Browsing by Author "Rattiyaporn Kanlaya"
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- PublicationAlterations in actin cytoskeletal assembly and junctional protein complexes in human endothelial cells induced by dengue virus infection and mimicry of leukocyte transendothelial migration(2009-05-01) Rattiyaporn Kanlaya; Sa Nga Pattanakitsakul; Supachok Sinchaikul; Shui Tein Chen; Visith Thongboonkerd; Mahidol University; Genomics Research Center, Academia Sinica; National Taiwan University; Faculty of Medicine, Siriraj Hospital, Mahidol UniversityVascular leakage is a hallmark of severe dengue infection. Although extensive studies have been conducted during the past several decades, the molecular mechanisms underlying vascular leakage in dengue shock syndrome (DSS) remain unclear. We thus performed a proteomics study to characterize responses in human endothelial cells (EA.hy926) after DEN-2 virus infection (MOI = 10). Comparative 2-D PAGE analysis revealed significantly altered abundance levels of 15 proteins, which were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/ MS). These altered proteins were involved in several biological processes, for example, mRNA stability/ processing, transcription and translation regulation, molecular chaperoning, oxidative stress response/ regulation, cytoskeletal assembly, protein degradation, and cellular metabolisms. We also performed functional analyses of alterations in actin cytoskeletal assembly and endothelial integrity focusing on adherens junction (VE-cadherin), tight junction (ZO-1) and adhesive molecule (PECAM-1) after 24-h of DEN-2 infection and simulation of transendothelial migration by PECAM-1 cross-linking. Decreased expression and disorganization of the actin-cytoskeleton were observed in the infected cells, whereas the increase in actin stress fibers was found in adjacent noninfected cells. Additionally, a decrease in adhesive protein PECAM-1 was observed. Furthermore, DEN-2 infection caused decreased expression and redistribution of both VE-cadherin and ZO-1, whose changes were enhanced by PECAM-1 engagement. These alterations may potentially be a molecular basis explaining increased endothelial permeability or vascular leakage in DSS. © 2009 American Chemical Society.
- PublicationAltered proteome in Burkholderia pseudomallei rpoE operon knockout mutant: Insights into mechanisms of rpoE operon in stress tolerance, survival, and virulence(2007-04-01) Visith Thongboonkerd; Muthita Vanaporn; Napat Songtawee; Rattiyaporn Kanlaya; Supachok Sinchaikul; Shui Tein Chen; Anna Easton; Karen Chu; Gregory J. Bancroft; Sunee Korbsrisate; Mahidol University; Academia Sinica Taiwan; National Taiwan University; London School of Hygiene & Tropical Medicine; Faculty of Medicine, Siriraj Hospital, Mahidol UniversityWe have previously shown that the alternative sigma factor σE (RpoE), encoded by rpoE, is involved in stress tolerance and survival of Burkholderia pseudomallei. However, its molecular and pathogenic mechanisms remain unclear. In the present study, we applied gel-based, differential proteomics to compare the cellular proteome of an rpoE operon knockout mutant (RpoE Mut) to that of wild-type (K96243 WT) B. pseudomallei. Quantitative intensity analysis (n = 5 gels from 5 individual culture flasks in each group) revealed significantly differential expression of 52 proteins, which were subsequently identified by Q-TOF MS/MS. These included oxidative, osmotic, and other stress response proteins; chaperones; transcriptional/translational regulators; metabolic enzymes; proteins involved in cell wall synthesis, fatty synthesis, glycogen synthesis, and storage; exported proteins; secreted proteins; adhesion molecule; protease/peptidase; protease inhibitor; signaling proteins; and other miscellaneous proteins. The down-regulation of several stress response proteins, chaperones, transcriptional/translational regulators, and proteins involved in cell wall synthesis in RpoE Mut provided some new insights into the mechanisms of the rpoE operon for the stress tolerance and survival of B. pseudomallei. In addition, the proteomic data and in vivo study indicated that the rpoE operon is also involved in the virulence of B. pseudomallei. Our findings underscore the usefulness of proteomics for unraveling pathogenic mechanisms of diseases at the molecular level. © 2007 American Chemical Society.
