localized to the membrane. Serum-starved MCs cultured in high-glucose medium exhibit a predominantly cytosolic localization of PKC-βIand both p50 and p65 NF-κB. However, phorbol 12-myristate 13-acetate (PMA) stimulation of cells grown in the presence of high glucose resulted in membrane translocation of PKC-βIthat was associated with nuclear translocation of NF-κB p65, but not NF-κB p50. Moreover, the translocation to the nucleus for NF-κB p65 was significantly higher in MCs exposed to high glucose compared with those exposed to normal glucose. These observations indicate that the NF-κB p65, but not NF-κB p50, expression and translocation pattern mirrors that of PKC-βI, which may be one important pathway by which signaling is enhanced in the high-glucose state.

Mesangial cell-reduced Ca2+ signaling in high glucose is due to inactivation of phospholipase C-β3 by protein kinase C

2005 ◽  
Vol 289 (5) ◽  
pp. F1078-F1087 ◽  
Author(s):  
Helena Frecker ◽  
Snezana Munk ◽  
Hong Wang ◽  
Catharine Whiteside
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
High Glucose ◽  
Mesangial Cells ◽  
Fold Increase ◽  
Glomerular Mesangial Cells ◽  
Kinase C ◽  
Normal Glucose ◽  
Intracellular Ca

In high glucose, glomerular mesangial cells (MCs) demonstrate impaired Ca2+ signaling in response to seven-transmembrane receptor stimulation. To identify the mechanism, we first postulated decreased release from intracellular stores. Intracellular Ca2+ was measured in fluo-3-loaded primary cultured rat MCs using confocal fluorescence microscopy. In high glucose (HG) 30 mM for 48 h, the 25 nM ionomycin-stimulated intracellular Ca2+ response was reduced to 82% of that observed in normal glucose (NG). In NG 5.6 mM, Ca2+ responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF) were unchanged in cells cultured in 50 nM Ca2+ vs. 1.8 mM Ca2+. Depletion of intracellular Ca2+ stores with thapsigargin eliminated ET-1-stimulated Ca2+ responses. Incubation in 30 mM glucose (HG) for 48 h or stimulation with phorbol myristate acetate (PMA) for 10 min eliminated the Ca2+ response to ET-1 but had no effect on the PDGF response. Downregulation of protein kinase C (PKC) with 24-h PMA or inhibition with Gö6976 in HG normalized the Ca2+ response to ET-1. Because ET-1 and PDGF stimulate Ca2+ signaling through different phospholipase C pathways, we hypothesized that, in HG, PKC selectively phosphorylates and inhibits PLC-β3. Using confocal immunofluorescence imaging, in NG, a 1.6- to 1.7-fold increase in PLC-β3 Ser1105 phosphorylation was observed following PMA or ET-1 stimulation for 10 min. In HG, immunofluorescent imaging and immunoblotting showed increased PLC-β3 phosphorylation, without change in total PLC-β3, which was reversed with 24-h PMA or Gö6976. We conclude that reduced Ca2+ signaling in HG cannot be explained by reduced Ca2+ stores but is due to conventional PKC-dependent phosphorylation and inactivation of PLC-β3.

scholarly journals Regulation of angiotensin II receptors and PKC isoforms by glucose in rat mesangial cells

1999 ◽  
Vol 276 (5) ◽  
pp. F691-F699 ◽  
Author(s):  
Farhad Amiri ◽  
Raul Garcia
Keyword(s):  
Angiotensin Ii ◽  
High Glucose ◽  
Glucose Concentration ◽  
Mesangial Cells ◽  
Competitive Binding ◽  
Ang Ii ◽  
Pkc Isoforms ◽  
Normal Glucose ◽  
Elevated Glucose ◽  
In Cells

It has been shown that glomerular angiotensin II (ANG II) receptors are downregulated and protein kinase C (PKC) is activated under diabetic conditions. We, therefore, investigated ANG II receptor and PKC isoform regulation in glomerular mesangial cells (MCs) under normal and elevated glucose concentrations. MCs were isolated from collagenase-treated rat glomeruli and cultured in medium containing normal or high glucose concentrations (5.5 and 25.0 mM, respectively). Competitive binding experiments were performed using the ANG II antagonists losartan and PD-123319, and PKC analysis was conducted by Western blotting. Competitive binding studies showed that the AT1 receptor was the only ANG II receptor detected on MCs grown to either subconfluence or confluence under either glucose concentration. AT1 receptor density was significantly downregulated in cells grown to confluence in high-glucose medium. Furthermore, elevated glucose concentration enhanced the presence of all MC PKC isoforms. In addition, PKCβ, PKCγ and PKCε were translocated only in cells cultured in elevated glucose concentrations following 1-min stimulation by ANG II, whereas PKCα, PKCθ, and PKCλ were translocated by ANG II only in cells grown in normal glucose. Moreover, no changes in the translocation of PKCδ, PKCι, PKCζ, and PKCμ were detected in response to ANG II stimulation under euglycemic conditions. We conclude that MCs grown in high glucose concentration show altered ANG II receptor regulation as well as PKC isoform translocation compared with cells grown in normal glucose concentration.

