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1.
Mol Cell Proteomics ; : 100151, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34562649

RESUMO

The incidence/prevalence of kidney stone disease has been increasing around the globe but its pathogenic mechanisms remained unclear. We evaluated effects of oxidative modifications of urinary proteins on calcium oxalate (CaOx) stone formation processes. Urinary proteins derived from 20 healthy individuals were modified by performic oxidation and the presence of oxidatively modified urinary proteins was verified, quantified and characterized by Oxyblot assay and tandem mass spectrometry (nanoLC-ESI-LTQ-Orbitrap-MS/MS). Subsequently, activities of oxidatively modified urinary proteins on CaOx stone formation processes were examined. Oxyblot assay confirmed the marked increase in protein oxidation level in the modified urine. NanoLC-ESI-LTQ-Orbitrap-MS/MS identified a total of 193 and 220 urinary proteins in non-modified and modified urine samples, respectively. Among these, there were 1,121 and 5,297 unambiguous oxidatively modified peptides representing 42 and 136 oxidatively modified proteins in the non-modified and modified urine samples, respectively. Crystal assays revealed that oxidatively modified urinary proteins significantly promoted CaOx crystallization, crystal growth and aggregation. By contrast, the non-modified urinary proteins had inhibitory activities. This is the first direct evidence demonstrating that oxidative modifications of urinary proteins increase the risk of kidney stone disease by switching their modulatory activities from inhibiting to promoting CaOx crystallization, crystal growth and aggregation.

2.
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
3.
Front Physiol ; 11: 566506, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33192563

RESUMO

Mitochondrion is a pivotal intracellular organelle that plays crucial roles in regulation of energy production, oxidative stress, calcium homeostasis, and apoptosis. Kidney stone disease (nephrolithiasis/urolithiasis), particularly calcium oxalate (CaOx; the most common type), has been shown to be associated with oxidative stress and tissue inflammation/injury. Recent evidence has demonstrated the involvement of mitochondrial dysfunction in CaOx crystal retention and aggregation as well as Randall's plaque formation, all of which are the essential mechanisms for kidney stone formation. This review highlights the important roles of mitochondria in renal cell functions and provides the data obtained from previous investigations of mitochondria related to kidney stone disease. In addition, mechanisms for the involvement of mitochondrial dysfunction in the pathophysiology of kidney stone disease are summarized. Finally, future perspectives on the novel approach to prevent kidney stone formation by mitochondrial preservation are discussed.

4.
Sci Rep ; 10(1): 5843, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32246012

RESUMO

Mitochondrial dysfunction has been thought to play roles in the pathogenesis of diabetic nephropathy (DN). However, precise mechanisms underlying mitochondrial dysfunction in DN remained unclear. Herein, mitochondria were isolated from renal tubular cells after exposure to normal glucose (5.5 mM glucose), high glucose (25 mM glucose), or osmotic control (5.5 mM glucose + 19.5 mM mannitol) for 96 h. Comparative proteomic analysis revealed six differentially expressed proteins among groups that were subsequently identified by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and confirmed by Western blotting. Several various types of post-translational modifications (PTMs) were identified in all of these identified proteins. Interestingly, phosphorylation and oxidation were most abundant in mitochondrial proteins whose levels were exclusively increased in high glucose condition. The high glucose-induced increases in phosphorylation and oxidation of mitochondrial proteins were successfully confirmed by various assays including MS/MS analyses. Moreover, high glucose also increased levels of phosphorylated ezrin, intracellular ATP and ROS, all of which could be abolished by a p38 MAPK inhibitor (SB239063), implicating a role of p38 MAPK-mediated phosphorylation in high glucose-induced mitochondrial dysfunction. These data indicate that phosphorylation and oxidation of mitochondrial proteins are, at least in part, involved in mitochondrial dysfunction in renal tubular cells during DN.