- PublicationApplication of tandem fast protein liquid chromatography to purify intact native monomeric/aggregated Tamm-Horsfall protein from human urine and systematic comparisons with diatomaceous earth adsorption and salt precipitation: Yield, purity and time-consumption(2021-08-14) Chadanat Noonin; Chompunoot Kapincharanon; Kanyarat Sueksakit; Rattiyaporn Kanlaya; Visith Thongboonkerd; Siriraj HospitalTamm-Horsfall protein (THP) is a high-abundance urinary protein. Although its functions have been studied for years, several aspects of these remain unclear. To achieve more knowledge on THP functions, an effective isolation/purification method providing a high yield and high purity is required. This is the first report that applied tandem fast protein liquid chromatography (FPLC) (by combining Mono Q anion-exchange with Superdex 200 size-exclusion columns in a tandem manner) to isolate intact THP from human urine. Its efficiency was then systematically compared with that of two conventional methods, diatomaceous earth (DE) adsorption and salt precipitation. The first ever systematic comparisons among the three methods revealed that, while Mono Q-Superdex 200 tandem FPLC offered the lowest %yield and was most time-consuming, it provided substantially high %purity and could selectively purify the monomeric and aggregated forms of urinary THP. On the other hand, DE adsorption provided the highest %yield and %purity, whereas salt precipitation offered the lowest %purity. In summary, the tandem FPLC system is most useful for selective purification of the monomeric and aggregated forms of urinary THP for further functional study, whereas DE adsorption remains the method of choice for general purification of THP from human urine.
- PublicationAssociation of Alix with late endosomal lysobisphosphatidic acid is important for dengue virus infection in human endothelial cells(2010-09-03) Sa Nga Pattanakitsakul; Jesdaporn Poungsawai; Rattiyaporn Kanlaya; Supachok Sinchaikul; Shui Tein Chen; Visith Thongboonkerd; Mahidol University; Academia Sinica Taiwan; National Taiwan UniversityThe most severe form of dengue virus (DENV) infection is dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is accompanied by increased vascular permeability indicating that endothelial cells are the targets of DENV infection. However, molecular mechanisms underlying DENV replication in endothelial cells remained poorly understood. We therefore examined changes in subcellular proteomes of different cellular compartments (including cytosolic, membrane/organelle, nucleus, and cytoskeleton) of human endothelial (EA.hy926) cells upon DENV2 infection using a 2-DE-based proteomics approach followed by Q-TOF MS and MS/MS. A total of 35 altered proteins were identified in these subcellular locales, including an increase in the level of Alix (apoptosis-linked gene-2-interacting protein X) in the cytosolic fraction of DENV2-infected cells compared to mock control cells. Double immunofluorescence staining revealed colocalization of Alix with late endosomal lysobisphosphatidic acid (LBPA). This complex has been proposed to be involved in the export of DENV proteins from late endosomes to the cytoplasm. Subsequent functional study revealed that pretreatment with an anti-LBPA antibody prior to DENV challenge significantly reduced the level of viral envelope protein synthesis and DENV replication. Our data indicate that Alix plays a pivotal role in the early phase of DENV replication, particularly when it arrives at the late endosome stage. Blocking this step may lead to a novel therapeutic approach to reducing the level of DENV replication in vivo. © 2010 American Chemical Society.
