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1.
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).

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

RESUMO

Several lines of evidence have demonstrated anti-fibrotic property of epigallocatechin-3-gallate (EGCG) in many tissues/organs but with unclear mechanisms. This study thus aimed to define cellular mechanisms underlying such protective effect of EGCG. HK-2 renal cells were treated with 5 ng/ml TGF-ß1 for 24 h with/without pretreatment by 5 µM EGCG for 1 h. The cells were then evaluated by morphological examination, immunofluorescence study, semi-quantitative RT-PCR, Western blotting, and atomic force microscopy (AFM). The results showed that TGF-ß1-treated cells underwent epithelial mesenchymal transition (EMT) as evidenced by morphological change into fibroblast-like and increases in spindle index, mesenchymal markers (Snail1 and vimentin), extracellular matrix (fibronectin), cell stiffness (by AFM measurement) and actin stress fibers, whereas the epithelial markers (E-cadherin and ZO-1) were decreased. All of these features were abolished by EGCG pretreatment. Functional studies revealed that the anti-fibrotic property of EGCG was, at least in part, due to de-activation/stabilization of GSK-3ß/ß-catenin/Snail1 (EMT-triggering) signaling pathway that was activated by TGF-ß1 as shown by maintaining phosphorylated GSK-3ß, ß-catenin and Snail1 to their basal levels. Additionally, Nrf2 knockdown by small interfering RNA could abolish the EGCG effect on ß-catenin expression. These data indicate that EGCG attenuates TGF-ß1-induced EMT in renal tubular cells through GSK-3ß/ß-catenin/Snail1 and Nrf2 pathways.


Assuntos
Catequina/análogos & derivados , Transição Epitelial-Mesenquimal , Glicogênio Sintase Quinase 3 beta/metabolismo , Rim/citologia , Transdução de Sinais , Animais , Catequina/farmacologia , Cães , Fibronectinas/metabolismo , Fibrose , Humanos , Rim/patologia , Células Madin Darby de Rim Canino , Microscopia de Força Atômica , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , beta Catenina/metabolismo
5.
J Biol Inorg Chem ; 24(7): 973-983, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31342142

RESUMO

Finasteride (a 5α-reductase inhibitor) has been widely used for treatment of several testosterone-related disorders. However, its beneficial role in kidney stone disease had not been previously investigated. This study thus addressed whether finasteride has any protective effects against testosterone-induced calcium oxalate monohydrate (COM) kidney stone formation. Renal tubular cells were treated with testosterone with/without finasteride for 72 h. Western blotting revealed the increased level of α-enolase (a known COM crystal receptor) in whole-cell lysate, apical membrane, and cytosolic fraction of the testosterone-treated cells. Immunofluorescence staining also showed the increased levels of surface and intracellular α-enolase in the testosterone-treated cells. In addition, testosterone significantly increased the number of adherent COM crystals on the cell surface. All of these effects were completely abolished by finasteride treatment. Interestingly, the secreted proteins from testosterone-treated cells significantly increased COM crystallization, but did not affect crystal growth and aggregation. Again, such promoting effect of testosterone on COM crystallization was completely abolished by finasteride. These data indicate that finasteride effectively protects testosterone-induced kidney stone formation by restoring apical surface expression of α-enolase and COM crystal-cell adhesion to their basal levels. Moreover, finasteride can also neutralize the promoting effect of testosterone on COM crystallization.


Assuntos
Oxalato de Cálcio/química , Adesão Celular/efeitos dos fármacos , Finasterida/farmacologia , Cálculos Renais/induzido quimicamente , Cálculos Renais/prevenção & controle , Testosterona/efeitos adversos , Animais , Cristalização , Cães , Cálculos Renais/patologia , Células Madin Darby de Rim Canino
6.
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
7.
Cell Death Discov ; 4: 53, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29760951

