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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.
Expert Rev Proteomics ; 18(7): 557-569, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34320328

RESUMO

INTRODUCTION: Main problems of kidney stone disease are its increasing prevalence and high recurrence rate after calculi removal in almost all areas around the globe. Despite enormous efforts in the past, its pathogenic mechanisms remain unclear and need further elucidations. Proteomics has thus become an essential tool to unravel such sophisticated disease mechanisms at cellular, subcellular, molecular, tissue, and whole organism levels. AREAS COVERED: This review provides abrief overview of kidney stone disease followed by updates on proteomics for investigating urinary stone modulators, matrix proteins, cellular responses to different types/doses of calcium oxalate (CaOx) crystals, sex hormones and other stimuli, crystal-cell interactions, crystal receptors, secretome, and extracellular vesicles (EVs), all of which lead to better understanding of the disease mechanisms. Finally, the future challenges and translation of these obtained data to the clinic are discussed. EXPERT OPINION: Knowledge from urinary proteomics for exploring the important stone modulators (either inhibitors or promoters) will be helpful for early detection of asymptomatic cases for prompt prevention of symptoms, complications, and new stone formation. Moreover, these modulators may serve as the new therapeutic targets in the future for successful treatment and prevention of kidney stone disease by medications or other means of intervention.

3.
Chem Biol Interact ; 345: 109557, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34147488

RESUMO

Tight junction is an intercellular protein complex that regulates paracellular permeability and epithelial cell polarization. This intercellular barrier is associated with actin filament. Calcium oxalate monohydrate (COM), the major crystalline composition in kidney stones, has been shown to disrupt tight junction but with an unclear mechanism. This study aimed to address whether COM crystal disrupts tight junction via actin deregulation. MDCK distal renal tubular epithelial cells were treated with 100 µg/ml COM crystals for 48 h. Western blot analysis revealed that level of a tight junction protein, zonula occludens-1 (ZO-1), significantly decreased, whereas that of ß-actin remained unchanged after exposure to COM crystals. Immunofluorescence study showed discontinuation and dissociation of ZO-1 and filamentous actin (F-actin) expression at the cell border. In addition, clumping of F-actin was found in some cytoplasmic areas of the COM-treated cells. Moreover, transepithelial resistance (TER) was reduced by COM crystals, indicating the defective barrier function of the polarized cells. All of these COM-induced defects could be completely abolished by pretreatment with 20 µM phalloidin, an F-actin stabilizer, 2-h prior to the 48-h crystal exposure. These findings indicate that COM crystal does not reduce the total level of actin but causes tight junction disruption via F-actin reorganization.


Assuntos
Actinas/metabolismo , Oxalato de Cálcio/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Animais , Cães , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Túbulos Renais/citologia , Células Madin Darby de Rim Canino
4.
Int J Biol Macromol ; 180: 1-13, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33675830

RESUMO

AT-rich interactive domain 1A (ARID1A) is a novel tumor suppressor gene found in several human cells and its loss/defect is commonly observed in many cancers. However, its roles in angiogenesis, which is one of the hallmarks for tumor progression, remained unclear. Herein, we demonstrated the direct effects of ARID1A knockdown in human endothelial cells by lentivirus-based short-hairpin RNA (shRNA) (shARID1A) on angiogenesis. Functional assays revealed that shARID1A significantly enhanced cell proliferation and migration/invasion and endothelial tube formation compared with the control cells transfected with scramble shRNA (shControl). Additionally, the shARID1A-transfected cells had significantly increased podosome formation and secretion of angiopoietin-2 (ANG2), a key angiogenic factor. Moreover, neutralization of ANG2 with monoclonal anti-ANG2 antibody strongly reduced cell proliferation and migration/invasion and endothelial tube formation in the shARID1A-transfected cells. These findings indicate that down-regulation of ARID1A in human endothelial cells directly induces angiogenesis by regulating angiopoietin-2 secretion and endothelial cell activity.