- PublicationC-terminal hemocyanin from hemocytes of penaeus vannamei interacts with ERK1/2 and undergoes serine phosphorylation(2009-05-01) Phattaraorn Havanapan; Rattiyaporn Kanlaya; Apichai Bourchookarn; Chartchai Krittanai; Visith Thongboonkerd; Mahidol University; Faculty of Medicine, Siriraj Hospital, Mahidol UniversityTo understand molecular immune response of Penaeus vannamei during Taura syndrome virus (TSV) infection, expression and functional proteomics studies were performed on hemocyanin, which is a major abundant protein in shrimp hemocytes. Two-dimensional electrophoresis (2-DE) revealed up- regulation of several C-terminal fragments of hemocyanin, whereas the N-terminal fragments were down-regulated during TSV infection. 2-D Western blot analysis showed that the C-terminal hemocyanin fragments had more acidic isoelectric points (p/), whereas the N-terminal fragments had less acidic p/. Further analysis by NetPhos showed a greater number of serine phosphorylation sites in the C-terminal hemocyanin. Additionally, motif scan using Scansite revealed ERK D-domain, which is required for activation of ERK1/2 effector kinase, as a kinase-binding site at the 527th valine in the C-terminal hemocyanin, whereas neither motif nor functional domain was found in the N-terminus. Co- immunoprecipitation confirmed the interaction between the C-terminal hemocyanin and ERK1/2. 1-D Western blot analysis showed that ERK1/2 was also up-regulated during TSV infection. Our findings demonstrate for the first time that ERK1/2 signaling pathway may play an important role in molecular immune response of P. vannamei upon TSV infection through its interaction with the C-terminal hemocyanin. © 2009 American Chemical Society.
- PublicationCaffeine inhibits hypoxia-induced renal fibroblast activation by antioxidant mechanism(2019-01-01) Angkhana Nilnumkhum; Rattiyaporn Kanlaya; Sunisa Yoodee; Visith Thongboonkerd; Faculty of Medicine, Siriraj Hospital, Mahidol University© 2019, © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Caffeine has been demonstrated to possess anti-fibrotic activity against liver fibrosis. However, its role in renal fibrosis remained unclear. This study investigated the effects of caffeine on renal fibroblast activation induced by hypoxia (one of the inducers for renal fibrosis). BHK-21 fibroblasts were cultured under normoxia or hypoxia with or without caffeine treatment. Hypoxia increased levels of fibronectin, α-smooth muscle actin, actin stress fibers, intracellular reactive oxygen species (ROS), and oxidized proteins. However, caffeine successfully preserved all these activated fibroblast markers to their basal levels. Cellular catalase activity was dropped under hypoxic condition but could be reactivated by caffeine. Hif1a gene and stress-responsive Nrf2 signaling molecule were elevated/activated by hypoxia, but only Nrf2 could be partially recovered by caffeine. These data suggest that caffeine exhibits anti-fibrotic effect against hypoxia-induced renal fibroblast activation through its antioxidant property to eliminate intracellular ROS, at least in part, via downstream catalase and Nrf2 mechanisms.
- PublicationCaffeine prevents oxalate-induced epithelial-mesenchymal transition of renal tubular cells by its anti-oxidative property through activation of Nrf2 signaling and suppression of Snail1 transcription factor(2021-09-01) Rattiyaporn Kanlaya; Chonnicha Subkod; Supanan Nanthawuttiphan; Visith Thongboonkerd; Siriraj HospitalCaffeine is an active ingredient found in coffee and energy beverages. Its hepatoprotective effects against liver fibrosis are well-documented. Nonetheless, its renoprotective effects against renal fibrogenesis and epithelial-mesenchymal transition (EMT) processes remain unclear and under-investigated. In this study, the protective effects of caffeine against oxalate-induced EMT in renal tubular cells were evaluated by various assays to measure expression levels of epithelial and mesenchymal markers, cell migrating activity, level of oxidized proteins, and expression of Nrf2 and Snail1. Oxalate at sublethal dose significantly suppressed cell proliferation but increased cell elongation, spindle index and migration. Oxalate also decreased expression of epithelial markers (zonula occludens-1 (ZO-1) and E-cadherin) but increased expression of mesenchymal markers (fibronectin, vimentin and α-smooth muscle actin (α-SMA)). All of these EMT-inducing effects of oxalate could be prevented by pretreatment with caffeine. While oxalate increased oxidized proteins and Snail1 levels, it decreased Nrf2 expression. Caffeine could preserve all these molecules to their basal (control) levels. Finally, silencing of Nrf2 expression by small interfering RNA (siRNA) could abolish such protective effects of caffeine on oxalate-induced EMT. Our data indicate that the renoprotective effects of caffeine against oxalate-induced EMT is mediated, at least in part, by its anti-oxidative property through activation of Nrf2 signaling and suppression of Snail1 transcription factor.
