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1.
Structure ; 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34492227

RESUMO

R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2-based complexes are identified: R2TP, RUVBL1/2-RPAP3 (R2T), and RUVBL1/2-PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N-terminal domain mediating interactions with substrates whose sequences are primarily enriched for Armadillo repeat domains and other helical-type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.

2.
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.

3.
Expert Rev Proteomics ; 18(8): 643-659, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34517741

RESUMO

INTRODUCTION: With available genomic data and related information, it is becoming possible to better highlight mutations or genomic alterations associated with a particular disease or disorder. The advent of high-throughput sequencing technologies has greatly advanced diagnostics, prognostics, and drug development. AREAS COVERED: Peptidomics and proteogenomics are the two post-genomic technologies that enable the simultaneous study of peptides and proteins/transcripts/genes. Both technologies add a remarkably large amount of data to the pool of information on various peptides associated with gene mutations or genome remodeling. Literature search was performed in the PubMed database and is up to date. EXPERT OPINION: This article lists various techniques used for peptidomic and proteogenomic analyses. It also explains various bioinformatics workflows developed to understand differentially expressed peptides/proteins and their role in disease pathogenesis. Their role in deciphering disease pathways, cancer research, and biomarker discovery using biofluids is highlighted. Finally, the challenges and future requirements to overcome the current limitations for their effective clinical use are also discussed.

4.
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
5.
Expert Rev Proteomics ; 18(7): 527-556, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34343059

RESUMO

INTRODUCTION: Peptidomics is an emerging field of omics sciences using advanced isolation, analysis, and computational techniques that enable qualitative and quantitative analyses of various peptides in biological samples. Peptides can act as useful biomarkers and as therapeutic molecules for diseases. AREAS COVERED: The use of therapeutic peptides can be predicted quickly and efficiently using data-driven computational methods, particularly artificial intelligence (AI) approach. Various AI approaches are useful for peptide-based drug discovery, such as support vector machine, random forest, extremely randomized trees, and other more recently developed deep learning methods. AI methods are relatively new to the development of peptide-based therapies, but these techniques already become essential tools in protein science by dissecting novel therapeutic peptides and their functions (Figure 1). EXPERT OPINION: Researchers have shown that AI models can facilitate the development of peptidomics and selective peptide therapies in the field of peptide science. Biopeptide prediction is important for the discovery and development of successful peptide-based drugs. Due to their ability to predict therapeutic roles based on sequence details, many AI-dependent prediction tools have been developed (Figure 1).

6.
Int J Med Sci ; 18(14): 3271-3279, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34400896

RESUMO

Several artificial urine (AU) formulas have been developed to mimic the normal urine. Most of them are protein-free, particularly when secreted proteins (secretome) is to be analyzed. However, the normal urine actually contains a tiny amount of proteins. We hypothesized that urinary proteins at physiologic level play a role in preservation of renal cell biology and function. This study evaluated the effects from supplementation of 0-10% fetal bovine serum (FBS) into the well-established AU-Siriraj protocol on MDCK renal tubular cells. Time to deformation (TD) was reduced by both native urine and AU-Siriraj without/with FBS compared with complete culture medium (control). Among the native urine and AU-Siriraj without/with FBS, the cells in AU-Siriraj+2.5% FBS had the longest TD. Supplementation of FBS increased cell death in a dose-dependent manner (but still <10%). Transepithelial electrical resistance (TER) of the polarized cells in the native urine was comparable to the control, whereas that of the cells in AU-Siriraj+2.5% FBS had the highest TER. These data indicate that supplementation of 2.5% FBS into AU-Siriraj can prolong time to deformation and enhance polarization of renal tubular cells. Therefore, AU-Siriraj+2.5% FBS is highly recommended for in vitro study of cell biology and function (when secretome is not subjected to analysis).

7.
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.

8.
Biomed Pharmacother ; 141: 111903, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34328112

RESUMO

Diosmin is a natural flavone glycoside (bioflavonoid) found in fruits and plants with several pharmacological activities. It has been widely used as a dietary supplement or therapeutic agent in various diseases/disorders. Although recommended, evidence of its protective mechanisms against kidney stone disease (nephrolithiasis/urolithiasis), especially calcium oxalate (CaOx) monohydrate (COM) that is the most common type, remained unclear. In this study, we thus systematically evaluated the effects of diosmin (at 2.5-160 nM) on various stages of kidney stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion, internalization into renal tubular cells and invasion through extracellular matrix (ECM). The results showed that diosmin had dose-dependent modulatory effects on all the mentioned COM kidney stone processes. Diosmin significantly increased COM crystal number and mass during crystallization, but reduced crystal size and growth. While diosmin promoted crystal aggregation, it inhibited crystal-cell adhesion and internalization into renal tubular cells. Finally, diosmin promoted crystal invasion through the ECM. Our data provide evidence demonstrating both inhibiting and promoting effects of diosmin on COM kidney stone formation processes. Based on these dual modulatory activities of diosmin, its anti-urolithiasis role is doubtful and cautions should be made for its use in kidney stone disease.

9.
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.