Assuntos
Angiopoietina-2/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Inativação Gênica , Neovascularização Fisiológica/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes/métodos , Células HEK293 , Humanos , Podossomos/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção
5.
Int J Biol Macromol ; 163: 2210-2223, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32956748

RESUMO

Adhesion of calcium oxalate (CaOx) crystals onto renal tubular epithelial cells is one of the critical steps in kidney stone formation. However, effects of crystal size on the crystal adhesive capability remained unclear. This study compared the adhesive capabilities of CaOx monohydrate (COM) crystals with various sizes (<10 µm, 20-30 µm, 50-60 µm, and > 80 µm). Crystal-cell adhesion assay showed size-dependent increase of COM crystal adhesion onto epithelial cell surface using the larger crystals. Identification of apical membrane proteins that could bind to COM crystals by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) demonstrated size-specific sets of the COM crystal-binding proteins. Among these, numbers of known oxalate-binding proteins and COM crystal receptors were greatest in the set of the largest size (>80 µm). Atomic force microscopy (AFM) revealed that adhesive forces between carboxylic-immobilized AFM tip and COM crystal surface and between COM-mounted AFM tip and renal epithelial cell surface were size-dependent (greater for the larger crystals). In summary, the adhesive capability of COM crystals is size-dependent - the larger the greater adhesive capability. These data may help better understanding of the pathogenic mechanisms of kidney stone formation at an initial stage when renal tubular cells are exposed to various sizes of COM crystals.


Assuntos
Adesivos/química , Oxalato de Cálcio/química , Células Epiteliais/química , Cálculos Renais/química , Adesivos/farmacologia , Proteínas de Transporte/química , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Cristalização , Células Epiteliais/efeitos dos fármacos , Humanos , Cálculos Renais/patologia , Cálculos Renais/ultraestrutura , Microscopia de Força Atômica , Espectrometria de Massas em Tandem
6.
Sci Rep ; 10(1): 15109, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934277

RESUMO

Better understanding of molecular mechanisms for kidney stone formation is required to improve management of kidney stone disease with better therapeutic outcome. Recent kidney stone research has indicated critical roles of a group of proteins, namely 'stone modulators', in promotion or inhibition of the stone formation. Nevertheless, such information is currently dispersed and difficult to obtain. Herein, we present the kidney stone modulator database (StoneMod), which is a curated resource by obtaining necessary information of such stone modulatory proteins, which can act as stone promoters or inhibitors, with experimental evidence from previously published studies. Currently, the StoneMod database contains 10, 16, 13, 8 modulatory proteins that affect calcium oxalate crystallization, crystal growth, crystal aggregation, and crystal adhesion on renal tubular cells, respectively. Informative details of each modulatory protein and PubMed links to the published articles are provided. Additionally, hyperlinks to other protein/gene databases (e.g., UniProtKB, Swiss-Prot, Human Protein Atlas, PeptideAtlas, and Ensembl) are made available for the users to obtain additional in-depth information of each protein. Moreover, this database provides a user-friendly web interface, in which the users can freely access to the information and/or submit their data to deposit or update. Database URL: https://www.stonemod.org .


Assuntos
Oxalato de Cálcio/metabolismo , Bases de Dados de Proteínas , Cálculos Renais/metabolismo , Cálculos Renais/patologia , Proteínas/metabolismo , Software , Cristalização , Humanos , Cálculos Renais/genética , Proteínas/genética
7.
Anal Biochem ; 590: 113518, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31786226