- PublicationCellufine sulfate column chromatography as a simple, rapid, and effective method to purify dengue virus(2016-08-01) Rattiyaporn Kanlaya; Visith Thongboonkerd; Mahidol University© 2016 Elsevier B.V. Conventional method to purify/concentrate dengue virus (DENV) is time-consuming with low virus recovery yield. Herein, we applied cellufine sulfate column chromatography to purify/concentrate DENV based on the mimicry between heparan sulfate and DENV envelope protein. Comparative analysis demonstrated that this new method offered higher purity (as determined by less contamination of bovine serum albumin) and recovery yield (as determined by greater infectivity). Moreover, overall duration used for cellufine sulfate column chromatography to purify/concentrate DENV was approximately 1/20 of that of conventional method. Therefore, cellufine sulfate column chromatography serves as a simple, rapid, and effective alternative method for DENV purification/concentration.
- PublicationCellular adaptive response of distal renal tubular cells to high-oxalate environment highlights surface alpha-enolase as the enhancer of calcium oxalate monohydrate crystal adhesion(2013-03-07) Rattiyaporn Kanlaya; Kedsarin Fong-ngern; Visith Thongboonkerd; Mahidol UniversityHyperoxaluria is one of etiologic factors of calcium oxalate kidney stone disease. However, response of renal tubular cells to high-oxalate environment remained largely unknown. We applied a gel-based proteomics approach to characterize changes in cellular proteome of MDCK cells induced by 10. mM sodium oxalate. A total of 14 proteins were detected as differentially expressed proteins. The oxalate-induced up-regulation of alpha-enolase in whole cell lysate was confirmed by 2-D Western blot analysis. Interaction network analysis revealed that cellular adaptive response under high-oxalate condition involved stress response, energy production, metabolism and transcriptional regulation. Down-regulation of RhoA, which was predicted to be associated with the identified proteins, was confirmed by immunoblotting. In addition, the up-regulation of alpha-enolase on apical surface of renal tubular epithelial cells was also confirmed by immunoblotting of the isolated apical membranes and immunofluorescence study. Interestingly, blockage of alpha-enolase expressed on the cell surface by antibody neutralization significantly reduced the number of calcium oxalate monohydrate (COM) crystals adhered on the cells. These results strongly suggest that surface alpha-enolase plays an important role as the enhancer of COM crystal binding. The increase of alpha-enolase expressed on the cell surface may aggravate kidney stone formation in patients with hyperoxaluria. © 2013 Elsevier B.V.
- PublicationChromosome-centric human proteome project (C-HPP): Chromosome 12(2014-07-03) Sakdithep Chaiyarit; Nilubon Singhto; Yi Ju Chen; Chia Ying Cheng; Wararat Chiangjong; Rattiyaporn Kanlaya; Henry H.N. Lam; Paleerath Peerapen; Ting Yi Sung; Phornpimon Tipthara; Akhilesh Pandey; Terence C.W. Poon; Yu Ju Chen; Ravi Sirdeshmukh; Maxey C.M. Chung; Visith Thongboonkerd; Mahidol University; Institute of Chemistry Academia Sinica Taiwan; Institute of Information Science Academia Sinica Taiwan; Hong Kong University of Science and Technology; Institute of Bioinformatics; The Johns Hopkins School of Medicine; Prince of Wales Hospital Hong Kong; National University of Singapore; Yong Loo Lin School of MedicineFollowing an official announcement of the Chromosome-centric Human Proteome Project (C-HPP), the Chromosome 12 (Ch12) Consortium has been established by five representative teams from five Asian countries including Thailand (Siriraj Hospital, Mahidol University), Singapore (National University of Singapore), Taiwan (Academia Sinica), Hong Kong (The Chinese University of Hong Kong), and India (Institute of Bioinformatics). We have worked closely together to extensively and systematically analyze all missing and known proteins encoded by Ch12 for their tissue/cellular/subcellular localizations. The target organs/tissues/cells include kidney, brain, gastrointestinal tissues, blood/immune cells, and stem cells. In the later phase, post-translational modifications and functional significance of Ch12-encoded proteins as well as their associations with human diseases (i.e., immune diseases, metabolic disorders, and cancers) will be defined. We have collaborated with other chromosome teams, Human Kidney and Urine Proteome Project (HKUPP), AOHUPO Membrane Proteomics Initiative, and other existing HUPO initiatives in the Biology/Disease-Based Human Proteome Project (B/D-HPP) to delineate functional roles and medical implications of Ch12-encoded proteins. The data set to be obtained from this multicountry consortium will be an important piece of the jigsaw puzzle to fulfill the missions and goals of the C-HPP and the global Human Proteome Project (HPP). © 2014 American Chemical Society.