10.
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
11.
Biomed Pharmacother ; 141: 111837, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34175818

RESUMO

A recent expression proteomics study has reported changes in cellular proteome (set of proteins) of human endothelial cells (ECs) induced by caffeine and epigallocatechin-3-gallate (EGCG), the most abundant bioactive compounds in coffee and green tea, respectively. Although both common and differential changes were highlighted by bioinformatics prediction, no experimental validation was performed. Herein, we reanalyzed these proteome datasets and performed protein-protein interactions network analysis followed by functional investigations using various assays to address the relevance of such proteome changes in human ECs functions. Protein-protein interactions network analysis revealed actin-crosslink formation, ubiquitin-proteasome activity and glycolysis as the three main networks among those significantly altered proteins induced by caffeine and EGCG. The experimental data showed predominant increases of actin-crosslink formation, ubiquitin-proteasome activity, and glycolysis (as reflected by increased F-actin and ß-actin, declined ubiquitinated proteins and increased intracellular ATP, respectively) in the EGCG-treated cells. Investigations on angiogenesis features revealed that EGCG predominantly reduced ECs proliferation, migration/invasion, endothelial tube formation (as determined by numbers of nodes/junctions and meshes), barrier function (as determined by levels of VE-cadherin, zonula occludens-1 (ZO-1) and transendothelial resistance (TER)), and angiopoietin-2 secretion. However, both caffeine and EGCG had no effects on matrix metalloproteinase-2 (MMP-2) secretion. These data indicate that EGCG exhibits more potent effects on human ECs functions to induce actin-crosslink, ubiquitin-proteasome activity and glycolysis, and to suppress angiogenesis processes that commonly occur in various diseases, particularly cancers.

12.
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
13.
J Extracell Vesicles ; 10(7): e12093, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34035881

RESUMO

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

14.
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
15.
Theranostics ; 11(9): 4436-4451, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754070

RESUMO

Inflammasome is a complex of multiple proteins found in cytoplasm of the cells activated by infectious and/or non-infectious stimuli. This complex involves caspase-1 activation, leading to unconventional secretion of interleukin-1ß (IL-1ß) and IL-18 and inflammatory cascade. Exosome is the nanoscale membrane-bound extracellular vesicle that plays significant roles in intercellular communications by carrying bioactive molecules, e.g., proteins, RNAs, microRNAs (miRNAs), DNAs, from one cell to the others. In this review, we provide the update information on the crosstalk between exosome and inflammasome and their roles in inflammatory responses. The effects of inflammasome activation on exosomal secretion are summarized. On the other hand, the (dual) effects of exosomes on inhibiting and promoting inflammasome activation are discussed. Finally, perspectives on therapeutic roles of exosomes in human diseases and future direction of the research on exosome-inflammasome crosstalk are provided.


Assuntos
Exossomos/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Animais , Comunicação Celular/fisiologia , Humanos
16.
Cell Mol Life Sci ; 78(7): 3265-3283, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33507324

RESUMO

Exosomes are involved in a wide variety of biochemical processes in human body homeostasis. Exosomes also provide important information regarding communications among several organ systems. Additionally, they can serve as molecular vehicles to deliver drugs. Therefore, exosomes have received wide attention in current biomedical research for unraveling pathogenic mechanisms of diseases, searching for novel biomarkers, and discovering new drugs. This paper reviews and discusses the significance of urinary exosomes for a better understanding of human disease pathophysiology and their potential use as therapeutic targets. Isolation methods of exosomes and the latest technological advances are also discussed. Furthermore, novel urinary exosomal biomarkers are highlighted with special emphasis on their clinical applicability (particularly sensitivity, specificity, reliability, and other aspects). Finally, future trends for this field are analyzed and our perspectives are provided.


Assuntos
Biomarcadores/urina , Nefropatias Diabéticas/diagnóstico , Exossomos/metabolismo , Cardiopatias/diagnóstico , Nefropatias/diagnóstico , Nefrite Lúpica/diagnóstico , Neoplasias/diagnóstico , Animais , Nefropatias Diabéticas/urina , Cardiopatias/urina , Humanos , Nefropatias/urina , Nefrite Lúpica/urina , Neoplasias/urina
17.
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.

18.
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
19.
Chem Biol Interact ; 331: 109270, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32991862

RESUMO

Hyperuricosuria is associated with kidney stone disease, especially uric acid (UA) and calcium oxalate (CaOx) types. Nevertheless, detailed mechanisms of hyperuricosuria-induced kidney stone formation remained unclear. This study examined changes in cellular proteome and function of renal tubular cells after treatment with high-dose UA for 48-h. Quantitative proteomics using 2-DE followed by nanoLC-ESI-ETD MS/MS tandem mass spectrometry revealed significant changes in levels of 22 proteins in the UA-treated cells. These proteomic data could be confirmed by Western blotting. Functional assays revealed an increase in intracellular ATP level and enhancement of tissue repairing capability in the UA-treated cells. Interestingly, levels of HSP70 and HSP90 (the known receptors for CaOx crystals) were increased in apical membranes of the UA-treated cells. CaOx crystal-cell adhesion assay revealed significant increase in CaOx-binding capability of the UA-treated cells, whereas neutralization of the surface HSP70 and/or HSP90 using their specific monoclonal antibodies caused significant reduction in such binding capability. These findings highlighted changes in renal tubular cells in response to high-dose UA that may, at least in part, explain the pathogenic mechanisms of hyperuricosuria-induced mixed kidney stone disease.


Assuntos
Trifosfato de Adenosina/metabolismo , Oxalato de Cálcio/metabolismo , Proteoma/efeitos dos fármacos , Ácido Úrico/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Oxalato de Cálcio/química , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cristalização , Cães , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/metabolismo , Cálculos Renais/etiologia , Cálculos Renais/patologia , Células Madin Darby de Rim Canino/citologia , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/metabolismo , Mapas de Interação de Proteínas , Proteoma/análise , Espectrometria de Massas em Tandem , Ácido Úrico/urina
20.
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
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