RESEARCH ARTICLE
GSTA3 Attenuates Renal Interstitial Fibrosis by Inhibiting TGF-Beta-Induced Tubular Epithelial-Mesenchymal Transition and Fibronectin Expression Yun Xiao1,2, Jishi Liu1, Yu Peng1, Xuan Xiong1, Ling Huang1, Huixiang Yang4, Jian Zhang5, Lijian Tao1,3*
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1 Division of Nephrology, Xiangya Hospital, Central South University, Changsha, China, 410078, 2 Division of Nephrology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China, 510120, 3 State Key Laboratory of Medical Genetics of China, Central South University, Changsha, China, 410078, 4 Division of Digestive Medicine, Xiangya Hospital, Central South University, Changsha, China, 410078, 5 Department of Microbial Infection and Immunity, The Ohio State University, Columbus, Ohio, United States of America *
[email protected]
OPEN ACCESS Citation: Xiao Y, Liu J, Peng Y, Xiong X, Huang L, Yang H, et al. (2016) GSTA3 Attenuates Renal Interstitial Fibrosis by Inhibiting TGF-Beta-Induced Tubular Epithelial-Mesenchymal Transition and Fibronectin Expression. PLoS ONE 11(9): e0160855. doi:10.1371/journal.pone.0160855 Editor: Giovanni Camussi, Universita degli Studi di Torino, ITALY Received: April 6, 2016 Accepted: July 26, 2016 Published: September 7, 2016 Copyright: © 2016 Xiao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the National Natural Science Foundation of China (Grants NO. 81370547), and the CHINA-CANADA Joint Health Research Initiative Foundation (Grants NO. 81061120524/H07).
Abstract Tubular epithelial-mesenchymal transition (EMT) has been widely accepted as the underlying mechanisms of renal interstitial fibrosis (RIF). The production of reactive oxygen species (ROS) plays a vital role in tubular EMT process. The purpose of this study was to investigate the involved molecular mechanisms in TGF-beta-induced EMT and identify the potential role of glutathione S-transferase alpha 3 (GSTA3) in this process. The iTRAQ screening was performed to identify protein alterations of the rats underwent unilateral-ureteral obstruction (UUO). Protein expression of GSTA3 in patients with obstructive nephropathy and UUO rats was detected by immunohistochemistry. Protein and mRNA expression of GSTA3 in UUO rats and NRK-52E cells were determined by Western blot and RT-PCR. siRNA and overexpression plasmid were transfected specifically to assess the role of GSTA3 in RIF. The generation of ROS was measured by dichlorofluorescein fluorescence analysis. GSTA3 protein and mRNA expression was significantly reduced in UUO rats. Immunohistochemical analysis revealed that GSTA3 expression was reduced in renal cortex in UUO rats and patients with obstructive nephropathy. Treating with TGF-β1 down-regulated GSTA3 expression in NRK-52E cells, which have been found to be correlated with the decreased expression in E-cadherin and megalin and increased expression in α-smooth muscle actin. Furthermore, knocking down GSTA3 in NRK-52 cells led to increased production of ROS and tubular EMT, whereas overexpressing GSTA3 ameliorated ROS production and prevented the occurrence of tubular EMT. GSTA3 plays a protective role against tubular EMT in renal fibrosis, suggesting GSTA3 is a potential therapeutic target for RIF.
Competing Interests: The authors have declared that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0160855 September 7, 2016
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Potential Role of Glutathione S-Transferase Alpha 3 (GSTA3) in TGF-Beta-Induced EMT
Introduction Progressive renal interstitial fibrosis (RIF) is the final common pathologic change for a number of independent and overlapping cellular and molecular pathways in tubulointerstitial injury of chronic kidney diseases (CKD) [1]. The extent of tubulointerstitial injury correlates closely with long-term renal function and is an important predictor of renal impairments. However, how to prevent CKD remains elusive. Understanding the mechanisms of RIF is essential in establishing novel interventional strategies to halt or even reverse renal fibrosis. Renal tubular epithelial to mesenchymal transition (EMT) has been recognized as the fact of loss of epithelial cell phenotype and a concomitant development of mesenchymal phenotype [2]. Therefore, tubular EMT is believed to be the core part of RIF [3] Phenotypically, tubular EMT are associated with down-regulation of expression of intercellular epithelial adhesion molecules such as E-cadherin and up-regulation of the robust markers of mesenchymal cells such as α-smooth muscle actin (α-SMA) and vimentin [4,5]. It is believed that tubular EMT is responsible for the dissociation of renal tubular epithelial cells, tubular atrophy, accumulation in the interstitium of fibroblasts with the phenotypic appearance of myofibroblasts, and secretion of large amounts of extra cellular matrix (ECM) [6]. A number of factors have been shown to affect the occurrence of renal tubular EMT including oxidative stress, inflammation, fibroblasts proliferation and activation, and apoptosis [7,8]. Among these factors, oxidative stress in the pathogenesis of RIF has grown in scope and understanding in recent years [9]. Mice deficient for endogenous antioxidant enzyme CAT are more susceptible to unilateral ureteral obstruction (UUO)-induced renal damage than normal wild type (WT) mice. Furthermore, increased renal concentrations of ROS have been observed in obstructed kidneys, together with decreased activities of the major protective antioxidant enzymes SOD, CAT, and glutathione peroxidase [10]. It has been shown that progression of RIF correlates with increased activity of ROS, augmented expression of collagen deposition and α-SMA, and loss of E-cadherin and megalin in renal tissue in animals undergoing UUO, suggesting that oxidative stress plays an important role in promoting the tubular EMT [11]. However, the mechanisms underlying this process are still unclear. The intracellular signaling pathways leading to initiation of EMT remain largely unknown. In the present study, we have used isobaric tags for relative and absolute quantitation (ITRAQ), a new tool for quantitative mass spectrometry, to analyze the differential expression of proteins in the kidneys of sham and UUO rats. Glutathione S-transferase alpha-3 (GSTA3), a member of an important family of detoxifying and cytoprotective enzymes [12] was identified to be the most significantly down-regulated protein in the renal cortex of UUO rats. GSTA3 plays a critical role in various diseases associated with oxidation-regulating proteins [13]. However, whether GSTA3 plays a role in the development of RIF is currently unknown. In this study, we would examine the molecular and cellular basis of RIF and report the role of GSTA3 in the progression of tubulointerstitial fibrosis.