RESUMO

Heat shock protein 90 (HSP90) plays essential roles in the normal physiology and comprises four distinct domains, including NH2-terminal (N), charged linker region (LR), middle (M), and COOH-terminal (C) domains, all of which regulate HSP90 biological functions. We reported herein detailed protocols to produce recombinant full-length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET-32b(+) expression vector. All HSP90 constructs were expressed as soluble Trx-His-S tagged proteins after induction with 0.25 mM isopropyl-ß-d-thiogalactopyranoside (IPTG) at 18 °C overnight and further purified by affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) resin. The enterokinase (EK) digestion was optimized for efficient cleavage of the Trx-His-S tag from each HSP90 construct by varying concentrations of EK (0.5-1 U) and urea (0-3 M). Each HSP90 construct was highly purified and approximately 0.1-1 mg proteins were obtained from 100 ml of bacterial culture. All the purified HSP90 constructs were successfully confirmed by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and their secondary structure was quantified using attenuated total reflection - Fourier-transform infrared (ATR-FTIR) spectroscopy. Our expression and purification protocols would facilitate further structural and functional studies of human HSP90.


Assuntos
Proteínas de Choque Térmico HSP90/biossíntese , Proteínas Recombinantes/biossíntese , Clonagem Molecular , Escherichia coli/genética , Proteínas de Choque Térmico HSP90/isolamento & purificação , Humanos , Domínios Proteicos , Proteínas Recombinantes/isolamento & purificação
8.
Food Chem Toxicol ; 135: 110915, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31669600

RESUMO

Fibrogenesis is a common feature for all types of chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the main processes involving renal fibrosis and its inhibition is considered as a preventive/therapeutic strategy for CKD. Trigonelline (TRIG), a plant alkaloid commonly found in herbs, coffee bean, soy bean and other edible food plants, has several beneficial effects on human health and has been proposed to reduce renal fibrosis but with unclear mechanisms. This study thus addressed cellular mechanism underlying the anti-fibrogenic effects of TRIG in renal tubular epithelial cells grown in vitro. EMT was successfully induced by oxalate treatment as indicated by morphological changes into spindle-shape cells, increased expression of mesenchymal proteins (fibronectin, vimentin and α-smooth muscle actin (α-SMA)), decreased expression of epithelial proteins (E-cadherin and zonula occludens-1 (ZO-1)) and increased activity of a profibrotic factor (matrix metalloproteinase-9 (MMP-9)). Interestingly, these oxalate-induced EMT features could be attenuated by TRIG pretreatment. Moreover, TRIG also prevented oxalate-induced cell migration, reactive oxygen species (ROS) overproduction, and down-regulation of Nrf-2 signaling molecule. These data indicated that TRIG could attenuate the effects of oxalate-induced EMT and thus may serve as the anti-fibrotic compound for prevention and/or treatment of CKD.


Assuntos
Alcaloides/farmacologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Oxalatos/efeitos adversos , Substâncias Protetoras/farmacologia , Actinas/metabolismo , Animais , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cães , Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Células Madin Darby de Rim Canino , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vimentina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
9.
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
10.
Proteomics ; 19(19): e1900095, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31475403

RESUMO

Females have less incidence/prevalence of kidney stone disease than males. Estrogen thus may serve as the protective factor but with unclear mechanism. This study explores cellular mechanism underlying such stone preventive mechanism of estrogen. Madin darby canine kidney (MDCK) renal tubular cells are incubated with or without 20 nm 17ß-estradiol for 7 days. Comparative proteomics reveals 58 differentially expressed proteins in estrogen-treated versus control cells that are successfully identified by nanoLC-ESI-Q-TOF-MS/MS. Interestingly, these altered proteins are involved mainly in "binding and receptor," "metabolic process," and "migration and healing" networks. Functional investigations demonstrate reduction of calcium oxalate (CaOx) crystal-binding capability of the estrogen-treated cells consistent with the decreased levels of annexin A1 and α-enolase (the known CaOx crystal-binding receptors) on the cell surface. High-calcium and high-oxalate challenge initially enhances surface expression of annexin A1 and α-enolase, respectively, both of which return to their basal levels by estrogen. Additionally, estrogen reduces intracellular ATP level and promotes cell migration and tissue healing. Taken together, estrogen causes changes in cellular proteome of renal tubular cells that lead to decreased surface expression of CaOx crystal receptors, reduced intracellular metabolism, and enhanced cell proliferation and tissue healing, all of which may contribute, at least in part, to stone prevention.


