Epithelial-mesenchymal plasticity in kidney fibrosis.

Epithelial-mesenchymal transition (EMT) is an important biological process contributing to kidney fibrosis and chronic kidney disease. This process is characterized by decreased epithelial phenotypes/markers and increased mesenchymal phenotypes/markers. Tubular epithelial cells (TECs) are commonly susceptible to EMT by various stimuli, for example, transforming growth factor-ß (TGF-ß), cellular communication network factor 2, angiotensin-II, fibroblast growth factor-2, oncostatin M, matrix metalloproteinase-2, tissue plasminogen activator (t-PA), plasmin, interleukin-1ß, and reactive oxygen species. Similarly, glomerular podocytes can undergo EMT via these stimuli and by high glucose condition in diabetic kidney disease. EMT of TECs and podocytes leads to tubulointerstitial fibrosis and glomerulosclerosis, respectively. Signaling pathways involved in EMT-mediated kidney fibrosis are diverse and complex. TGF-ß1/Smad and Wnt/ß-catenin pathways are the major venues triggering EMT in TECs and podocytes. These two pathways thus serve as the major therapeutic targets against EMT-mediated kidney fibrosis. To date, a number of EMT inhibitors have been identified and characterized. As expected, the majority of these EMT inhibitors affect TGF-ß1/Smad and Wnt/ß-catenin pathways. In addition to kidney fibrosis, these EMT-targeted antifibrotic inhibitors are expected to be effective for treatment against fibrosis in other organs/tissues.

Proteomic investigations of dengue virus infection: key discoveries over the last 10 years.

INTRODUCTION: Dengue virus (DENV) infection remains one of the most significant infectious diseases in humans. Several efforts have been made to address its molecular mechanisms. Over the last 10 years, proteomics has been widely applied to investigate various aspects of DENV infection. AREAS COVERED: In this review, we briefly introduce common proteomics approaches using various mass spectrometric modalities followed by summarizing all the discoveries obtained from proteomic investigations of DENV infection over the last 10 years. These include the data on DENV-vector interactions and host responses to address the DENV biology and disease mechanisms. Moreover, applications of proteomics to disease prevention, diagnosis, vaccine design, development of anti-DENV agents and other new treatment strategies are discussed. EXPERT OPINION: Despite efforts on disease prevention, DENV infection is still a significant global healthcare burden that affects the general population. As summarized herein, proteomic technologies with high-throughput capabilities have provided more in-depth details of protein dynamics during DENV infection. More extensive applications of proteomics and other powerful research tools would provide a promise to better cope and prevent this mosquito-borne infectious disease.

Identification of inositol monophosphatase as a broad-spectrum antiviral target of ivermectin.

Ivermectin has broad-spectrum antiviral activities. Despite the failure in clinical application of COVID-19, it can serve as a lead compound for the development of more effective broad-spectrum antivirals, for which a better understanding of its antiviral mechanisms is essential. We thus searched for potential novel targets of ivermectin in host cells by label-free thermal proteomic profiling using Huh-7 cells. Inositol monophosphatase (IMPase) was found among the proteins with shifted thermal stability by ivermectin. Ivermectin could inhibit IMPase activity and reduce cellular myo-inositol and phosphatidylinositol-4-phosphate levels. On the other hand, inositol could impair the antiviral activity of ivermectin and lithium, an IMPase inhibitor with known antiviral activity. As phosphatidylinositol phosphate is crucial for the replication of many RNA viruses, inhibition of cellular myo-inositol biosynthesis may be an important antiviral mechanism of ivermectin. Hence, inhibition of IMPase could serve as a potential target for broad-spectrum antiviral development.

Mitochondria-derived vesicles and their potential roles in kidney stone disease.

Recent evidence has shown significant roles of mitochondria-derived vesicles (MDVs) in mitochondrial quality control (MQC) system. Under mild stress condition, MDVs are formed to carry the malfunctioned mitochondrial components, such as mitochondrial DNA (mtDNA), peptides, proteins and lipids, to be eliminated to restore normal mitochondrial structure and functions. Under severe oxidative stress condition, mitochondrial dynamics (fission/fusion) and mitophagy are predominantly activated to rescue mitochondrial structure and functions. Additionally, MDVs generation can be also triggered as the major MQC machinery to cope with unhealthy mitochondria when mitophagy is unsuccessful for eliminating the damaged mitochondria or mitochondrial fission/fusion fail to recover the mitochondrial structure and functions. This review summarizes the current knowledge on MDVs and discuss their roles in physiologic and pathophysiologic conditions. In addition, the potential clinical relevance of MDVs in therapeutics and diagnostics of kidney stone disease (KSD) are emphasized.

Identification and characterization of ARID1A-interacting proteins in renal tubular cells and their molecular regulation of angiogenesis.