Materials and Methods This study was approved by the ethical committee of Central South University.
Antibodies and reagents NRK-52E cells were purchased from American Type Culture Collection (Rockville, Md., USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and Carboxy-DCFDA were obtained from Invitrogen (Carlsbad, Calif., USA). SB431542 and antibodies against α-SMA and β-tubulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibody against E-cadherin was purchased from BD Biosciences
PLOS ONE | DOI:10.1371/journal.pone.0160855 September 7, 2016
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Potential Role of Glutathione S-Transferase Alpha 3 (GSTA3) in TGF-Beta-Induced EMT
(San Jose, CA, USA). Antibodies against GSTA3 and Fibronectin (FN) were obtained from Abcam (Cambridge, UK). Horseradish-peroxideconjugated secondary antibodies for Western blot were from the Jackson ImmunoResearch Laboratories (West Grove, PA, USA), and secondary antibodies for immunohistochemistry were from GBI (Mukilteo, WA, USA). The enhanced chemiluminescence kit for Western blotting was from GE Healthcare (Buckinghamshire, UK). Recombinant human TGF-β1 was purchased from Peprotech (Rocky Hill, NJ, USA). iTRAQ™ Reagents were purchased from Applied Biosystems (USA). DCFH-DA was purchased from Invitrogen (Carlsbad, CA, USA)
Animal model Male Sprague-Dawley rats (180-220g, female, 6–8 weeks old, n = 10) were obtained from Slac Laboratory Animal (Shanghai, China). They were given free access to commercial standard rat chow (Hunan Shi Lai Ke Jing Da Experimental Animal Co. Ltd., China) and tap water and housed in stable conditions at 22°C with a 12 h light/dark cycle during the entire study unless mentioned somewhere else. Sprague-Dawley rats were divided into 2 groups (5/group): Sham operation (SHM) and UUO. Under anesthesia with 10% chloral hydrate (0.4 ml/100 g), UUO was induced by ligation of the left ureter according to the procedure described previously [14]. Heart rate was monitored to ensure adequate levels of anesthesia. Sham operations were performed without ligation. Immediately post-surgery, animals were administrated buprenorphine (0.03 mg/kg, s.c.) for post-operative pain. Animals were monitored twice/day, and weighed daily. All rats were sacrificed at day 14. Left ventricular blood was collected and tested for serum creatinine and uric acid. Half of the left kidneys were fixed in 10% neutral-buffered formalin for H&E and Masson’s trichrome staining or immunohistochemistry, and the remaining half was frozen in liquid nitrogen for Western blot or RT-PCR. All animal studies were approved by the Institutional Animal Care and Use Committee of Xiangya School of Medicine, Central South University.
Quantitative proteomics using iTRAQ labeling and mass spectrometry For iTRAQ screening, renal cortex isolated from SHM rats and UUO rats were lysed several times by freeze-thawing, followed by sonication. The extracted proteins were reduced, alkylated as described in the iTRAQ protocol (Applied Biosystems, Foster City, CA, USA). Isobaric tagging iTRAQ reagent (1 unit in ethanol) was added directly to the protein digest (70% ethanol final) and the mixture was incubated. Proteins were subjected to the conventional procedure with off-line 2D LC-MS/MS. Peptides were eluted with a linear gradient. Then the LC MS/MS analysis was conducted with samples injected into the trap column. To identify the peptides, database search were performed with ProteinPilot 2.0.1 software (Applied Biosystems) using Paragon and ProGroup algorithms against the Swiss-Prot rat database [15]. We selected differentially expressed proteins using peptide confidence >95% criteria, and fold-changes>1.2 or