Assuntos
Estradiol/farmacologia , Cálculos Renais/prevenção & controle , Proteoma/metabolismo , Proteômica/métodos , Animais , Oxalato de Cálcio/química , Oxalato de Cálcio/metabolismo , Células Cultivadas , Cromatografia Líquida/métodos , Cristalização , Cães , Estrogênios/farmacologia , Cálculos Renais/metabolismo , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Nanotecnologia/métodos , Substâncias Protetoras/farmacologia , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
11.
FASEB J ; 33(11): 12226-12239, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31424966

RESUMO

Down-regulation/mutation of AT-rich interactive domain 1A (ARID1A), a novel tumor suppressor gene, has been reported in various cancers. Nevertheless, its role in renal cell carcinoma (RCC) remained unclear and underinvestigated. We thus evaluated carcinogenesis effects of ARID1A knockdown in nonmalignant Madin-Darby canine kidney (MDCK) renal cells using small interfering RNA (siRNA) against ARID1A (siARID1A). The siARID1A-transfected cells had decreased cell death, increased cell proliferation, and cell cycle shift (from G0/G1 to G2/M) compared with those transfected with controlled siRNA (siControl). Additionally, the siARID1A-transfected cells exhibited epithelial-mesenchymal transition (EMT) shown by greater spindle index, increased mesenchymal markers (fibronectin/vimentin), and decreased epithelial markers (E-cadherin/zonula occludens-1). Moreover, the siARID1A-transfected cells had increases in migratory activity, nuclear size, self-aggregated multicellular spheroid size, invasion capability, chemoresistance (to docetaxel), Snail family transcriptional repressor 1 expression, and TGF-ß1 secretion. All of these siARID1A-knockdown effects on the carcinogenic features were reproducible in malignant RCC (786-O) cells, which exhibited a higher degree of carcinogenic phenotypes compared with the nonmalignant MDCK cells. Finally, immunohistochemistry showed obvious decrease in ARID1A protein expression in human RCC tissues (n = 23) compared with adjacent normal renal tissues (n = 23). These data indicate that ARID1A down-regulation triggers EMT and carcinogenesis features of renal cells in vitro, and its role in RCC could be proven in human tissues.-Somsuan, K., Peerapen, P., Boonmark, W., Plumworasawat, S., Samol, R., Sakulsak, N., Thongboonkerd, V. ARID1A knockdown triggers epithelial-mesenchymal transition and carcinogenesis features of renal cells: role in renal cell carcinoma.


Assuntos
Carcinogênese , Carcinoma de Células Renais/patologia , Proteínas de Ligação a DNA/fisiologia , Transição Epitelial-Mesenquimal , Neoplasias Renais/patologia , Fatores de Transcrição/fisiologia , Animais , Carcinoma de Células Renais/etiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Cães , Humanos , Neoplasias Renais/etiologia , Células Madin Darby de Rim Canino , Fatores de Transcrição da Família Snail/fisiologia , Fatores de Transcrição/antagonistas & inibidores , Fator de Crescimento Transformador beta1/fisiologia
12.
Adv Nutr ; 9(4): 419-424, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30032225

RESUMO

Kidney stone disease is a global health care problem, with a high recurrence rate after stone removal. It is thus crucial to develop effective strategies to prevent the formation of new or recurrent stones. Caffeine is one of the main components in caffeinated beverages worldwide (i.e., coffee, tea, soft drinks, and energy drinks). Previous retrospective and prospective studies have reported contradictory effects of caffeine on kidney stone risk. Although it has a diuretic effect on enhancing urinary output, it may slightly increase the stone risk index. However, 3 large cohorts have suggested a preventive role of caffeine in kidney stone disease. In addition, a recent in vitro study has addressed relevant mechanisms underlying the preventive role of caffeine against stone pathogenesis. This review summarizes the relevant data from previous evidence and discusses the association between caffeine consumption and kidney stone risk reduction.