Assuntos
Glucose/farmacologia , Túbulos Renais/efeitos dos fármacos , Proteínas Mitocondriais/efeitos dos fármacos , Animais , Western Blotting , Cães , Túbulos Renais/metabolismo , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/metabolismo , Espectrometria de Massas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredução/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteômica/métodos
5.
J Biol Inorg Chem ; 24(2): 235-246, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30701361

RESUMO

Fibronectin, an extracellular matrix (ECM) protein, has been thought to be involved in pathogenic mechanisms of kidney stone disease, especially calcium oxalate (CaOx) type. Nevertheless, its precise roles in modulation of CaOx crystal remained unclear. We thus performed a systematic evaluation of effects of fibronectin on CaOx monohydrate (COM) crystal (the major causative chemical crystal in kidney stone formation) in various stages of kidney stone pathogenesis, including crystallization, crystal growth, aggregation, adhesion onto renal tubular cells, and invasion through ECM in renal interstitium. The data showed that fibronectin significantly decreased crystallization, growth and adhesive capability of COM crystals in a dose-dependent manner. In contrast, COM crystal aggregation and invasion through ECM migration chamber were significantly enhanced by fibronectin in a dose-dependent fashion. Sequence analysis revealed three calcium-binding and six oxalate-binding domains in fibronectin. Immunofluorescence study confirmed binding of fibronectin to COM crystals. Additionally, calcium- and oxalate-affinity assays confirmed depletion of both calcium and oxalate ions after incubation with fibronectin. Moreover, calcium-saturated and oxalate-saturated forms of fibronectin markedly reduced the modulatory activities of fibronectin on COM crystallization, crystal growth, aggregation, and adhesion onto the cells. These data strongly indicate the dual functions of fibronectin, which serves as an inhibitor for COM crystallization, crystal growth and adhesion onto renal tubular cells, but on the other hand, acts as a promoter for COM crystal aggregation and invasion through ECM. Finally, its COM crystal modulatory activities are most likely mediated through binding with calcium and oxalate ions on the crystals and in their environment.


Assuntos
Oxalato de Cálcio/química , Matriz Extracelular/química , Fibronectinas/química , Túbulos Renais/química , Animais , Adesão Celular , Cristalização , Cães , Humanos , Túbulos Renais/citologia , Células Madin Darby de Rim Canino
6.
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
7.
Urolithiasis ; 46(3): 257-264, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28585182

RESUMO

Our previous study has shown that lime powder (LP) had an inhibitory effect against calcium oxalate stone formation. However, the precise mechanisms underlying such beneficial effect remained unclear. Our present study thus aimed to address the effect of LP on excretory level and compositions of urinary proteins using a proteomics approach. From a total of 80 calcium oxalate stone formers recruited into our 2-year randomized clinical trial of LP effect, 10 patients with comparable age and clinical parameters were selected for this proteomic study. 24-h urine specimens were collected from all subjects, at baseline (before) and after LP treatment for 6 months, and then subjected to quantitative proteomics analysis and subsequent validation by ELISA. Total urinary protein excretion was significantly decreased by LP treatment, but unaffected by placebo. Nanoflow liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) followed by quantitative analysis revealed 17 proteins whose levels were significantly altered (16 decreased and 1 increased) exclusively by LP treatment. Among these, the decrease of transferrin and increase of uromodulin were validated by ELISA. Moreover, there was a significant correlation between microalbuminuria and urinary transferrin level by Pearson's correlation test. In summary, LP treatment caused significant reduction in total urinary protein excretion and changes in urinary protein compositions that could be linked to stone inhibitory effects and might be relevant mechanisms responsible for the beneficial effects of LP to prevent kidney stone formation and recurrence.


Assuntos
Albuminúria/tratamento farmacológico , Compostos de Cálcio/farmacologia , Cálculos Renais/tratamento farmacológico , Óxidos/farmacologia , Eliminação Renal/efeitos dos fármacos , Transferrina/urina , Uromodulina/urina , Adulto , Albuminúria/urina , Compostos de Cálcio/uso terapêutico , Oxalato de Cálcio/química , Oxalato de Cálcio/urina , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Cálculos Renais/urina , Masculino , Pessoa de Meia-Idade , Óxidos/uso terapêutico , Pós , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Transferrina/metabolismo , Uromodulina/metabolismo
8.
Front Chem ; 5: 113, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29270403