- PublicationThe cultural divide: Exponential growth in classical 2D and metabolic equilibrium in 3D environments(2014-09-15) Krzysztof Wrzesinski; Adelina Rogowska-Wrzesinska; Rattiyaporn Kanlaya; Kamil Borkowski; Veit Schwämmle; Jie Dai; Kira Eyd Joensen; Katarzyna Wojdyla; Vasco Botelho Carvalho; Stephen J. Fey; Syddansk Universitet; Kobenhavns Universitet; Mahidol University© 2014 PLOS ONE. Introduction: Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different.Results: Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell - along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex.Summary: We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance.
- PublicationDifferential proteomics of lesional vs. non-lesional biopsies revealed non-immune mechanisms of alopecia areata(2018-12-01) Kanchalit Thanomkitti; Rattiyaporn Kanlaya; Kedsarin Fong-Ngern; Chompunoot Kapincharanon; Kanyarat Sueksakit; Prangwalai Chanchaem; Rattapon Thuangtong; Visith Thongboonkerd; Mahidol University; Faculty of Medicine, Siriraj Hospital, Mahidol University© 2018 The Author(s). Alopecia areata (AA) is one of the common hair disorders for which treatment is frequently ineffective and associated with relapsing episodes. Better understanding of disease mechanisms and novel therapeutic targets are thus required. From 10 AA patients, quantitative proteomics using LTQ-Orbitrap-XL mass spectrometer revealed 104 down-regulated, 4 absent, 3 up-regulated and 11 newly present proteins in lesional vs. non-lesional biopsies. Among these, the decreased levels of α-tubulin, vimentin, heat shock protein 70 (HSP70), HSP90, annexin A2 and α-enolase were successfully confirmed by Western blotting. Protein-protein interactions network analysis using STRING tool revealed that the most frequent biological processes/networks of the down-regulated proteins included tissue development, cell differentiation, response to wounding and catabolic process, whereas those for the up-regulated proteins included biological process, metabolic process, cellular transport, cellular component organization and response to stimulus. Interestingly, only 5 increased/newly present proteins were associated with the regulation of immune system, which may not be the predominant pathway in AA pathogenic mechanisms as previously assumed. In summary, we report herein the first proteome dataset of AA demonstrating a number of novel pathways, which can be linked to the disease mechanisms and may lead to discovery of new therapeutic targets for AA.
- PublicationEffects of secretome derived from macrophages exposed to calcium oxalate crystals on renal fibroblast activation(2021-12-01) Sunisa Yoodee; Chadanat Noonin; Kanyarat Sueksakit; Rattiyaporn Kanlaya; Sakdithep Chaiyarit; Paleerath Peerapen; Visith Thongboonkerd; Siriraj HospitalThe association between kidney stone disease and renal fibrosis has been widely explored in recent years but its underlying mechanisms remain far from complete understanding. Using label-free quantitative proteomics (nanoLC-ESI-LTQ-Orbitrap MS/MS), this study identified 23 significantly altered secreted proteins from calcium oxalate monohydrate (COM)-exposed macrophages (COM-MP) compared with control macrophages (Ctrl-MP) secretome. Functional annotation and protein-protein interactions network analysis revealed that these altered secreted proteins were involved mainly in inflammatory response and fibroblast activation. BHK-21 renal fibroblasts treated with COM-MP secretome had more spindle-shaped morphology with greater spindle index. Immunofluorescence study and gelatin zymography revealed increased levels of fibroblast activation markers (α-smooth muscle actin and F-actin) and fibrotic factors (fibronectin and matrix metalloproteinase-9 and -2) in the COM-MP secretome-treated fibroblasts. Our findings indicate that proteins secreted from macrophages exposed to COM crystals induce renal fibroblast activation and may play important roles in renal fibrogenesis in kidney stone disease.