scholarly journals Lack of miRNA-17 family mediates high glucose-induced PAR-1 up-regulation in glomerular mesangial cells

2021 ◽  
Author(s):  
Zhuang-Zhuang Tang ◽  
Pan-Pan Gu ◽  
Xiao-Fei An ◽  
Ling-Shan Gou ◽  
Yao-Wu Liu
Keyword(s):  
High Glucose ◽  
Mesangial Cells ◽  
Kidney Diseases ◽  
Mrna Level ◽  
Glomerular Mesangial Cells ◽  
Chronic Kidney Diseases ◽  
Protein Levels ◽  
Normal Glucose ◽  
Protease Activated Receptor 1

Abstract Up-regulation of thrombin receptor protease-activated receptor 1 (PAR-1) is verified to contribute to chronic kidney diseases, including diabetic nephropathy, however, the mechanisms are still unclear. In this study, we investigated the effect of PAR-1 on high glucose-induced proliferation of human glomerular mesangial cells (HMCs), and explored the mechanism of PAR-1 up-regulation from alteration of microRNAs. We found that high glucose stimulated proliferation of the mesangial cells whereas PAR-1 inhibition with vorapaxar attenuated the cell proliferation. Moreover, high glucose up-regulated PAR-1 in mRNA level and protein expression while did not affect the enzymatic activity of thrombin in HMCs after 48 h culture. Then high glucose induced PAR-1 elevation was likely due to the alteration of the transcription or post-transcriptional processing. It was found that miR-17 family members including miR-17-5p, -20a-5p, and − 93-5p were markedly decreased among the eight detected microRNAs only in high glucose-cultured HMCs, but miR-129-5p, miR-181a-5p, and miR-181b-5p were markedly decreased in both high glucose-cultured HMCs and osmotic press control compared with normal glucose culture. So miR-20a was selected to confirm the role of miR-17 family on PAR-1 up-regulation, finding that miR-20a-5p overexpression reversed the up-regulation of PAR-1 in mRNA and protein levels induced by high glucose in HMCs. In summary, our finding indicated that PAR-1 up-regulation mediated proliferation of glomerular mesangial cells induced by high glucose, and deficiency of miR-17 family resulted in PAR-1 up-regulation.

scholarly journals Translocation of protein kinase C ? and ? in rat glomerular mesangial cells cultured under high glucose conditions

Diabetologia ◽  
1994 ◽  
Vol 37 (8) ◽  
pp. 838-841 ◽  
Author(s):  
R. Kikkawa ◽  
M. Haneda ◽  
T. Uzu ◽  
D. Koya ◽  
T. Sugimoto ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
High Glucose ◽  
Mesangial Cells ◽  
Glomerular Mesangial Cells ◽  
Kinase C ◽  
Cells Cultured

AGEs induce oxidative stress and activate protein kinase C-βII in neonatal mesangial cells

2000 ◽  
Vol 278 (4) ◽  
pp. F676-F683 ◽  
Author(s):  
Vincenzo Scivittaro ◽  
Michael B. Ganz ◽  
Miriam F. Weiss
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Mesangial Cells ◽  
Specific Protein ◽  
Extracellular Proteins ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Β ◽  
Animal Models Of Diabetes

Increased activation of specific protein kinase C (PKC) isoforms and increased nonenzymatic glycation of intracellular and extracellular proteins [the accumulation of advanced glycation end products (AGEs)] are major mechanistic pathways implicated in the pathogenesis of diabetic complications. Blocking PKC-βII has been shown to decrease albuminuria in animal models of diabetes. To demonstrate a direct relationship between AGEs and the induction and translocation of PKC-βII, studies were carried out in rat neonatal mesangial cells, known to express PKC-βII in association with rapid proliferation in post-natal development. Oxidative stress was studied by using the fluorescent probe dichlorfluorescein diacetate. Translocation of PKC-βII was demonstrated by using immunofluorescence and Western blotting of fractionated mesangial cells. Induction of intracellular oxidative stress, increase in intracellular calcium, and cytosol to membrane PKC-βII translocation (with no change in PKC-α) were demonstrated after exposure to AGE-rich proteins. These data support the hypothesis that AGEs cause mesangial oxidative stress and alterations in PKC-βII, changes that may ultimately contribute to phenotypic abnormalities associated with diabetic nephropathy.