RESUMO

Crystal aggregation is one of the most crucial steps in kidney stone pathogenesis. However, previous studies of crystal aggregation were rarely done and quantitative analysis of aggregation degree was handicapped by a lack of the standard measurement. We thus performed an in vitro assay to generate aggregation of calcium oxalate monohydrate (COM) crystals with various concentrations (25-800 µg/ml) in saturated aggregation buffer. The crystal aggregates were analyzed by microscopic examination, UV-visible spectrophotometry, and GraphPad Prism6 software to define a total of 12 aggregation indices (including number of aggregates, aggregated mass index, optical density, aggregation coefficient, span, number of aggregates at plateau time-point, aggregated area index, aggregated diameter index, aggregated symmetry index, time constant, half-life, and rate constant). The data showed linear correlation between crystal concentration and almost all of these indices, except only for rate constant. Among these, number of aggregates provided the greatest regression coefficient (r = 0.997; p < 0.001), whereas the equally second rank included aggregated mass index and optical density (r = 0.993; p < 0.001 and r = -0.993; p < 0.001, respectively) and the equally forth were aggregation coefficient and span (r = 0.991; p < 0.001 for both). These five indices are thus recommended as the most appropriate indices for quantitative analysis of COM crystal aggregation in vitro.

9.
Chem Biol Interact ; 246: 30-5, 2016 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-26748311

RESUMO

Interaction between calcium oxalate crystals and renal tubular cells has been recognized as one of the key mechanisms for kidney stone formation. While crystal adhesion and internalization have been extensively investigated, subsequent phenomena (i.e. crystal degradation and dissolution) remained poorly understood. To explore these mechanisms, we used fluorescein isothiocyanate (FITC)-labelled calcium oxalate monohydrate (COM) crystals (1000 µg/ml of crystals/culture medium) to confirm crystal internalization into MDCK (Type II) renal tubular cells after exposure to the crystals for 1 h and to trace the internalized crystals. Crystal size, intracellular and extracellular fluorescence levels were measured using a spectrofluorometer for up to 48 h after crystal internalization. Moreover, markers for early endosome (Rab5), late endosome (Rab7) and lysosome (LAMP-2) were examined by laser-scanning confocal microscopy. Fluorescence imaging and flow cytometry confirmed that FITC-labelled COM crystals were internalized into MDCK cells (14.83 ± 0.85%). The data also revealed a reduction of crystal size in a time-dependent manner. In concordance, intracellular and extracellular fluorescence levels were decreased and increased, respectively, indicating crystal degradation/dissolution inside the cells and the degraded products were eliminated extracellularly. Moreover, Rab5 and Rab7 were both up-regulated and were also associated with the up-regulated LAMP-2 to form large endolysosomes in the COM-treated cells at 16-h after crystal internalization. We demonstrate herein, for the first time, that COM crystals could be degraded/dissolved by endolysosomes inside renal tubular cells. These findings will be helpful to better understand the crystal fate and protective mechanism against kidney stone formation.


Assuntos
Oxalato de Cálcio/química , Oxalato de Cálcio/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Lisossomos/metabolismo , Animais , Transporte Biológico , Biomarcadores/metabolismo , Cristalização , Cães , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Células Madin Darby de Rim Canino , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
10.
PLoS One ; 9(9): e106779, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25215595

RESUMO

Leber's Hereditary Optic Neuropathy (LHON) is one of the commonest mitochondrial diseases. It causes total blindness, and predominantly affects young males. For the disease to develop, it is necessary for an individual to carry one of the primary mtDNA mutations 11778G>A, 14484T>C or 3460G>A. However these mutations are not sufficient to cause disease, and they do not explain the characteristic features of LHON such as the higher prevalence in males, incomplete penetrance, and relatively later age of onset. In order to explore the roles of nuclear encoded mitochondrial proteins in development of LHON, we applied a proteomic approach to samples from affected and unaffected individuals from 3 pedigrees and from 5 unrelated controls. Two-dimensional electrophoresis followed by MS/MS analysis in the mitochondrial lysate identified 17 proteins which were differentially expressed between LHON cases and unrelated controls, and 24 proteins which were differentially expressed between unaffected relatives and unrelated controls. The proteomic data were successfully validated by western blot analysis of 3 selected proteins. All of the proteins identified in the study were mitochondrial proteins and most of them were down regulated in 11778G>A mutant fibroblasts. These proteins included: subunits of OXPHOS enzyme complexes, proteins involved in intermediary metabolic processes, nucleoid related proteins, chaperones, cristae remodelling proteins and an anti-oxidant enzyme. The protein profiles of both the affected and unaffected 11778G>A carriers shared many features which differed from those of unrelated control group, revealing similar proteomic responses to 11778G>A mutation in both affected and unaffected individuals. Differentially expressed proteins revealed two broad groups: a cluster of bioenergetic pathway proteins and a cluster involved in protein quality control system. Defects in these systems are likely to impede the function of retinal ganglion cells, and may lead to the development of LHON in synergy with the primary mtDNA mutation.