Assuntos
Cafeína/administração & dosagem , Cafeína/efeitos adversos , Cálculos Renais/epidemiologia , Cálculos Renais/prevenção & controle , Bebidas , Cafeína/farmacocinética , Bebidas Gaseificadas , Café , Dieta , Diuréticos , Relação Dose-Resposta a Droga , Bebidas Energéticas , Humanos , Rim/efeitos dos fármacos , Estudos Prospectivos , Recidiva , Estudos Retrospectivos , Fatores de Risco , Chá
14.
Proteomics ; 18(8): e1800008, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29464862

RESUMO

Our previous expression study has reported a set of proteins with altered levels in renal tubular cells after exposure to calcium oxalate monohydrate (COM) crystals, which are the main composition of kidney stones. However, their functional significance remained largely unknown. In this study, protein network analysis revealed that the significantly altered proteins induced by COM crystals were involved mainly in three main functional networks, including i) cell proliferation and wound healing; ii) oxidative stress and mitochondrial function; and iii) cellular junction complex and integrity. Cell proliferation and wound healing assays showed that the COM-treated cells had defective proliferation and tissue healing capability, respectively. Oxyblot analysis demonstrated accumulation of the oxidized proteins, whereas intracellular ATP level was significantly increased in the COM-treated cells. Additionally, level of zonula occludens-1 (ZO-1), a tight junction protein, was significantly decreased, consistent with the significant declines in transepithelial resistance (TER) and level of RhoA signaling molecule in the COM-treated cells. These findings indicate significant perturbations in mitochondrial and oxidative stress axis that cause defective cell proliferation, tissue healing capability, junctional protein complex, and cellular integrity of renal tubular epithelial cells exposed to COM crystals that may play important roles in kidney stone pathogenesis.


Assuntos
Oxalato de Cálcio/metabolismo , Células Epiteliais/citologia , Túbulos Renais/citologia , Mapas de Interação de Proteínas , Trifosfato de Adenosina/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Cristalização , Cães , Células Epiteliais/metabolismo , Túbulos Renais/metabolismo , Células Madin Darby de Rim Canino , Estresse Oxidativo , Proteína da Zônula de Oclusão-1/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
15.
Sci Rep ; 7(1): 1798, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28496123

RESUMO

Urine pH has been thought to be an important factor that can modulate kidney stone formation. Nevertheless, there was no systematic evaluation of such pH effect. Our present study thus addressed effects of differential urine pH (4.0-8.0) on calcium oxalate (CaOx) crystallization, crystal-cell adhesion, crystal internalization into renal tubular cells, and binding of apical membrane proteins to the crystals. Microscopic examination revealed that CaOx monohydrate (COM), the pathogenic form, was crystallized with greatest size, number and total mass at pH 4.0 and least crystallized at pH 8.0, whereas COD was crystallized with the vice versa order. Fourier-transform infrared (FT-IR) spectroscopy confirmed such morphological study. Crystal-cell adhesion assay showed the greatest degree of crystal-cell adhesion at the most acidic pH and least at the most basic pH. Crystal internalization assay using fluorescein isothiocyanate (FITC)-labelled crystals and flow cytometry demonstrated that crystal internalization into renal tubular cells was maximal at the neutral pH (7.0). Finally, there were no significant differences in binding capacity of the crystals to apical membrane proteins at different pH. We concluded that the acidic urine pH may promote CaOx kidney stone formation, whereas the basic urine pH (i.e. by alkalinization) may help to prevent CaOx kidney stone disease.