RESUMO

Previous expression study using quantitative proteomics has shown that immune-mediated pathway may not be the main mechanism inducing alopecia areata (AA). Nevertheless, functional impact of such expression data set remained unknown and unexplored. This study thus aimed to define potentially novel mechanisms of the AA pathogenesis by functional investigations of the differentially expressed proteins previously identified from lesional biopsies. From 122 altered proteins, protein-protein interactions network analysis revealed that downregulated heat shock protein 90 (HSP90) and lamin A/C served as the central nodes of protein-protein interactions involving in several crucial biological functions, including cytoskeleton organization, extracellular matrix organization, and tissue development. Interaction between HSP90 and lamin A/C in dermal papilla cells (DPCs) was confirmed by reciprocal immunoprecipitation and immunofluorescence co-staining. Small-interfering RNA (siRNA) targeting to HSP90 (siHSP90) and lamin A/C (siLamin A/C) effectively reduced levels of HSP90 and lamin A/C, respectively and vice versa, comparing to non-transfected and siControl-transfected cells, strengthening their interactive roles in DPCs. Functional investigations revealed that DPCs transfected with siHSP90 and siLamin A/C had defective cell proliferation and growth, prolonged doubling time, cell cycle arrest at G0/G1 phase, and defective self-aggregation formation. Moreover, siHSP90-transfected cells had less spindle index, reduced levels of vimentin (mesenchymal marker) and fibronectin (extracellular matrix), and defective migratory activity. Our data have demonstrated for the first time that HSP90 and lamin A/C physically interact with each other. Moreover, both of them are essential for growth, migration, and self-aggregation of DPCs and can be linked to the disease mechanisms of AA.

8.
Sci Rep ; 8(1): 521, 2018 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-29323127

RESUMO

Alopecia areata (AA) is one of the common hair disorders for which treatment is frequently ineffective and associated with relapsing episodes. Better understanding of disease mechanisms and novel therapeutic targets are thus required. From 10 AA patients, quantitative proteomics using LTQ-Orbitrap-XL mass spectrometer revealed 104 down-regulated, 4 absent, 3 up-regulated and 11 newly present proteins in lesional vs. non-lesional biopsies. Among these, the decreased levels of α-tubulin, vimentin, heat shock protein 70 (HSP70), HSP90, annexin A2 and α-enolase were successfully confirmed by Western blotting. Protein-protein interactions network analysis using STRING tool revealed that the most frequent biological processes/networks of the down-regulated proteins included tissue development, cell differentiation, response to wounding and catabolic process, whereas those for the up-regulated proteins included biological process, metabolic process, cellular transport, cellular component organization and response to stimulus. Interestingly, only 5 increased/newly present proteins were associated with the regulation of immune system, which may not be the predominant pathway in AA pathogenic mechanisms as previously assumed. In summary, we report herein the first proteome dataset of AA demonstrating a number of novel pathways, which can be linked to the disease mechanisms and may lead to discovery of new therapeutic targets for AA.


Assuntos
Alopecia em Áreas/patologia , Proteoma/metabolismo , Adulto , Alopecia em Áreas/metabolismo , Anexina A2/metabolismo , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Proteoma/análise , Proteômica , Espectrometria de Massas em Tandem , Regulação para Cima , Vimentina/metabolismo
9.
FASEB J ; 31(5): 2157-2167, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28196897

RESUMO

Because underlying mechanisms of diabetic nephropathy/tubulopathy remained poorly understood, we aimed to define a key protein involving in hyperglycemia-induced renal tubular dysfunction. All altered renal proteins identified from previous large-scale proteome studies were subjected to global protein network analysis, which revealed heat shock protein 60 (HSP60, also known as HSPD1) as the central node of protein-protein interactions. Functional validation was performed using small interfering RNA (siRNA) to knock down HSP60 (siHSP60). At 48 h after exposure to high glucose (HG) (25 mM), Madin-Darby canine kidney (MDCK) renal tubular cells transfected with controlled siRNA (siControl) had significantly increased level of HSP60 compared to normal glucose (NG) (5.5 mM), whereas siHSP60-transfected cells showed a dramatically decreased HSP60 level. siHSP60 modestly increased intracellular protein aggregates in both NG and HG conditions. Luciferin-luciferase assay showed that HG modestly increased intracellular ATP, and siHSP60 further enhanced such an increase. OxyBlot assay showed significantly increased level of oxidized proteins in HG-treated siControl-transfected cells, whereas siHSP60 caused marked increase of oxidized proteins under the NG condition. However, the siHSP60-induced accumulation of oxidized proteins was abolished by HG. In summary, our data demonstrated that HSP60 plays roles in regulation of intracellular protein aggregation, ATP production, and oxidative stress in renal tubular cells. Its involvement in HG-induced tubular cell dysfunction was most likely via regulation of intracellular ATP production.-Aluksanasuwan, S., Sueksakit, K., Fong-ngern, K., Thongboonkerd, V. Role of HSP60 (HSPD1) in diabetes-induced renal tubular dysfunction: regulation of intracellular protein aggregation, ATP production, and oxidative stress.


