scholarly journals Knockdown of NLRP3 alleviates high glucose or TGFB1-induced EMT in human renal tubular cells

2018 ◽  
Vol 61 (3) ◽  
pp. 101-113 ◽  
Author(s):  
Shan Song ◽  
Duojun Qiu ◽  
Fengwei Luo ◽  
Jinying Wei ◽  
Ming Wu ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
P38 Mapk ◽  
Nlrp3 Inflammasome ◽  
High Glucose ◽  
Matrix Protein ◽  
Transforming Growth Factor ◽  
Inflammasome Activation ◽  
Mesenchymal Transition ◽  
Tubular Cells ◽  
Renal Tubular Cells

Tubular injury is one of the crucial determinants of progressive renal failure in diabetic nephropathy (DN), while epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the accumulation of matrix protein in the diabetic kidney. Activation of the nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome leads to the maturation of interleukin (IL)-1B and is involved in the pathogenic mechanisms of diabetes. In this study, we explored the role of NLRP3 inflammasome on high glucose (HG) or transforming growth factor-B1 (TGFB1)-induced EMT in HK-2 cells. We evaluated EMT through the expression of α-smooth muscle actin (α-SMA) and E-cadherin as well as the induction of a myofibroblastic phenotype. Reactive oxygen species (ROS) was observed using the confocal microscopy. HG was shown to induce EMT at 48 h, which was blocked byNLRP3silencing or antioxidant N-acetyl-L-cysteine (NAC). We found thatNLRP3interference could inhibit HG-induced ROS. Knockdown ofNLRP3could prevent HG-induced EMT by inhibiting the phosphorylation of SMAD3, P38 MAPK and ERK1/2. In addition, P38 MAPK and ERK1/2 might be involved in HG-induced NLRP3 inflammasome activation. Besides, TGFB1 induced the activation of NLRP3 inflammasome and the generation of ROS, which were blocked byNLRP3interference or NAC. Tubular cells exposed to TGFB1 also underwent EMT, and this could be inhibited byNLRP3shRNA or NAC. These results indicated that knockdown ofNLRP3antagonized HG-induced EMT by inhibiting ROS production, phosphorylation of SMAD3, P38MAPK and ERK1/2, highlighting NLRP3 as a potential therapy target for diabetic nephropathy.

scholarly journals De-Glycyrrhizinated Licorice Extract Attenuates High Glucose-Stimulated Renal Tubular Epithelial–Mesenchymal Transition via Suppressing the Notch2 Signaling Pathway

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 125 ◽  
Author(s):  
Yung-Chien Hsu ◽  
Pey-Jium Chang ◽  
Chun-Wu Tung ◽  
Ya-Hsueh Shih ◽  
Wen-Chiu Ni ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
High Glucose ◽  
Cultured Cells ◽  
Cell Clone ◽  
Tubulointerstitial Fibrosis ◽  
Mesenchymal Transition ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Licorice Extract ◽  
Renal Tubular

Tubulointerstitial fibrosis is a major pathological hallmark of diabetic nephropathy. Increasing evidence has shown that epithelial-to-mesenchymal transition (EMT) of renal proximal tubular cells plays a crucial role in tubulointerstitial fibrosis. Herein, we aimed to elucidate the detailed mechanism of EMT in renal tubular cells under high glucose (HG) conditions, and to investigate the potential of licorice, a medicinal herb, to inhibit HG-induced EMT. Our results showed that renal tubular epithelial cells (normal rat kidney cell clone 52E; NRK-52E) exposed to HG resulted in EMT induction characterized by increased fibronectin and α-SMA (alpha-smooth muscle actin) but decreased E-cadherin. Elevated levels of cleaved Notch2, MAML-1 (mastermind-like transcriptional coactivator 1), nicastrin, Jagged-1 and Delta-like 1 were also concomitantly detected in HG-cultured cells. Importantly, pharmacological inhibition, small interfering RNA (siRNA)-mediated depletion or overexpression of the key components of Notch2 signaling in NRK-52E cells supported that the activated Notch2 pathway is essential for tubular EMT. Moreover, we found that licorice extract (LE) with or without glycyrrhizin, one of bioactive components in licorice, effectively blocked HG-triggered EMT in NRK-52E cells, mainly through suppressing the Notch2 pathway. Our findings therefore suggest that Notch2-mediated renal tubular EMT could be a therapeutic target in diabetic nephropathy, and both LE and de-glycyrrhizinated LE could have therapeutic potential to attenuate renal tubular EMT and fibrosis.

