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1.
Commun Biol ; 4(1): 959, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381146

RESUMO

The 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.


Assuntos
Oxalato de Cálcio/metabolismo , Fibroblastos/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Animais , Oxalato de Cálcio/química , Cricetinae , Humanos , Mapas de Interação de Proteínas , Células U937
2.
Anal Methods ; 13(30): 3359-3367, 2021 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-34296239

RESUMO

Tamm-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.


Assuntos
Terra de Diatomáceas , Cloreto de Sódio , Adsorção , Cromatografia Líquida de Alta Pressão , Humanos , Uromodulina
3.
Biomed Pharmacother ; 141: 111870, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34246192

RESUMO

Caffeine 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.

4.
J Nutr Biochem ; 76: 108266, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31760226

RESUMO

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-ß1 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-ß1-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-3ß/ß-catenin/Snail1 (EMT-triggering) signaling pathway that was activated by TGF-ß1 as shown by maintaining phosphorylated GSK-3ß, ß-catenin and Snail1 to their basal levels. Additionally, Nrf2 knockdown by small interfering RNA could abolish the EGCG effect on ß-catenin expression. These data indicate that EGCG attenuates TGF-ß1-induced EMT in renal tubular cells through GSK-3ß/ß-catenin/Snail1 and Nrf2 pathways.


Assuntos
Catequina/análogos & derivados , Transição Epitelial-Mesenquimal , Glicogênio Sintase Quinase 3 beta/metabolismo , Rim/citologia , Transdução de Sinais , Animais , Catequina/farmacologia , Cães , Fibronectinas/metabolismo , Fibrose , Humanos , Rim/patologia , Células Madin Darby de Rim Canino , Microscopia de Força Atômica , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , beta Catenina/metabolismo
5.
Front Microbiol ; 10: 2507, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31749785

RESUMO

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.

6.
Curr Dev Nutr ; 3(9): nzz101, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31555758

RESUMO

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.

7.
Cell Adh Migr ; 13(1): 260-272, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31271106

RESUMO

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.


Assuntos
Antioxidantes/farmacologia , Cafeína/farmacologia , Hipóxia Celular/efeitos dos fármacos , Fibrose/tratamento farmacológico , Rim/patologia , Actinas/metabolismo , Animais , Catalase/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular , Cricetinae , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Fibrose/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Rim/citologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
8.
Adv Nutr ; 10(1): 112-121, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615092

RESUMO

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.


Assuntos
Catequina/análogos & derivados , Nefropatias/terapia , Substâncias Protetoras/farmacologia , Chá/química , Catequina/farmacologia , Humanos , Rim/efeitos dos fármacos
9.
Cell Death Discov ; 4: 9, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29531806

RESUMO

TGF-ß1 is a key fibrotic factor mediating epithelial mesenchymal transition (EMT) of epithelial cells through various signaling pathways. However, roles of proteolytic cleavage and endogenous peptide dynamics in TGF-ß1-induced EMT remain unknown. We therefore performed quantitative peptidomics of TGF-ß1-induced EMT in renal tubular epithelial cells. The acquired mesenchymal characteristics were confirmed, including morphological change (from cobblestone-like to fibroblast-like), decreased epithelial marker (ZO-1), and increased mesenchymal marker (vimentin). Quantitative peptidomics using stable isotope labeling revealed significantly altered levels of 70 unique endogenous peptides (derived from internal and C-terminal parts of 39 unique precursor proteins) after EMT induction. Interestingly, the majority of these peptides were derived from non-short-lived proteins, and analysis of P1 position revealed predominance of hydrophobic residues, suggesting that these endogenous peptides were generated mainly from proteasome cleavage. This hypothesis was confirmed by treating the cells with MG132 (a proteasome inhibitor), which provided almost identical endogenous peptide pattern as of the TGF-ß1-treated cells. Moreover, validation assay showed marked reduction of proteasome peptidase activity in both TGF-ß1-treated and MG132-treated cells. This is the first peptidome dataset that provides several novel aspects of mechanisms for TGF-ß1-induced EMT. Our data also suggest that TGF-ß1 exerts inhibitory effect against proteasome activity during EMT induction.