- PublicationEGCG decreases binding of calcium oxalate monohydrate crystals onto renal tubular cells via decreased surface expression of alpha-enolase(2016-06-01) Rattiyaporn Kanlaya; Nilubon Singhto; Visith Thongboonkerd; Mahidol University© 2016, SBIC. Crystal retention on tubular cell surface inside renal tubules is considered as the earliest and crucial step for kidney stone formation. Therapeutics targeting this step would cease the development of kidney stone. This study thus aimed to investigate the potential role of epigallocatechin-3-gallate (EGCG), a major antioxidant found in green tea leaves, in the reduction of calcium oxalate monohydrate (COM) crystal binding onto renal tubular cells. Pretreatment of the cells with EGCG for up to 6 h significantly diminished crystal-binding capability in a dose-dependent manner. Indirect immunofluorescence assay without and with cell permeabilization followed by laser-scanning confocal microscopy revealed that EGCG significantly reduced surface expression of alpha-enolase, whereas its intracellular level was increased. Western blot analysis confirmed such contradictory changes in membrane and cytosolic fractions of EGCG-treated cells, whereas the total level in whole cell lysate remained unchanged. Moreover, overexpression of surface alpha-enolase and enhancement of cell–crystal adhesion induced by 10 mM sodium oxalate were completely abolished by EGCG. Taken together, these data indicate that EGCG decreases binding of COM crystals onto renal tubular cells by decreasing the surface expression of alpha-enolase via re-localization or inhibition of alpha-enolase shuttling from the cytoplasm to the plasma membrane. These findings may also explain the effects of EGCG in reducing COM crystal deposition in previous animal models of kidney stone disease. Thus, EGCG may be useful for the prevention of new or recurrent stone formation.
- PublicationEpigallocatechin-3-gallate prevents TGF-β1-induced epithelial-mesenchymal transition and fibrotic changes of renal cells via GSK-3β/β-catenin/Snail1 and Nrf2 pathways(2020-02-01) Rattiyaporn Kanlaya; Paleerath Peerapen; Angkhana Nilnumkhum; Sirikanya Plumworasawat; Kanyarat Sueksakit; Visith Thongboonkerd; Faculty of Medicine, Siriraj Hospital, Mahidol University© 2019 Several lines of evidence have demonstrated anti-fibrotic property of epigallocatechin-3-gallate (EGCG) in many tissues/organs but with unclear mechanisms. This study thus aimed to define cellular mechanisms underlying such protective effect of EGCG. HK-2 renal cells were treated with 5 ng/ml TGF-β1 for 24 h with/without pretreatment by 5 μM EGCG for 1 h. The cells were then evaluated by morphological examination, immunofluorescence study, semi-quantitative RT-PCR, Western blotting, and atomic force microscopy (AFM). The results showed that TGF-β1-treated cells underwent epithelial mesenchymal transition (EMT) as evidenced by morphological change into fibroblast-like and increases in spindle index, mesenchymal markers (Snail1 and vimentin), extracellular matrix (fibronectin), cell stiffness (by AFM measurement) and actin stress fibers, whereas the epithelial markers (E-cadherin and ZO-1) were decreased. All of these features were abolished by EGCG pretreatment. Functional studies revealed that the anti-fibrotic property of EGCG was, at least in part, due to de-activation/stabilization of GSK-3β/β-catenin/Snail1 (EMT-triggering) signaling pathway that was activated by TGF-β1 as shown by maintaining phosphorylated GSK-3β, β-catenin and Snail1 to their basal levels. Additionally, Nrf2 knockdown by small interfering RNA could abolish the EGCG effect on β-catenin expression. These data indicate that EGCG attenuates TGF-β1-induced EMT in renal tubular cells through GSK-3β/β-catenin/Snail1 and Nrf2 pathways.