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.

Abstract 132: Hypoxia-inducible Factor Prolyl-hydroxylase-2 Mediates Transforming Growth Factor Beta 1-induced Epithelial-mesenchymal Transition In Renal Tubular Cells

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Weiqing Han ◽  
Jun-Pin Hu ◽  
Pin-Lan Li ◽  
Ningjun Li
Keyword(s):  
Signaling Pathway ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Epithelial Mesenchymal Transition ◽  
Kidney Diseases ◽  
Hypoxia Inducible Factor ◽  
Mesenchymal Transition ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Renal Tubular

Transforming growth factor beta 1 (TGFβ1)-induced epithelial-mesenchymal transition (EMT) in kidney epithelial cells plays a key role in renal tubulointerstitial fibrosis in chronic kidney diseases. As hypoxia-inducible factor (HIF)-1α is found to mediate TGFβ1 signaling pathway, we tested the hypothesis that HIF-1α and its upstream regulator prolyl hydroxylase domain-containing proteins (PHDs) are involved in TGFβ1-induced EMT in renal tubular cells. Our results showed that TGFβ1 treatment for 48 h stimulated EMT in cultured renal tubular cells as indicated by the decrease in epithelial marker P-cadherin from 1.0 ± 0.02 to 0.40 ± 0.05 ( P < 0.05), and the increase in mesenchymal markers α-smooth muscle actin (2.14 ± 0.32 fold, P < 0.05) and fibroblast-specific protein (2.0 ± 0.17 fold, P < 0.05) as shown in Western blot assay. Meanwhile, TGFβ1 time-dependently increased HIF-1α, which reached its maximum value (2.36 ± 0.2 fold, P < 0.05) at 24 h, and that HIF-1α siRNA significantly inhibited TGFβ1-induced EMT, suggesting that HIF-1α mediated TGFβ1 induced-EMT. Real-time PCR showed that PHD1 and PHD2, rather than PHD3, could be detected, with PHD2 as the predominant form of PHDs (PHD1 : PHD2 = 0.21:1.0). Importantly, TGFβ1 time-dependently decreased PHD2 mRNA and protein level, which reached their maximum value from 1.0 ± 0.15 to 0.45 ± 0.08 ( P < 0.05) for mRNA at 16 h and from 1.0 ± 0.08 to 0.26 ± 0.08 ( P < 0.05) for protein at 24 h, respectively. In contrast, TGFβ1 had no effect on PHD1 mRNA and protein levels. Furthermore, over-expression of PHD2 transgene almost fully prevented TGFβ1-induced HIF-1α accumulation and EMT marker changes, indicating that PHD2 is involved in TGFβ1-induced EMT. Finally, Smad2 inhibitor SB431542 prevented TGFβ1-induced PHD2 decrease, suggesting that Smad2 may mediate TGFβ1-induced EMT through PHD2/HIF-1α. It is concluded that TGFβ1 decreased PHD2 expression via a Smad2-dependent signaling pathway, thereby leading to HIF-1α accumulation and EMT in renal tubular cells. The present study suggests that PHD2/HIF-1α is a novel signaling pathway mediating the fibrogenic effect of TGFβ1 and that manipulating PHD2/HIF-1α pathway may be used as a therapeutic strategy in chronic kidney diseases. (support: NIH grant HL89563 and HL106042)

ERK and p38 mediate high-glucose-induced hypertrophy and TGF-β expression in renal tubular cells

