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Dynamic transdifferentiation of epithelial cells from epithelial-mesenchymal transition (EMT) to its reverse process, mesenchymal-epithelial transition (MET), has gained wide attention for management of cancers and tissue fibrosis. In this study, we addressed beneficial effects of epigallocatechin-3-gallate (EGCG) on EMT-MET reversion using an in vitro EMT model by overexpressing SNAI1 gene encoding Snail1, an EMT-inducing transcription factor, into renal tubular epithelial cells (pcDNA6.2-SNAI1 cells). The cells transfected with empty vector (pcDNA6.2 cells) served as the control. Titrating EGCG concentrations revealed its optimal dose at 25 µM for 24-h, which was used throughout. pcDNA6.2-SNAI1 cells had increased spindle index and typical morphology of EMT, whereas EGCG could restore the normal index and morphology. Increased nuclear Snail1 and β-catenin; increased cytoplasmic Snail1, p-GSK-3β, vimentin, fibronectin and F-actin; and decreased occludin, ZO-1, transepithelial resistance (TER), E-cadherin and cell cluster size were observed in the pcDNA6.2-SNAI1 cells. These pcDNA6.2-SNAI1 cells also had increased migrating activity associated with increased forward but decreased non-forward α-tubulin filaments, G

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+ = 5.3 mmol/L), low-K+ (CLK) (K+ = 2.5 mmol/L), or K+-depleted (CKD) (K+ = 0 mmol/L) medium for 10 days (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.

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.

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 20 nM testosterone for 7 days. 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).

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.

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.

Hyperoxaluria is one of etiologic factors of calcium oxalate kidney stone disease. However, response of renal tubular cells to high-oxalate environment remained largely unknown. We applied a gel-based proteomics approach to characterize changes in cellular proteome of MDCK cells induced by 10mM sodium oxalate. A total of 14 proteins were detected as differentially expressed proteins. The oxalate-induced up-regulation of alpha-enolase in whole cell lysate was confirmed by 2-D Western blot analysis. Interaction network analysis revealed that cellular adaptive response under high-oxalate condition involved stress response, energy production, metabolism and transcriptional regulation. Down-regulation of RhoA, which was predicted to be associated with the identified proteins, was confirmed by immunoblotting. In addition, the up-regulation of alpha-enolase on apical surface of renal tubular epithelial cells was also confirmed by immunoblotting of the isolated apical membranes and immunofluorescence study. Interestingly, blockage of alpha-enolase expressed on the cell surface by antibody neutralization significantly reduced the number of calcium oxalate monohydrate (COM) crystals adhered on the cells. These results strongly suggest that surface alpha-enolase plays an important role as the enhancer of COM crystal binding. The increase of alpha-enolase expressed on the cell surface may aggravate kidney stone formation in patients with hyperoxaluria.

To search for a strategy to prevent kidney stone formation/recurrence, this study addressed the role of α-enolase on apical membrane of renal tubular cells in mediating calcium oxalate monohydrate (COM) crystal adhesion. Its presence on apical membrane and in COM crystal-bound fraction was confirmed by Western blotting and immunofluorescence staining. Pretreating MDCK cells with anti-α-enolase antibody, not isotype-controlled IgG, dramatically reduced cell-crystal adhesion. Immunofluorescence staining also confirmed the direct binding of purified α-enolase to COM crystals at {121} > {100} > {010} crystal faces. Coating COM crystals with urinary proteins diminished the crystal binding capacity to cells and purified α-enolase. Moreover, α-enolase selectively bound to COM, not other crystals. Chemico-protein interactions analysis revealed that α-enolase interacted directly with Ca2+ and Mg2+. Incubating the cells with Mg2+ prior to cell-crystal adhesion assay significantly reduced crystal binding on the cell surface, whereas preincubation with EDTA, a divalent cation chelator, completely abolished Mg2+ effect, indicating that COM and Mg2+ competitively bind to α-enolase. Taken together, we successfully confirmed the role of α-enolase as a COM crystal receptor to mediate COM crystal adhesion at apical membrane of renal tubular cells. It may also serve as a target for stone prevention by blocking cell-crystal adhesion and stone nidus formation.

Chronic potassium (KMDCK cells were maintained in normal-KProteomic 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.In conclusion, prolonged K

Pathogenic mechanisms of kidney stone disease remained unclear. This study investigated its initial cellular/molecular mechanisms when calcium oxalate monohydrate (COM) crystal adhered to renal tubular cells. Transmission electron microscopy revealed decreased length and density of microvilli, whereas Western blot analysis showed that whole-cell ezrin (a microvillus-stabilizing protein), not β-actin, was decreased in COM-treated cells. Immunofluorescence staining, followed by laser-scanning confocal microscopy and subcellular fractionations, revealed decreases in both ezrin and F-/β-actin at apical membrane. Cytoskeletal extraction by Triton X-100 showed reduced cytoskeleton-associated ezrin, consistent with colocalization data of ezrin/F-actin. Thr