10.
Front Immunol ; 9: 316, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29535716

RESUMO

In kidney stone disease, macrophages secrete various mediators via classical secretory pathway and cause renal interstitial inflammation. However, whether their extracellular vesicles, particularly exosomes, are involved in kidney stone pathogenesis remained unknown. This study investigated alterations in exosomal proteome of U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) after exposure to calcium oxalate monohydrate (COM) crystals for 16-h using 2-DE-based proteomics approach. Six significantly altered proteins in COM-treated exosomes were successfully identified by nanoscale liquid chromatography-electrospray ionization-electron transfer dissociation tandem mass spectrometry as proteins involved mainly in immune processes, including T-cell activation and homeostasis, Fcγ receptor-mediated phagocytosis, interferon-γ (IFN-γ) regulation, and cell migration/movement. The decreased heat shock protein 90-beta (HSP90ß) and increased vimentin were confirmed by Western blotting. ELISA showed that the COM-treated macrophages produced greater level of interleukin-1ß (IL-1ß), one of the markers for inflammasome activation. Functional studies demonstrated that COM-treated exosomes enhanced monocyte and T-cell migration, monocyte activation and macrophage phagocytic activity, but on the other hand, reduced T-cell activation. In addition, COM-treated exosomes enhanced production of proinflammatory cytokine IL-8 by monocytes that could be restored to its basal level by small-interfering RNA targeting on vimentin (si-Vimentin). Moreover, si-Vimentin could also abolish effects of COM-treated exosomes on monocyte and T-cell migration as well as macrophage phagocytic activity. These findings provided some implications to the immune response during kidney stone pathogenesis via exosomal pathway of macrophages after exposure to COM crystals.


Assuntos
Oxalato de Cálcio/farmacologia , Movimento Celular/efeitos dos fármacos , Exossomos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/imunologia , Linfócitos T/imunologia , Movimento Celular/imunologia , Exossomos/patologia , Proteínas de Choque Térmico HSP90/imunologia , Humanos , Interleucina-1beta/imunologia , Interleucina-8/imunologia , Células Jurkat , Macrófagos/patologia , Linfócitos T/patologia , Células U937
11.
Sci Rep ; 8(1): 521, 2018 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-29323127

RESUMO

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.


Assuntos
Alopecia em Áreas/patologia , Proteoma/metabolismo , Adulto , Alopecia em Áreas/metabolismo , Anexina A2/metabolismo , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Proteoma/análise , Proteômica , Espectrometria de Massas em Tandem , Regulação para Cima , Vimentina/metabolismo
12.
Diagn Microbiol Infect Dis ; 86(4): 340-344, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27717648

RESUMO

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.


Assuntos
Staphylococcus aureus Resistente à Meticilina/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Proteoma/análise , Resistência a Vancomicina , Proteínas de Bactérias/análise , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/microbiologia , Espectrometria de Massas em Tandem
13.
Sci Rep ; 6: 31855, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27546060

RESUMO

Vascular leakage is a life-threatening complication of dengue virus (DENV) infection. Previously, association between "paracellular" endothelial hyperpermeability and plasma leakage had been extensively investigated. However, whether "transcellular" endothelial leakage is involved in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) remained unknown. We thus investigated effects of DENV (serotype 2) infection on transcellular transport of albumin, the main oncotic plasma protein, through human endothelial cell monolayer by Western blotting, immunofluorescence staining, fluorescence imaging, and fluorometry. The data showed that Alexa488-conjugated bovine serum albumin (Alexa488-BSA) was detectable inside DENV2-infected cells and its level was progressively increased during 48-h post-infection. While paracellular transport could be excluded using FITC-conjugated dextran, Alexa488-BSA was progressively increased and decreased in lower and upper chambers of Transwell, respectively. Pretreatment with nystatin, an inhibitor of caveolae-dependent endocytic pathway, significantly decreased albumin internalization into the DENV2-infected cells, whereas inhibitors of other endocytic pathways showed no significant effects. Co-localization of the internalized Alexa488-BSA and caveolin-1 was also observed. Our findings indicate that DENV infection enhances caveolae-mediated albumin transcytosis through human endothelial cells that may ultimately induce plasma leakage from intravascular compartment. Further elucidation of this model in vivo may lead to effective prevention and better therapeutic outcome of DHF/DSS.