Assuntos
Regulação para Baixo , Metabolismo Energético , Fibroblastos/patologia , Proteínas Mitocondriais/metabolismo , Mutação/genética , Atrofia Óptica Hereditária de Leber/metabolismo , Proteoma/metabolismo , Adulto , Biópsia , Western Blotting , Estudos de Casos e Controles , Bases de Dados de Proteínas , Família , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteômica , Reprodutibilidade dos Testes , Frações Subcelulares/metabolismo , Tailândia , Adulto Jovem
11.
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
12.
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
13.
Biochem Biophys Res Commun ; 424(3): 629-34, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22796524

RESUMO

Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) to resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed "L-x(3,5)-R-x(2)-[AGILPV]" as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone modulators.


Assuntos
Cálculos Renais/metabolismo , Rim/metabolismo , Oxalatos/metabolismo , Proteínas/isolamento & purificação , Proteínas/metabolismo , Animais , Cromatografia de Afinidade/métodos , Cálculos Renais/química , Espectrometria de Massas , Oxalatos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Suínos
14.
Eur J Pharmacol ; 689(1-3): 219-25, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22713548

RESUMO

Dissolution therapy of calcium oxalate monohydrate (COM) kidney stone disease has not yet been implemented due to a lack of well characterized COM dissolution agents. The present study therefore aimed to identify potential COM crystal dissolution compounds. COM crystals were treated with deionized water (negative control), 5 mM EDTA (positive control), 5 mM sodium citrate, or 5mM sodium phosphate. COM crystal dissolution activities of these compounds were evaluated by phase-contrast and video-assisted microscopic examinations, semi-quantitative analysis of crystal size, number and total mass, and spectrophotometric oxalate-dissolution assay. In addition, effects of these compounds on detachment of COM crystals, which adhered tightly onto renal tubular cell surface, were also investigated. The results showed that citrate, not phosphate, had a significant dissolution effect on COM crystals as demonstrated by significant reduction of crystal size (approximately 37% decrease), crystal number (approximately 53% decrease) and total crystal mass (approximately 72% decrease) compared to blank and negative controls. Spectrophotometric oxalate-dissolution assay successfully confirmed the COM crystal dissolution property of citrate. Moreover, citrate could detach up to 85% of the adherent COM crystals from renal tubular cell surface. These data indicate that citrate is better than phosphate for dissolution and detachment of COM crystals.


Assuntos
Oxalato de Cálcio/química , Citratos/química , Túbulos Renais/química , Fosfatos/química , Animais , Citratos/fisiologia , Cristalização , Cães , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Células Madin Darby de Rim Canino , Fosfatos/fisiologia , Citrato de Sódio , Solubilidade
15.
J Proteomics ; 76 Spec No.: 239-50, 2012 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-22705320

RESUMO

Aggregatability and oxidative damage of red blood cells (RBCs), platelet activation and increased amount of blood cells-derived microparticles (MPs) are thought to be the etiologies for the thrombotic risk in thalassemia, but with unclear mechanisms. Here we report cellular origins and increases in number, oxidative stress status, and procoagulant activity, as well as altered proteome of MPs isolated from ß-thal/HbE patients. Flow cytometric analysis revealed that ß-thal/HbE patients had significantly higher levels of phosphatidylserine (PS)-bearing MPs in platelet-free plasma (PFP) as compared to normal subjects. The high levels of MPs correlated with not only the increased procoagulant activity but also the increased platelet counts. Additionally, these PS-bearing MPs were originated mostly from platelets and RBCs, both of which had increased levels of reactive oxygen species. Proteome analysis of MPs by 2-DE followed by Q-TOF MS and MS/MS analyses identified 29 proteins with significantly altered levels in MPs derived from ß-thal/HbE patients (e.g. the increased levels of peroxiredoxin 6, apolipoprotein E, cyclophilin A and heat shock protein 90). These findings suggest that the oxidative damage in platelets and RBCs potentially induces production of MPs with altered proteome that may, in turn, facilitate thromboembolic complications, which are commonly found in thalassemic patients. This article is part of a Special Issue entitled: Integrated omics.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Hemoglobina E , Hemoglobinúria/sangue , Plasma/metabolismo , Proteoma/metabolismo , Talassemia beta/sangue , Adulto , Proteínas Sanguíneas/metabolismo , Feminino , Humanos , Masculino , Proteômica/métodos , Tromboembolia/sangue
16.
J Proteome Res ; 11(6): 3269-80, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22512661