A previous study reported that lamin A/C (LMNA) expression was increased in renal tubular cells adhered with calcium oxalate monohydrate (COM) crystals; however, its functional significance in kidney stone disease remained unknown. In the present study, increased levels of LMNA and its partner, nesprin-1 (SYNE1), in Madin-Darby canine kidney cells upon COM crystal adhesion were confirmed by Western blotting and immunofluorescence staining. LMNA was then knocked down by small interfering RNA. Immunofluorescence staining confirmed the efficiency of small interfering RNA of LMNA (si-LMNA), which also reduced expression of its partner, SYNE1. Scratch assay and total cell count revealed defects in tissue repair and cell proliferation, respectively, whereas cell death quantitation showed no cytotoxicity in si-LMNA-transfected cells. Crystal-binding assay highlighted the role of LMNA in crystal adhesion, whereas protein network analysis revealed interactions between LMNA and potential COM crystal receptors. Their associations were confirmed by reduced levels of these proteins, including vimentin, tubulin, enolase, S100, and annexin A2, in si-LMNA-transfected cells. These data have demonstrated for the first time, to our knowledge, that LMNA in renal tubular cells is important for tissue repair, cell proliferation, and COM crystal adhesion and is associated with potential COM crystal receptors. Therefore, LMNA may serve as a potential target for prevention of kidney stone disease and its recurrence.-Pongsakul, N., Vinaiphat, A., Chanchaem, P., Fong-ngern, K., Thongboonkerd, V. Lamin A/C in renal tubular cells is important for tissue repair, cell proliferation, and calcium oxalate crystal adhesion, and is associated with potential crystal receptors.

Hypercalciuria is associated with kidney stone formation and impaired renal function. However, responses of renal tubular cells upon exposure to high-calcium environment remain largely unknown. We thus performed a proteomic analysis of altered proteins in renal tubular cells induced by high-calcium and evaluated functional significance of these changes. MDCK cells were maintained with or without 20 mM CaCl(2) for 72 h. Cellular proteins were then analyzed by two-dimensional electrophoresis (2-DE) (n = 5 gels derived from 5 independent culture flasks per group). Spot matching and quantitative intensity analysis revealed 20 protein spots (from a total of 700) that were differentially expressed between the two groups. These altered proteins were then identified by Q-TOF-MS and MS/MS analyses, including those involved in calcium binding, protein synthesis, carbohydrate metabolism, lipid metabolism, cell proliferation, mitosis regulation, apoptosis, cell migration, oxidative stress, and ion transport. Protein network analysis and functional validation revealed that high-calcium-exposed cells had 36.5% increase in calcium oxalate monohydrate (COM) crystal-binding capacity. This functional change was consistent to the expression data in which annexin A1 (ANXA1), a membrane-associated calcium-binding protein, was markedly increased on the apical surface of high-calcium-exposed cells. Pretreatment with anti-ANXA1 antibody could neutralize this increasing crystal-binding capacity. Moreover, high-calcium exposure caused defects in cell proliferation and wound healing. These expression and functional data demonstrate the enhanced crystal-binding capacity but impaired cell proliferation and wound healing in renal tubular cells induced by high-calcium. Taken together, these phenomena may contribute, at least in part, to the pathogenic mechanisms of hypercalciuria-induced nephrolithiasis and impaired renal function. Our in vitro study offers several candidates for further targeted functional studies to confirm their relevance in hypercalciuria and kidney stone disease in vivo.

Adhesion of calcium oxalate monohydrate (COM) crystals onto apical surface of renal tubular epithelial cells is a crucial mechanism for crystal retention, leading to kidney stone formation. Various proteins on apical membrane may bind to COM crystals; however, these crystal-binding proteins remained unidentified. The present study therefore aimed to identify COM crystal-binding proteins on apical membrane of distal renal tubular epithelial cells. Madin-Darby Canine Kidney (MDCK) cells were cultivated to be polarized epithelial cells and apical membrane was isolated from these cells using a peeling method established recently. Enrichment and purity of isolated apical membrane were confirmed by Western blot analysis for specific markers of apical (gp135) and basolateral (Na(+)/K(+)-ATPase) membranes. Proteins derived from the isolated apical membrane were then resuspended in artificial urine and incubated with COM crystals. The bound proteins were eluted, resolved by SDS-PAGE, and analyzed by Q-TOF MS and MS/MS, which identified 96 proteins. Among these, expression and localization of annexin II on apical surface of MDCK cells were confirmed by Western blot analysis, immunofluorescence staining, and laser-scanning confocal microscopic examination. Finally, the function of annexin II as the COM crystal-binding protein was successfully validated by COM crystal-binding assay. This large data set offers many opportunities for further investigations of kidney stone disease and may lead to the development of new therapeutic targets.

Apical membrane of polarized epithelial cells is generally isolated by physicochemical methods, that is, precipitation with polyethylene glycol (PEG) or MgCl(2) followed by differential centrifugation or sucrose density gradient centrifugation. However, these protocols are considerably sophisticated and frequently accompanied by impurities (e.g., contaminations of basolateral membrane and intracellular organelles), particularly by inexperienced investigators. We have developed a simple and effective method for isolation of apical membrane from intact polarized renal tubular epithelial cells. On the basis of hydrous affinity and/or ionic interaction, the apical membrane could be efficiently peeled from the cells by four different materials-Whatman filter paper, nitrocellulose membrane, cellophane, and glass coverslip-all of which are available in most research laboratories. Phase-contrast and laser-scanning confocal microscopic examinations using anti-ZO-1 antibody showed that other parts of the cells, particularly tight junction complex, remained intact after peeling by all four of these surfaces. Western blot analyses of gp135 (apical membrane marker) and of Na(+)/K(+)-ATPase, LAMP-2, COX-4, and calpain-1 (markers of basolateral membrane, lysosome, mitochondria, and cytosolic compartment, respectively) revealed that peeling with Whatman filter paper and glass coverslip was most and second-most effective, respectively, without any contaminations from basolateral membrane and other intracellular organelles that could be detected in the samples isolated by peeling with nitrocellulose membrane and cellophane and by conventional methods (i.e., precipitation with PEG or MgCl(2) followed by differential centrifugation or sucrose density gradient centrifugation). Our physical method is very simple, easy to follow (even by inexperienced investigators), time-saving, and cost-effective with a higher efficiency (as compared with conventional methods) for isolation of apical membrane from polarized epithelial cells.