Assuntos
Cavéolas/metabolismo , Células Endoteliais/metabolismo , Albumina Sérica Humana/metabolismo , Dengue Grave/metabolismo , Permeabilidade Capilar , Linhagem Celular , Vírus da Dengue/fisiologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/virologia , Humanos , Modelos Biológicos , Nistatina/farmacologia , Transcitose/efeitos dos fármacos
14.
Sci Rep ; 6: 30233, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27452398

RESUMO

This study evaluated effect of oxalate on epithelial mesenchymal transition (EMT) and potential anti-fibrotic property of epigallocatechin-3-gallate (EGCG). MDCK renal tubular cells were incubated with 0.5 mM sodium oxalate for 24-h with/without 1-h pretreatment with 25 µM EGCG. Microscopic examination, immunoblotting and immunofluorescence staining revealed that oxalate-treated cells gained mesenchymal phenotypes by fibroblast-like morphological change and increasing expression of vimentin and fibronectin, while levels of epithelial markers (E-cadherin, occludin, cytokeratin and ZO-1) were decreased. EGCG pretreatment could prevent all these changes and molecular mechanisms underlying the prevention by EGCG were most likely due to reduced production of intracellular ROS through activation of Nrf2 signaling and increased catalase anti-oxidant enzyme. Knockdown of Nrf2 by small interfering RNA (siRNA) abrogated all the effects of EGCG, confirming that the EGCG protection against oxalate-induced EMT was mediated via Nrf2. Taken together, our data indicate that oxalate turned on EMT of renal tubular cells that could be prevented by EGCG via Nrf2 pathway. These findings also shed light onto development of novel therapeutics or preventive strategies of renal fibrosis in the future.


Assuntos
Catequina/análogos & derivados , Fibrose/tratamento farmacológico , Túbulos Renais/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Animais , Catequina/administração & dosagem , Cães , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Fibrose/genética , Fibrose/patologia , Humanos , Túbulos Renais/patologia , Células Madin Darby de Rim Canino , Oxalatos/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos
15.
J Virol Methods ; 234: 174-7, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27155240

RESUMO

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.


Assuntos
Cromatografia/métodos , Vírus da Dengue/isolamento & purificação , Cromatografia/instrumentação , Dengue/diagnóstico , Dengue/virologia , Polímeros , Proteínas do Envelope Viral/isolamento & purificação
16.
J Biol Inorg Chem ; 21(3): 339-46, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26898643

RESUMO

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.


Assuntos
Oxalato de Cálcio/metabolismo , Catequina/análogos & derivados , Fosfopiruvato Hidratase/biossíntese , Fosfopiruvato Hidratase/deficiência , Animais , Sítios de Ligação/efeitos dos fármacos , Oxalato de Cálcio/química , Catequina/administração & dosagem , Catequina/farmacologia , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Células Madin Darby de Rim Canino , Fosfopiruvato Hidratase/química , Relação Estrutura-Atividade , Propriedades de Superfície
17.
PLoS One ; 9(9): e106973, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25222612

RESUMO

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.


Assuntos
Técnicas de Cultura de Células , Metaboloma , Proteoma , Actinas/metabolismo , Comunicação Celular , Fenômenos Fisiológicos Celulares , Proliferação de Células , Células Cultivadas , Cromatografia Líquida , Células Hep G2 , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Espectrometria de Massas em Tandem
18.
J Proteome Res ; 13(7): 3160-5, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24831074

RESUMO

Following 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).


Assuntos
Cromossomos Humanos Par 12/genética , Proteoma/genética , Cromossomos Humanos Par 12/metabolismo , Humanos , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Especificidade de Órgãos , Proteoma/metabolismo , Projetos de Pesquisa
19.
Cell Biochem Biophys ; 67(3): 1207-15, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23700163

RESUMO

Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to "epithelial mesenchymal transition/transdifferentiation" (EMT) of renal tubular cells. Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with "secreted products from CaOx-exposed macrophages" (CaOx-M-Sup). Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-ß1 (TGF-ß1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-ß1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with a proteasome inhibitor (MG132) could restore RhoA to its basal level, most likely through ubiquitin-proteasome pathway (UPP). Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-ß1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease.


Assuntos
Oxalato de Cálcio/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Cães , Humanos , Queratinas/metabolismo , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Leupeptinas/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Células Madin Darby de Rim Canino , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Vimentina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
20.
Cell Biochem Biophys ; 67(3): 1171-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23695784

RESUMO

During 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.


Assuntos
Oxalato de Cálcio/metabolismo , Endocitose , Animais , Oxalato de Cálcio/química , Clorpromazina/farmacologia , Cristalização , Citocalasina D/farmacologia , Cães , Endocitose/efeitos dos fármacos , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Células Madin Darby de Rim Canino , Microscopia Confocal , Nistatina/farmacologia
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