Assuntos
Oxalato de Cálcio/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais/metabolismo , Nefrolitíase/etiologia , Nefrolitíase/metabolismo , Animais , Oxalato de Cálcio/química , Adesão Celular , Morte Celular , Proliferação de Células , Cristalização , Cães , Endocitose , Concentração de Íons de Hidrogênio , Células Madin Darby de Rim Canino , Proteínas de Membrana/metabolismo , Nefrolitíase/urina , Urinálise
16.
Clin Chem Lab Med ; 55(7): 993-1002, 2017 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-27987354

RESUMO

BACKGROUND: Diurnal variations and physiologic changes of urinary proteome have been suggested in the urinary proteomics field. However, no clear evidence has been demonstrated. The present study thus aimed to define changes in urinary proteome by physiological stimuli, i.e. caffeine intake and excessive water drinking, both of which cause physiologic diuresis. METHODS: Urine samples were collected from 30 healthy individuals under three different conditions: (i) morning void as the control; (ii) after drinking a cup of coffee; and (iii) after drinking 1 L of water within 20 min. Thereafter, differentially excreted proteins were analyzed by 2-DE proteomics approach and validated by Western blotting and ELISA. RESULTS: Spot matching, quantitative intensity analysis, and ANOVA followed by Tukey's post-hoc multiple comparisons and the Bonferroni correction revealed significant differences in levels of five protein spots among three different conditions. These proteins were identified by quadrupole time-of-flight mass spectrometry (Q-TOF MS) and/or MS/MS analyses as kininogen 1 isoform 3, ß-actin, prostaglandin D synthase (PGDS), fibrinogen α-chain and immunoglobulin light chain. Among these, the decreased level of immunoglobulin was successfully validated by Western blotting and ELISA. CONCLUSIONS: These data indicated that caffeine intake and excessive water drinking could affect urinary excretion of some proteins and may affect urinary proteome analysis.


Assuntos
Cafeína/farmacologia , Ingestão de Líquidos , Proteoma/efeitos dos fármacos , Urinálise , Água/farmacologia , Adulto , Artefatos , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Fatores de Tempo
17.
Cell Adh Migr ; 11(4): 360-366, 2017 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-27627890

RESUMO

Acute mountain sickness (AMS) can cause capillary hyper-permeability and vasogenic edema. However, its underlying mechanisms remained unclear and there is no previous in vitro study on AMS. We therefore conducted an in vitro study and examined whether continuous hypobaric hypoxia (CHH) could alter expression of junctional protein complex of vascular endothelial cells, causing hyper-permeabilization. EA.hy926 human endothelial cells were exposed to either CHH or normoxia for up to 24 h. Flow cytometry using annexin V/propidium iodide co-staining demonstrated that cell death had no significant difference at 12-h, but was increased by CHH at 24-h. Transendothelial resistance (TER) of endothelial cell monolayer was progressively decreased by CHH from 1-h to 24-h. Western blot analysis and immunofluorescence study demonstrated decreased expression levels of VE-cadherin, PECAM-1 and ZO-1 junctional proteins at both 12-h and 24-h exposure time-points. Interestingly, while the main form of ZO-1 (220 kDa) was decreased, its degraded form (100 kDa) was increased by 24-h CHH that might be linked to the increased cell death. Our data have demonstrated that CHH caused vascular endothelial hyper-permeability and defective junctional protein complex by reducing expression levels of VE-cadherin, PECAM-1, and ZO-1. Taken together, these data may explain pathophysiology underlying vascular hyper-permeability in AMS.


Assuntos
Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Regulação para Baixo , Hipóxia/metabolismo , Junções Intercelulares/metabolismo , Proteínas de Membrana/metabolismo , Permeabilidade Capilar , Morte Celular , Linhagem Celular , Células Endoteliais/metabolismo , Humanos
18.
Sci Rep ; 6: 38536, 2016 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-27924845