BACKGROUND: Defects and deficiency of AT-rich interactive domain-containing protein 1A (ARID1A) encoded by a tumor suppressor gene ARID1A have recently been suggested to get involved in angiogenesis, a crucial process in carcinogenesis. However, molecular mechanisms of ARID1A deficiency to induce angiogenesis in kidney cancer remain underinvestigated. METHODS: We performed large-scale identification of ARID1A protein interactors in renal tubular epithelial cells (RTECs) using immunoprecipitation (IP) followed by nanoLC-ESI-LTQ-Orbitrap tandem mass spectrometry (MS/MS). Their roles in angiogenesis were investigated using various assays. RESULTS: A total of 74 ARID1A-interacting proteins were identified. Protein-protein interactions analysis revealed that these identified proteins interacted directly or indirectly with ARID1A. Among them, the direct interaction between ARID1A and ß-actin was validated by IP and reciprocal IP followed by Western blotting. Small interfering RNA (siRNA) was used for single and double knockdowns of ARID1A and ACTB. Semi-quantitative RT-PCR demonstrated that deficiency of ARID1A, but not ACTB, significantly affected expression of angiogenesis-related genes in RTECs (VEGF and FGF2 were increased, whereas PDGF and EGF were decreased). However, the knockdowns did not affect TGFB1 and FGF1 levels. The quantitative mRNA expression data of VEGF and TGFB1 were consistent with the secreted levels of their protein products as measured by ELISA. Only secreted products derived from ARID1A-deficient RTECs significantly increased endothelial cells (ECs) migration and tube formation. Some of the other carcinogenic features could also be confirmed in the ARID1A-deficient RTECs, including increased cell migration and chemoresistance. Double knockdowns of both ARID1A and ACTB did not enhance the effects of single ARID1A knockdown in all assays. CONCLUSIONS: We report herein a large dataset of the ARID1A-interacting proteins in RTECs using an IP-MS/MS approach and confirm the direct interaction between ARID1A and ß-actin. However, the role of ARID1A deficiency in angiogenesis is independent of ß-actin.

Persistent Escherichia coli infection in renal tubular cells enhances calcium oxalate crystal-cell adhesion by inducing ezrin translocation to apical membranes via Rho/ROCK pathway.

Recent evidence has suggested that recurrent urinary tract infection (UTI) can cause not only infection stones but also metabolic stones (e.g., those containing calcium oxalate monohydrate or COM). However, precise mechanisms underlying UTI-induced metabolic stones remained unknown. In this study, Escherichia coli, the most common bacterium found in recurrent UTI was used to establish the in vitro model for persistent infection of renal epithelial cells. The promoting effects of persistent E. coli infection on kidney stone formation were validated by COM crystal-cell adhesion assay, followed by immunofluorescence study for changes in surface expression of the known COM crystal receptors. Among the five receptors examined, only ezrin had significantly increased level on the surface of persistently infected cells without change in its total level. Such translocation of ezrin to apical membranes was confirmed by Western blotting of apical membrane and cytosolic fractions and confocal microscopic examination. Additionally, persistent infection increased phosphorylation (Thr567) of ezrin. However, all of these changes induced by persistent E. coli infection were significantly inhibited by small-interfering RNA (siRNA) specific for ezrin or a Rho-associated kinase (ROCK)-specific inhibitor (Y-27632). In summary, this study provides a piece of evidence demonstrating that persistent infection by E. coli, one of the non-urease-producing bacteria, may contribute to COM metabolic stone formation by translocation of ezrin to apical membranes, thereby promoting COM crystal-cell adhesion. Such ezrin translocation was mediated via Rho/ROCK signaling pathway. These findings may, at least in part, explain the pathogenic mechanisms underlying recurrent UTI-induced metabolic kidney stone disease.

Roles of heat-shock protein 90 and its four domains (N, LR, M and C) in calcium oxalate stone-forming processes.

Human heat-shock protein 90 (HSP90) has four functional domains, including NH2-terminal (N), charged linker region (LR), middle (M) and COOH-terminal (C) domains. In kidney stone disease (or nephrolithiasis/urolithiasis), HSP90 serves as a receptor for calcium oxalate monohydrate (COM), which is the most common crystal to form kidney stones. Nevertheless, roles of HSP90 and its four domains in kidney stone formation remained unclear and under-investigated. We thus examined and compared their effects on COM crystals during physical (crystallization, growth and aggregation) and biological (crystal-cell adhesion and crystal invasion through extracellular matrix (ECM)) pathogenic processes of kidney stone formation. The analyses revealed that full-length (FL) HSP90 obviously increased COM crystal size and abundance during crystallization and markedly promoted crystal growth, aggregation, adhesion onto renal cells and ECM invasion. Comparing among four individual domains, N and C domains exhibited the strongest promoting effects, whereas LR domain had the weakest promoting effects on COM crystals. In summary, our findings indicate that FL-HSP90 and its four domains (N, LR, M and C) promote COM crystallization, crystal growth, aggregation, adhesion onto renal cells and invasion through the ECM, all of which are the important physical and biological pathogenic processes of kidney stone formation.