Extracellular glucose reduces the responsiveness of mesangial cell ion channels to angiotensin II

1995 ◽  
Vol 269 (3) ◽  
pp. F389-F397 ◽  
Author(s):  
E. E. Seal ◽  
D. C. Eaton ◽  
L. M. Gomez ◽  
H. Ma ◽  
B. N. Ling
Keyword(s):  
Angiotensin Ii ◽  
Ion Channels ◽  
High Glucose ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Ion Homeostasis ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Extracellular Glucose ◽  
Normal Glucose

Abnormal cellular ion homeostasis is a well-recognized component of diabetic glomerular disease. In cultured rat glomerular mesangial cells, we have previously shown that insulin regulates Ca(2+)-dependent activation of 4-pS Cl- channels and 27-pS nonselective cation channels (NSCC) by angiotensin II (ANG II). To assess whether extracellular glucose also affects mesangial ion channels, we applied patch-clamp techniques to cells incubated in constant insulin (100 mU/ml) and either "normal" (5 mM) or "high" (30 mM) glucose for 1 wk. In normal glucose, 100 nM ANG II increased Cl- and NSCC activity by > 16-fold and > 60-fold, respectivley. Direct release of intracellular Ca2+ ([Ca2+]i) stores (0.25 microM thapsigargin) mimicked ANG II-induced channel stimulation. In high glucose, Cl- and NSCC stimulation by ANG II was attenuated (< 7-fold), whereas channel activation by thapsigargin was unaffected. Protein kinase C (PKC) inhibition (30-min exposure to 0.5 microM calphostin) or downregulation (24-h exposure to 0.1 microM 4 beta-phorbol 12-myristate 13-acetate), but not aldose reductase inhibition (0.5 mM sorbinil), restored channel responsiveness to ANG II despite high glucose. Channel responsiveness was also restored if mesangial cells were coincubated in both high glucose and 500 microM myo-inositol. Acute exposure to a synthetic diacylglycerol (100 microM 1-oleoyl-2-acetyl glycerol) reestablished channel unresponsiveness to ANG II. We conclude the following in rat mesangial cell cultures: 1) Activation of Ca(2+)-dependent Cl- and NSCCs by ANG II is reduced by high extracellular glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Transient High-Glucose Stimulation Induces Persistent Inflammatory Factor Secretion from Rat Glomerular Mesangial Cells via an Epigenetic Mechanism

2018 ◽  
Vol 49 (5) ◽  
pp. 1747-1754 ◽  
Author(s):  
Deng Yunlei ◽  
Fan  Qiuling ◽  
Wang Xu ◽  
Zhao Qianwen ◽  
Cao Xu ◽  
...  
Keyword(s):  
High Glucose ◽  
Mesangial Cells ◽  
Inflammatory Factors ◽  
Control Group ◽  
Metabolic Memory ◽  
Glomerular Mesangial Cells ◽  
Glucose Stimulation ◽  
Normal Glucose ◽  
High Glucose Group ◽  
Glucose Group

Background/Aims: Diabetic nephropathy is the one of the most serious microvascular complications of diabetes mellitus, and “metabolic memory” plays a vital role in the development of diabetic complications. To investigate the effect of epigenetics on metabolic memory, we analyzed the impact of transient high-glucose stimulation on the secretion of inflammatory factors from rat glomerular mesangial cells. Methods: Rat glomerular mesangial cells (HBZY-1) were divided into three groups: high-glucose group (25 mM glucose), hypertonic group (5.5 mM glucose+19.5 mM mannitol), and normal-glucose control group (5.5 mM glucose). Mesangial cells were cultured in high-glucose, hypertonic, and normal-glucose media for 24 h and transitioned to normal-glucose culture for 24, 48, and 72 h. Then, protein, mRNA, and supernatants were harvested. The expression of monomethylated H3K4 was determined by western blot analysis, and the expression of the NF-κB subunit p65 and histone methyltransferase set7/9 was determined by quantitative real-time PCR. The expression of monocyte chemoattractant protein 1 (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1) was detected by an enzyme-linked immunosorbent assay. Results: Compared with the control group, H3K4me1 expression was upregulated after transient high-glucose stimulation, gradually downregulated in the following 48 h (P < 0.05), and reached the level of the control group at 72 h (P > 0.05). The expression of set7/9 was increased after 24 h of high-glucose stimulation and the following 24 h and 48 h (P < 0.05); it then returned to the level of the control group at 72 h. Compared with the control group, the increased expression of p65, VCAM-1, and MCP-1 was sustained for at least 72 h in the high-glucose group. Conclusion: Transient high-glucose stimulation can induce the persistent secretion of inflammatory factors from rat glomerular mesangial cells via histone modification.

scholarly journals Translocation of protein kinase C ? and ? in rat glomerular mesangial cells cultured under high glucose conditions