- PublicationFlagellum Is Responsible for Promoting Effects of Viable Escherichia coli on Calcium Oxalate Crystallization, Crystal Growth, and Crystal Aggregation(2019-11-05) Rattiyaporn Kanlaya; Orapan Naruepantawart; Visith Thongboonkerd; Faculty of Medicine, Siriraj Hospital, Mahidol University© Copyright © 2019 Kanlaya, Naruepantawart and Thongboonkerd. Urease-producing bacteria (especially Proteus mirabilis) can cause infection kidney stone. However, recent studies have shown that intact viable non-urease-producing bacteria such as Escherichia coli might also promote calcium oxalate (CaOx) kidney stone formation but with unclear mechanism. We thus hypothesized that some relevant bacterial components might be responsible for such promoting effects of the intact viable E. coli. Flagella, capsule, lipopolysaccharide (LPS), and outer membrane vesicles (OMVs) were isolated/purified and their stone modulatory activities were evaluated using CaOx crystallization, crystal growth, and crystal aggregation assays. Among these, flagella had the most potent promoting effects on CaOx crystallization, crystal growth, and crystal aggregation. Validation was performed by deflagellation demonstrating that the deflagellated intact viable E. coli had markedly reduced CaOx crystal modulatory activities in all aspects (comparable to those of the negative controls). Similarly, neutralization of the isolated/purified flagella using a specific anti-flagellin antibody, not an isotype control, could abolish the promoting effects of flagella. These findings provide direct evidence indicating that flagellum is responsible for the promoting effects of the viable E. coli on CaOx crystallization, crystal growth and aggregation.
- PublicationMacropinocytosis is the Major Mechanism for Endocytosis of Calcium Oxalate Crystals into Renal Tubular Cells(2013-12-01) Rattiyaporn Kanlaya; Kitisak Sintiprungrat; Sakdithep Chaiyarit; Visith Thongboonkerd; Mahidol UniversityDuring an initial phase of kidney stone formation, the internalization of calcium oxalate (CaOx) crystals by renal tubular cells has been thought to occur via endocytosis. However, the precise mechanism of CaOx crystal endocytosis remained unclear. In the present study, MDCK renal tubular cells were pretreated with inhibitors specific to individual endocytic pathways, including nystatin (lipid raft/caveolae-mediated), cytochalasin D (actin-dependent or macropinocytosis), and chlorpromazine (CPZ; clathrin-mediated) before exposure to plain (non-labeled), or fluorescence-labeled CaOx monohydrate (COM) crystals. Quantitative analysis by flow cytometry revealed that pretreatment with nystatin and CPZ slightly decreased the crystal internalization, whereas the cytochalasin D pretreatment caused a marked decrease in crystal uptake. Immunofluorescence study and laser-scanning confocal microscopic examination confirmed that the cytochalasin D-pretreated cells had dramatic decrease of the internalized crystals, whereas the total number of crystals interacted with the cells was unchanged (crystals could adhere but were not internalized). These data have demonstrated for the first time that renal tubular cells endocytose COM crystals mainly via macropinocytosis. These novel findings will be useful for further tracking the endocytosed crystals inside the cells during the course of kidney stone formation. © 2013 Springer Science+Business Media New York.
