title: TGR5 suppresses high glucose-induced upregulation of fibronectin and transforming growth factor-β1 in rat glomerular mesangial cells by inhibiting RhoA/ROCK signaling.
abstract: 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.
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.|National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Guangzhou, 510006, China.|Guangzhou Key Laboratory of Druggability Assessment for Biologically Active Compounds, Guangzhou, 510006, China.
authors.forename: Fengxiao
authors.initials: F
authors.lastname: Xiong
authors.affiliation: Dept of Pharmacy, Shenzhen Children's Hospital, Shenzhen, Guangdong, 518026, China.
authors.forename: Xuejuan
authors.initials: X
authors.lastname: Li
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.
authors.forename: Wenyan
authors.initials: W
authors.lastname: Gong
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.
authors.forename: Junying
authors.initials: J
authors.lastname: Huang
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Chen
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.|National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Guangzhou, 510006, China.|Guangzhou Key Laboratory of Druggability Assessment for Biologically Active Compounds, Guangzhou, 510006, China.
authors.forename: Peiqing
authors.initials: P
authors.lastname: Liu
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. huangheq@mail.sysu.edu.cn.|National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Guangzhou, 510006, China. huangheq@mail.sysu.edu.cn.|Guangzhou Key Laboratory of Druggability Assessment for Biologically Active Compounds, Guangzhou, 510006, China. huangheq@mail.sysu.edu.cn.
authors.forename: Heqing
authors.initials: H
authors.lastname: Huang
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D003928:Diabetic Nephropathies / Q000188:drug therapy; D005260:Female; D005353:Fibronectins / Q000378:metabolism*; D005947:Glucose; D008297:Male; D050527:Mesangial Cells / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D058990:Molecular Targeted Therapy; D016328:NF-kappa B / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D016207:Cytokines; D005353:Fibronectins; C518268:Gpbar1 protein, rat; D016328:NF-kappa B; D043562:Receptors, G-Protein-Coupled; D053773:Transforming Growth Factor beta1; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose
keywords: Diabetic nephropathy; Glomerular mesangial cells; RhoA/ROCK; TGR5