RESUMO

Calcium oxalate monohydrate (COM) crystals, the major crystalline compound in kidney stones, have been suggested to induce oxidative stress by overproduction of reactive oxygen species (ROS) and renal tubular cell injury. Our present study aimed to examine changes in mitochondrial proteome in distal renal tubular cells induced by COM crystals (100 µg of crystals/mL of culture medium). Adhesion and internalization of COM crystals by MDCK cells were examined by fluorescent and laser-scanning confocal microscopy. Moreover, the internalized COM crystals were quantified by flow cytometry. Thereafter, mitochondria were isolated from controlled and COM-treated cells, and mitochondrial proteins were subjected to 2-DE-based comparative proteomic analysis, which revealed 15 differentially expressed proteins. These significantly altered proteins were identified by Q-TOF MS and MS/MS analyses, including those involved in several biological processes, e.g., cellular structure, carbohydrate metabolism, and energy metabolism. 2-D Western blot analysis confirmed the increase of ezrin and decrease of ß-actin. Global protein network analysis was then performed to obtain additional functional significance of the identified proteins and to guide for subsequent functional analysis. The results implicated that the altered proteins were involved in energy production and might contribute to mitochondrial dysfunction. The loss of ROS regulation by mitochondria was finally confirmed by OxyBlot assay, which demonstrated markedly increased levels of the oxidatively modified mitochondrial proteins in the COM-treated cells in a dose-dependent manner. Our data may lead to a better understanding of molecular mechanisms of mitochondrial dysfunction underlying the overt oxidative stress induced by COM crystals in kidney stone disease.


Assuntos
Oxalato de Cálcio/farmacologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Oxalato de Cálcio/metabolismo , Adesão Celular , Cristalização , Cães , Túbulos Renais/citologia , Células Madin Darby de Rim Canino , Mitocôndrias/efeitos dos fármacos , Oxirredução , Estresse Oxidativo , Proteoma/metabolismo
17.
Anal Biochem ; 394(2): 249-58, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19622339

RESUMO

One of the most crucial steps in mitochondrial isolation is disruption of intact cells to denude intracellular organelles, but the yield and purity of different disruption protocols have not been well addressed. In the present study, MDCK cells were disrupted by mechanical (sonication and homogenization), physical (repeated freeze/thaw cycles and hypoosmotic burst), and chemical (using Triton X-100, NP-40, or CHAPS) methods. Efficacy of cell disruption was evaluated by trypan blue staining and mitochondria were subsequently isolated by standardized differential centrifugation. The yield of isolation was also determined by measuring protein concentrations, whereas the purity was examined by Janus green B staining, Western blot analyses of markers for mitochondria (COX-4) and other subcellular organelles/locales (i.e., nucleus, cytoplasm, endoplasmic reticulum, and lysosome), transmission electron microscopy, two-dimensional electrophoresis, and Q-TOF MS and/or MS/MS analyses. Our data demonstrated that sonication is the method of choice for disruption of cells prior to mitochondrial isolation for proteome analysis.


Assuntos
Fracionamento Celular/métodos , Mitocôndrias/química , Proteômica/métodos , Animais , Linhagem Celular , Centrifugação com Gradiente de Concentração/métodos , Cães , Congelamento , Rim/citologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/metabolismo , Octoxinol/metabolismo , Concentração Osmolar , Polietilenoglicóis/metabolismo , Proteoma/análise , Proteoma/metabolismo , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Ultrassom/efeitos adversos
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