- PublicationMolecular Mechanisms of Epigallocatechin-3-Gallate for Prevention of Chronic Kidney Disease and Renal Fibrosis: Preclinical Evidence(2019-09-19) Rattiyaporn Kanlaya; Visith Thongboonkerd; Faculty of Medicine, Siriraj Hospital, Mahidol UniversityCopyright © American Society for Nutrition 2019. Chronic kidney disease (CKD) is a common public health problem worldwide characterized by gradual decline of renal function over months/years accompanied by renal fibrosis and failure in tissue wound healing after sustained injury. Patients with CKD frequently present with profound signs/symptoms that require medical treatment, mostly culminating in hemodialysis and renal transplantation. To prevent CKD more efficiently, there is an urgent need for better understanding of the pathogenic mechanisms and molecular pathways of the disease pathogenesis and progression, and for developing novel therapeutic targets. Recently, several lines of evidence have shown that epigallocatechin-3-gallate (EGCG), an abundant phytochemical polyphenol derived from Camellia sinensis, might be a promising bioactive compound for prevention of CKD development/progression. This review summarizes current knowledge of molecular mechanisms underlying renoprotective roles of EGCG in CKD based on available preclinical evidence (from both in vitro and in vivo animal studies), particularly its antioxidant property through preservation of mitochondrial function and activation of Nrf2 (nuclear factor erythroid 2-related factor 2)/HO-1 (heme oxygenase-1) signaling, anti-inflammatory activity, and protective effect against epithelial mesenchymal transition. Finally, future perspectives, challenges, and concerns regarding its clinical use in CKD and renal fibrosis are discussed.
- PublicationPhenotypic characteristics and comparative proteomics of Staphylococcus aureus strains with different vancomycin-resistance levels(2016-12-01) Auttawit Sirichoat; Aroonlug Lulitanond; Rattiyaporn Kanlaya; Ratree Tavichakorntrakool; Aroonwadee Chanawong; Sujintana Wongthong; Visith Thongboonkerd; Khon Kaen University; Mahidol University© 2016 Elsevier Inc. Reduced vancomycin susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide problem. Unfortunately, its genetic marker and molecular mechanisms remained unknown. This study investigated differential phenotypic characteristic and protein expression profiles among three groups of MRSA isolates, including vancomycin-susceptible S. aureus (VSSA), heterogeneous vancomycin-intermediate S. aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) (n = 7 isolates/group). Phenotypic characteristic revealed significant greater number of isolates with non-spreading colony in VISA as compared to both VSSA and hVISA groups. 2-DE followed by nanoLC-MS/MS analyses revealed increased glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both hVISA and VISA, whereas 50S ribosomal protein L14 (RplN) and DNA-binding protein II (Hup) were increased only in VISA. The non-spreading colony and GAPDH level of MRSA may be used as the markers for differentiation of VSSA, hVISA and VISA.
- PublicationProtective Effects of Epigallocatechin-3-Gallate from Green Tea in Various Kidney Diseases(2019-01-01) Rattiyaporn Kanlaya; Visith Thongboonkerd; Faculty of Medicine, Siriraj Hospital, Mahidol University© 2019 American Society for Nutrition. All rights reserved. Kidney diseases are common health problems worldwide. Various etiologies (e.g., diabetes, hypertension, drug-induced nephrotoxicity, infection, cancers) can affect renal function and ultimately lead to development of chronic kidney disease (CKD) and end-stage renal disease (ESRD). The global rise in number of CKD/ESRD patients during recent years has led to tremendous concern to look for effective strategies to prevent or slow progression of CKD and ESRD. Natural compounds derived from herbs or medicinal plants have gained wide attention for scientific scrutiny to achieve such goals. One of such natural compounds that has been extensively investigated is epigallocatechin-3-gallate (EGCG), a major polyphenol found in the tea plant (Camellia sinensis). A growing body of recent evidence has shown that EGCG may be a promising therapeutic or protective agent in various kidney diseases. This article thus highlights recent progress in medical research on beneficial effects of EGCG against a broad spectrum of kidney diseases, including acute kidney injury, cisplatin-induced nephrotoxicity, kidney stone disease, glomerulonephritis, lupus nephritis, renal cell carcinoma, diabetic nephropathy, CKD, and renal fibrosis. The renoprotective mechanisms are also detailed. Finally, future perspectives of medical research on EGCG and its potential use in clinical practice for treatment and prevention of kidney diseases are discussed.