Assuntos
Trifosfato de Adenosina/biossíntese , Chaperonina 60/metabolismo , Hiperglicemia/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Citoplasma/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Cães , Túbulos Renais/fisiopatologia , Células Madin Darby de Rim Canino/metabolismo , RNA Interferente Pequeno/genética
10.
Metabolism ; 74: 47-61, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28095989

RESUMO

BACKGROUND: Chronic potassium (K+) deficiency can cause renal damage namely hypokalemic nephropathy with unclear pathogenic mechanisms. In the present study, we investigated expression and functional alterations in renal tubular cells induced by prolonged K+ deficiency. METHODS: MDCK cells were maintained in normal-K+ (CNK) (K+=5.3mmol/L), low-K+ (CLK) (K+=2.5mmol/L), or K+-depleted (CKD) (K+=0mmol/L) medium for 10days (n=5 independent cultures/condition). Differentially expressed proteins were identified by a proteomics approach followed by various functional assays. RESULTS: Proteomic analysis revealed 46 proteins whose levels significantly differed among groups. The proteomic data were confirmed by Western blotting. Gene Ontology (GO) classification and protein network analysis revealed that majority of the altered proteins participated in metabolic process, whereas the rest involved in cellular component organization/biogenesis, cellular process (e.g., cell cycle, regulation of cell death), response to stress, and signal transduction. Interestingly, ATP measurement revealed that intracellular ATP production was increased in CLK and maximum in CKD. Flow cytometry showed cell cycle arrest at S-phase and G2/M-phase in CLK and CKD, respectively, consistent with cell proliferation and growth assays, which showed modest and marked degrees of delayed growth and prolonged doubling time in CLK and CKD, respectively. Cell death quantification also revealed modest and marked degrees of increased cell death in CLK and CKD, respectively. CONCLUSIONS: In conclusion, prolonged K+ deficiency increased intracellular ATP, cell cycle arrest and cell death in renal tubular cells, which might be responsible for mechanisms underlying the development of hypokalemic nephropathy.


Assuntos
Trifosfato de Adenosina/metabolismo , Pontos de Checagem do Ciclo Celular , Morte Celular , Túbulos Renais/patologia , Deficiência de Potássio/patologia , Animais , Proliferação de Células , Cães , Células Madin Darby de Rim Canino , Deficiência de Potássio/metabolismo , Proteômica/métodos
11.
J Biol Inorg Chem ; 21(4): 463-74, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27115409

RESUMO

Adhesion of calcium oxalate monohydrate (COM) crystals on renal tubular epithelial cells is a crucial step in kidney stone formation. Finding potential crystal receptors on the apical membrane of the cells may lead to a novel approach to prevent kidney stone disease. Our previous study identified a large number of crystal-binding proteins on the apical membrane of MDCK cells. However, their functional role as potential crystal receptors had not been validated. The present study aimed to address the potential role of heat shock protein 90 (HSP90) as a COM crystal receptor. The apical membrane was isolated from polarized MDCK cells by the peeling method and recovered proteins were incubated with COM crystals. Western blot analysis confirmed the presence of HSP90 in the apical membrane and the crystal-bound fraction. Immunofluorescence staining without permeabilization and laser-scanning confocal microscopy confirmed the surface HSP90 expression on the apical membrane of the intact cells. Crystal adhesion assay showed that blocking surface HSP90 by specific anti-HSP90 antibody and knockdown of HSP90 by small interfering RNA (siRNA) dramatically reduced crystal binding on the apical surface of MDCK cells (by approximately 1/2 and 2/3, respectively). Additionally, crystal internalization assay revealed the presence of HSP90 on the membrane of endocytic vesicle containing the internalized COM crystal. Moreover, pretreatment of MDCK cells with anti-HSP90 antibody significantly reduced crystal internalization (by approximately 1/3). Taken together, our data indicate that HSP90 serves as a potential receptor for COM crystals on the apical membrane of renal tubular epithelial cells and is involved in endocytosis/internalization of the crystals into the cells.


Assuntos
Oxalato de Cálcio/química , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Túbulos Renais/citologia , Animais , Oxalato de Cálcio/metabolismo , Membrana Celular/química , Cristalização , Cães , Células Epiteliais/citologia , Células Madin Darby de Rim Canino , Propriedades de Superfície
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