Diabetologia ◽  
1994 ◽  
Vol 37 (8) ◽  
pp. 838-841 ◽  
Author(s):  
R. Kikkawa ◽  
M. Haneda ◽  
T. Uzu ◽  
D. Koya ◽  
T. Sugimoto ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
High Glucose ◽  
Mesangial Cells ◽  
Glomerular Mesangial Cells ◽  
Kinase C ◽  
Cells Cultured

scholarly journals High glucose and diabetes enhanced store-operated Ca2+ entry and increased expression of its signaling proteins in mesangial cells

2014 ◽  
Vol 306 (9) ◽  
pp. F1069-F1080 ◽  
Author(s):  
Sarika Chaudhari ◽  
Peiwen Wu ◽  
Yanxia Wang ◽  
Yanfeng Ding ◽  
Joseph Yuan ◽  
...  
Keyword(s):  
High Glucose ◽  
Mesangial Cells ◽  
Cyclopiazonic Acid ◽  
Diabetic Rats ◽  
Type I ◽  
Signaling Proteins ◽  
Glomerular Mesangial Cells ◽  
Rt Pcr ◽  
Protein Expressions ◽  
Normal Glucose

The present study was conducted to determine whether and how store-operated Ca2+ entry (SOCE) in glomerular mesangial cells (MCs) was altered by high glucose (HG) and diabetes. Human MCs were treated with either normal glucose or HG for different time periods. Cyclopiazonic acid-induced SOCE was significantly greater in the MCs with 7-day HG treatment and the response was completely abolished by GSK-7975A, a selective inhibitor of store-operated Ca2+ channels. Similarly, the inositol 1,4,5-trisphosphate-induced store-operated Ca2+ currents were significantly enhanced in the MCs treated with HG for 7 days, and the enhanced response was abolished by both GSK-7975A and La3+. In contrast, receptor-operated Ca2+ entry in MCs was significantly reduced by HG treatment. Western blotting showed that HG increased the expression levels of STIM1 and Orai1 in cultured MCs. A significant HG effect occurred at a concentration as low as 10 mM, but required a minimum of 7 days. The HG effect in cultured MCs was recapitulated in renal glomeruli/cortex of both type I and II diabetic rats. Furthermore, quantitative real-time RT-PCR revealed that a 6-day HG treatment significantly increased the mRNA expression level of STIM1. However, the expressions of STIM2 and Orai1 transcripts were not affected by HG. Taken together, these results suggest that HG/diabetes enhanced SOCE in MCs by increasing STIM1/Orai1 protein expressions. HG upregulates STIM1 by promoting its transcription but increases Orai1 protein through a posttranscriptional mechanism.

scholarly journals Lack of miRNA-17 family mediates high glucose-induced PAR-1 up-regulation in glomerular mesangial cells

2021 ◽  
Author(s):  
Zhuang-Zhuang Tang ◽  
Pan-Pan Gu ◽  
Xiao-Fei An ◽  
Ling-Shan Gou ◽  
Yao-Wu Liu
Keyword(s):  
High Glucose ◽  
Mesangial Cells ◽  
Kidney Diseases ◽  
Mrna Level ◽  
Glomerular Mesangial Cells ◽  
Chronic Kidney Diseases ◽  
Protein Levels ◽  
Normal Glucose ◽  
Protease Activated Receptor 1

Abstract Up-regulation of thrombin receptor protease-activated receptor 1 (PAR-1) is verified to contribute to chronic kidney diseases, including diabetic nephropathy, however, the mechanisms are still unclear. In this study, we investigated the effect of PAR-1 on high glucose-induced proliferation of human glomerular mesangial cells (HMCs), and explored the mechanism of PAR-1 up-regulation from alteration of microRNAs. We found that high glucose stimulated proliferation of the mesangial cells whereas PAR-1 inhibition with vorapaxar attenuated the cell proliferation. Moreover, high glucose up-regulated PAR-1 in mRNA level and protein expression while did not affect the enzymatic activity of thrombin in HMCs after 48 h culture. Then high glucose induced PAR-1 elevation was likely due to the alteration of the transcription or post-transcriptional processing. It was found that miR-17 family members including miR-17-5p, -20a-5p, and − 93-5p were markedly decreased among the eight detected microRNAs only in high glucose-cultured HMCs, but miR-129-5p, miR-181a-5p, and miR-181b-5p were markedly decreased in both high glucose-cultured HMCs and osmotic press control compared with normal glucose culture. So miR-20a was selected to confirm the role of miR-17 family on PAR-1 up-regulation, finding that miR-20a-5p overexpression reversed the up-regulation of PAR-1 in mRNA and protein levels induced by high glucose in HMCs. In summary, our finding indicated that PAR-1 up-regulation mediated proliferation of glomerular mesangial cells induced by high glucose, and deficiency of miR-17 family resulted in PAR-1 up-regulation.

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