2004 ◽  
Vol 286 (1) ◽  
pp. F120-F126 ◽  
Author(s):  
Hisayo Fujita ◽  
Sayu Omori ◽  
Kenji Ishikura ◽  
Mariko Hida ◽  
Midori Awazu
Keyword(s):  
High Glucose ◽  
Transforming Growth Factor ◽  
Mek Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Leucine Incorporation ◽  
Renal Tubules ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Renal Tubular ◽  
Sb 203580

We investigated the expression of ERK, p38 mitogen-activated protein kinase (p38), and JNK in renal tubules of diabetic rats following 3 wk after streptozotocin injection (DM). Although the expression of ERK was not different between controls and DM, phosphorylated ERK was expressed more intensely in DM. p38 And phosphorylated p38 were detected only in the diabetic kidney and were localized in all tubular segments. JNK and phosphorylated JNK were expressed similarly in controls and DM. Transforming growth factor (TGF)-β was expressed in all tubular segments of DM, coinciding with the localization of p38. In LLC-PK1 cells, phosphorylation of ERK and p38 increased after 24- to 72-h exposure to high glucose (HG). Coincubation with a p38 inhibitor SB-203580 or a MEK inhibitor, PD-98059, suppressed the HG-induced increases in protein content, [3H]leucine incorporation, and the protein-to-DNA ratio. SB-203580 or PD-98059 also abolished the HG-stimulated expression of TGF-β protein. These results demonstrate that ERK and p38 are activated in renal tubular cells of DM and may mediate HG-induced cellular hypertrophy and TGF-β expression.

PPARγ agonists exert antifibrotic effects in renal tubular cells exposed to high glucose

2005 ◽  
Vol 289 (5) ◽  
pp. F1153-F1158 ◽  
Author(s):  
U. Panchapakesan ◽  
S. Sumual ◽  
C. A. Pollock ◽  
X. Chen
Keyword(s):  
Extracellular Matrix ◽  
High Glucose ◽  
Matrix Protein ◽  
Collagen Iv ◽  
Extracellular Matrix Protein ◽  
Total Production ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Nuclear Binding ◽  
Pparγ Agonists

Peroxisome proliferator-activated receptor-γ (PPARγ) are ligand-activated transcription factors that regulate cell growth, inflammation, lipid metabolism, and insulin sensitivity. We recently demonstrated that PPARγ agonists limit high glucose-induced inflammation in a model of proximal tubular cells (PTC; Panchapakesan U, Pollock CA, and Chen XM. Am J Physiol Renal Physiol 287: F528–F534, 2004). However, the role of PPARγ in the excess extracellular matrix production is largely unknown. We evaluated the effect of 24- to 48-h 8 μM l-805645 or 10 μM pioglitazone on 25 mM d-glucose-induced markers of fibrosis in HK-2 cells. High d-glucose induced nuclear binding of activator protein-1 (AP-1) to 140.8 ± 10.9% ( P < 0.05), which was attenuated with L-805645 and pioglitazone to 82.3 ± 14.4 ( P < 0.01 vs. high d-glucose) and 99.3 ± 12.2% ( P < 0.05 vs. high d-glucose), respectively. High d-glucose increased total production of transforming growth factor (TGF)-β1 139.6 ± 6.5% ( P < 0.05), which was reversed with L-805645 and pioglitazone to 68.73 ± 5.7 ( P < 0.01 vs. high d-glucose) and 112 ± 13.6% ( P < 0.05 vs. high d-glucose). L-805645 and pioglitazone reduced high d-glucose-induced fibronectin from 156.0 ± 24.9 ( P < 0.05) to 81.9 ± 16.0 and 57.4 ± 12.7%, respectively (both P < 0.01 vs. high d-glucose). Collagen IV was not induced by high d-glucose. L-805645 and pioglitazone suppressed collagen IV to 68.0 ± 14.5 ( P < 0.05) and 46.5 ± 11.6% ( P < 0.01) vs. high d-glucose, respectively. High d-glucose increased the nuclear binding of NF-κB to 167 ± 22.4% ( P < 0.05), which was not modified with PPARγ agonists. In conclusion, PPARγ agonists exert antifibrotic effects in human PTC in high glucose by attenuating the increase in AP-1, TGF-β1, and the downstream production of the extracellular matrix protein fibronectin.

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