Oxidative Modifications Switch Modulatory Activities of Urinary Proteins From Inhibiting to Promoting Calcium Oxalate Crystallization, Growth, and Aggregation.

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 MS (nanoLC-electrospray ionization-linear trap quadrupole-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-electrospray ionization-linear trap quadrupole-Orbitrap-MS/MS identified a total of 193 and 220 urinary proteins in nonmodified and modified urine samples, respectively. Among these, there were 1121 and 5297 unambiguous oxidatively modified peptides representing 42 and 136 oxidatively modified proteins in the nonmodified and modified urine samples, respectively. Crystal assays revealed that oxidatively modified urinary proteins significantly promoted CaOx crystallization, crystal growth, and aggregation. By contrast, the nonmodified 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.

Systematic comparisons of various markers for mast cell activation in RBL-2H3 cells.

Mast cell activation plays a key role in various allergic diseases and anaphylaxis. Several methods/techniques can be used for detection of mast cell activation. However, there was no previous systematic evaluation to compare the efficacy of each method/technique. The present study thus systematically compared various markers for mast cell activation induced by IgE cross-linking. The widely used RBL-2H3 mast cells were sensitized with anti-DNP (dinitrophenyl) IgE overnight and activated with DNP-BSA (bovine serum albumin) for up to 4 h. The untreated cells and those with anti-DNP IgE sensitization but without DNP-BSA activation served as the controls. Intracellular calcium level gradually increased to ~2-fold at 1 h, reached its peak (~5-fold) at 2 h, and returned to the basal level at 3-h post-activation. The increases in cellular tryptase level (by Western blotting) (~0.3- to 0.4-fold) and average cell size (~2.5-fold) and decrease of nucleus/cytoplasm ratio (~0.4- to 0.5-fold) were marginal at all time-points. By contrast, ß-hexosaminidase release and CD63 expression (by both flow cytometry and immunofluorescence detection/localization), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence detection/localization) stably and obviously increased (~10-fold as compared with the untreated control and sensitized-only cells or detectable only after activation). Based on these data, the stably obvious increases (by ≥ 10-fold) in ß-hexosaminidase release, CD63 expression (by both flow cytometry and immunofluorescence staining), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence staining) are recommended as the markers of choice for the in vitro study of mast cell activation using RBL-2H3 cells.

What can urinary exosomes tell us?

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.

Kidney stone proteomics: an update and perspectives.

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.

Peptidomics and proteogenomics: background, challenges and future needs.

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.

How can artificial intelligence be used for peptidomics?

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).

Optimization of artificial urine formula for in vitro cellular study compared with native urine.

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).

Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N-LR-M-C) domains.

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

ARID1A knockdown triggers epithelial-mesenchymal transition and carcinogenesis features of renal cells: role in renal cell carcinoma.

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

Protective Cellular Mechanism of Estrogen Against Kidney Stone Formation: A Proteomics Approach and Functional Validation.

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

Protective effects of finasteride against testosterone-induced calcium oxalate crystallization and crystal-cell adhesion.

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.

Modulatory effects of fibronectin on calcium oxalate crystallization, growth, aggregation, adhesion on renal tubular cells, and invasion through extracellular matrix.

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.

Comparative proteomics reveals concordant and discordant biochemical effects of caffeine versus epigallocatechin-3-gallate in human endothelial cells.

Caffeine and epigallocatechin-3-gallate (EGCG) are the most abundant bioactive chemicals found in coffee and tea, respectively. While they are known to regulate normal physiology and body homeostasis, their biochemical effects, particularly at cellular and subcellular levels, remain under-investigated. We thus performed comparative proteomics study followed by bioinformatics analyses to investigate differential biochemical effects of these two chemicals on human endothelial cells. EA.hy926 cells were incubated with 100 µM caffeine or EGCG for 24-h and then subjected to label-free quantitative proteomics using nanoLC-ESI-Qq-TOF MS/MS compared to the control cells. A total of 142 and 152 significantly altered proteins were identified in caffeine-exposed and EGCG-exposed cells, respectively. Among these, 86 were the common changes found in both caffeine-exposed and EGCG-exposed cells. Bioinformatics revealed that both caffeine and EGCG mostly affected ribosomal proteins involving protein synthesis for membrane destination and secretion (e.g., SRP-dependent cotranslational protein targeting to membrane, protein targeting to endoplasmic reticulum, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, etc.). While the down-regulated proteins were similar in both groups, the up-regulated proteins in the EGCG-exposed cells highlighted the promoting effects of EGCG on actin-crosslink formation, glycolysis and ubiquitin-proteasome activity. These concordant and discordant changes in cellular proteome of human endothelial cells induced by caffeine and EGCG are useful for better understanding of biochemical/physiological effects of these two bioactive chemicals.