RESUMO

Recent large 3 cohorts have shown that caffeinated beverage consumption was associated with lower risk of kidney stone disease. However, its protective mechanisms remained unknown and had not been previously investigated. We thus evaluated protective effects of caffeine (1 µM-10 mM) on calcium oxalate monohydrate (COM) kidney stone formation, using crystallization, crystal growth, cell-crystal adhesion, Western blotting, and immunofluorescence assays. The results showed that caffeine reduced crystal number but, on the other hand, increased crystal size, resulting in unchanged crystal mass, consistent with crystal growth that was not affected by caffeine. However, caffeine significantly decreased crystal-binding capacity of MDCK renal tubular cells in a dose-dependent manner. Western blotting and immunofluorescence study of COM crystal-binding proteins revealed significantly decreased level of annexin A1 on apical surface and its translocation into cytoplasm of the caffeine-treated cells, but no significant changes in other COM crystal-binding proteins (annexin A2, α-enolase, HSP70, and HSP90) were observed. Moreover, caffeine decreased intracellular [Ca2+] but increased [Ca2+] secretory index. Taken together, our findings showed an in vitro evidence of the protective mechanism of caffeine against kidney stone formation via translocation of annexin A1 from apical surface into cytoplasm to reduce the crystal-binding capacity of renal tubular epithelial cells.


Assuntos
Anexina A1/metabolismo , Cafeína/uso terapêutico , Oxalato de Cálcio/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Cálculos Renais/tratamento farmacológico , Cálculos Renais/prevenção & controle , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Oxalato de Cálcio/química , Membrana Celular/efeitos dos fármacos , Cristalização , Cães , Espaço Intracelular/metabolismo , Cálculos Renais/metabolismo , Células Madin Darby de Rim Canino , Transporte Proteico/efeitos dos fármacos
19.
J Proteomics ; 144: 11-22, 2016 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-27260493

RESUMO

UNLABELLED: Incidence of kidney stone disease in males is 2- to 4-fold greater than in females. This study aimed to determine effects of testosterone on kidney stone disease using a proteomics approach. MDCK renal tubular cells were treated with or without 20nM testosterone for 7days. Cellular proteins were extracted, resolved by 2-DE, and stained with Deep Purple fluorescence dye (n=5 gels derived from 5 independent samples/group). Spot matching, quantitative intensity analysis, and statistics revealed significant changes in levels of nine protein spots after testosterone treatment. These proteins were then identified by nanoLC-ESI-Qq-TOF MS/MS. Global protein network analysis using STRING software revealed α-enolase as the central node of protein-protein interactions. The increased level of α-enolase was then confirmed by Western blotting analysis, whereas immunofluorescence study revealed the increased α-enolase on cell surface and intracellularly. Functional analysis confirmed the potential role of the increased α-enolase in enhanced calcium oxalate monohydrate (COM) crystal-cell adhesion induced by testosterone. Finally, neutralization of surface α-enolase using anti-α-enolase antibody successfully reduced the enhanced COM crystal-cell adhesion to the basal level. Our data provided in vitro evidence of promoting effect of testosterone on kidney stone disease via enhanced COM crystal-cell adhesion by the increased surface α-enolase. BIOLOGICAL SIGNIFICANCE: The incidence of kidney stone disease in male is 2- to 4-fold greater than in female. One of the possible factors of the male preference is the higher testosterone hormone level. However, precise molecular mechanisms that testosterone plays in kidney stone disease remained unclear. Our present study is the first exploratory investigation on such aspect using a proteomics approach. Our data also provide a novel mechanistic aspect of how testosterone can impact the risk of kidney stone formation (i.e. the discovery that testosterone increases alpha-enolase expression on the surface of renal tubular cells that is responsible, at least in part, for crystal-cell adhesion).


Assuntos
Cálculos Renais/induzido quimicamente , Proteoma/efeitos dos fármacos , Proteômica/métodos , Testosterona/farmacologia , Animais , Oxalato de Cálcio/metabolismo , Adesão Celular , Cães , Cálculos Renais/patologia , Túbulos Renais/química , Túbulos Renais/patologia , Células Madin Darby de Rim Canino , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/metabolismo , Mapas de Interação de Proteínas , Proteoma/análise
20.
Biochim Biophys Acta ; 1864(6): 683-696, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26976750

RESUMO

Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury.


Assuntos
Deficiência de Potássio/metabolismo , Proteínas/metabolismo , Animais , Cães , Eletroforese em Gel Bidimensional , Células Madin Darby de Rim Canino
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