Resveratrol inhibits calcium oxalate crystal growth, reduces adhesion to renal cells and induces crystal internalization into the cells, but promotes crystal aggregation.

Resveratrol is a natural phenolic compound that belongs to stilbenoid group found in diverse plants. Health benefits and therapeutic potentials of resveratrol have been widely recognized in various diseases. In kidney stone disease, it can alleviate oxalate-induced hyperproduction of free radicals in renal epithelial cells. Nevertheless, its direct effects on calcium oxalate (CaOx) crystal, which is the major stone component, remained unclear. This study therefore addressed the direct effects of resveratrol (at 1, 10 or 100 µM) on each step of CaOx kidney stone formation. The results revealed that resveratrol had no significant effects on CaOx crystallization. However, resveratrol significantly decreased CaOx crystal growth and adhesion to renal epithelial cells at all concentrations, and induced crystal internalization into the cells (a process related to crystal degradation by endolysosomes) in a concentration-dependent manner. On the other hand, resveratrol promoted crystal aggregation. These data indicate that resveratrol serves as a dual modulator on CaOx stone formation. While it inhibits CaOx stone development by reducing crystal growth and adhesion to renal cells and by inducing crystal internalization into the cells, resveratrol promotes crystal aggregation, which is one of the mechanisms leading to kidney stone formation.

Curcumin prevents high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation via mitigating intracellular free radicals and TGF-ß secretion.

Diabetic kidney disease (DKD) is a leading cause of kidney failure. However, the involvement of renal fibroblasts and their communications with renal epithelial cells during DKD remain poorly understood. We investigated the potential role of renal proximal tubular epithelial cells (PTECs) in renal fibroblast activation that might lead to DKD. Additionally, the protective effects of curcumin, a known antioxidant, against renal fibroblast activation induced by high glucose-treated PTECs were investigated. Secretome was collected from HK-2 PTECs under normal glucose, high glucose, high glucose pretreated/cotreated with curcumin, or osmotic control condition for 24 h. Such secretome was then used to treat BHK-21 renal fibroblasts for 24 h. BHK-21 cells treated with high glucose-induced secretome had increased levels of fibroblast activation markers, including spindle index, F-actin, α-smooth muscle actin (α-SMA), fibronectin, collagen I, matrix metalloproteinase-2 (MMP-2) and MMP-9, as compared with normal glucose and osmotic control conditions. However, all these increases were successfully mitigated by curcumin. In addition, high glucose markedly increased intracellular reactive oxygen species (ROS) and transforming growth factor-ß (TGF-ß) secretion, but did not affect the secretion of platelet-derived growth factor A (PDGFA) and interleukin-1ß (IL-1ß), in HK-2 renal cells as compared with normal glucose and osmotic control conditions. Both intracellular ROS and secreted TGF-ß levels were successfully mitigated by curcumin. Therefore, curcumin prevents the high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation, at least in part, via mitigating intracellular ROS and TGF-ß secretion.

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.

The modulatory effects of large and small extracellular vesicles from normal human urine on calcium oxalate crystallization, growth, aggregation, adhesion on renal cells, and invasion through extracellular matrix: An in vitro study.

Urinary extracellular vesicles (uEVs) play important roles in physiologic condition and various renal/urological disorders. However, their roles in kidney stone disease remain unclear. This study aimed to examine modulatory effects of large and small uEVs derived from normal human urine on calcium oxalate (CaOx) crystals (the main component in kidney stones). After isolation, large uEVs, small uEVs and total urinary proteins (TUPs) with equal (protein equivalent) concentration were added into various crystal assays to compare with the control (without uEVs or TUPs). TUPs strongly inhibited CaOx crystallization, growth, aggregation and crystal-cell adhesion. Large uEVs had lesser degree of inhibition against crystallization, growth and crystal-cell adhesion, and comparable degree of aggregation inhibition compared with TUPs. Small uEVs had comparable inhibitory effects as of TUPs for all these crystal assays. However, TUPs and large uEVs slightly promoted CaOx invasion through extracellular matrix, whereas small uEVs did not affect this. Matching of the proteins reported in six uEVs datasets with those in the kidney stone modulator (StoneMod) database revealed that uEVs contained 18 known CaOx stone modulators (mainly inhibitors). These findings suggest that uEVs derived from normal human urine serve as CaOx stone inhibitors to prevent healthy individuals from kidney stone formation.

Beneficial roles of gastrointestinal and urinary microbiomes in kidney stone prevention via their oxalate-degrading ability and beyond.

Formation of calcium oxalate (CaOx) crystal, the most common composition in kidney stones, occurs following supersaturation of calcium and oxalate ions in the urine. In addition to endogenous source, another main source of calcium and oxalate ions is dietary intake. In the intestinal lumen, calcium can bind with oxalate to form precipitates to be eliminated with feces. High intake of oxalate-rich foods, inappropriate amount of daily calcium intake, defective intestinal transporters for oxalate secretion and absorption, and gastrointestinal (GI) malabsorption (i.e., from gastric bypass surgery) can enhance intestinal oxalate absorption, thereby increasing urinary oxalate level and risk of kidney stone disease (KSD). The GI microbiome rich with oxalate-degrading bacteria can reduce intestinal oxalate absorption and urinary oxalate level. In addition to the oxalate-degrading ability, the GI microbiome also affects expression of oxalate transporters and net intestinal oxalate transport, cholesterol level, and short-chain fatty acids (SCFAs) production, leading to lower KSD risk. Recent evidence also shows beneficial effects of urinary microbiome in KSD prevention. This review summarizes the current knowledge on the aforementioned aspects. Potential benefits of the GI and urinary microbiomes as probiotics for KSD prevention are emphasized. Finally, challenges and future perspectives of probiotic treatment in KSD are discussed.

StoneMod 2.0: Database and prediction of kidney stone modulatory proteins.

Stone modulators are various kinds of molecules that play crucial roles in promoting/inhibiting kidney stone formation. Several recent studies have extensively characterized the stone modulatory proteins with the ultimate goal of preventing kidney stone formation. Herein, we introduce the StoneMod 2.0 database (https://www.stonemod.org), which has been dramatically improved from the previous version by expanding the number of the modulatory proteins in the list (from 32 in the initial version to 17,130 in this updated version). The stone modulatory proteins were recruited from solid experimental evidence (via PubMed) and/or predicted evidence (via UniProtKB, QuickGO, ProRule, STITCH and OxaBIND to retrieve calcium-binding and oxalate-binding proteins). Additionally, StoneMod 2.0 has implemented a scoring system that can be used to determine the likelihood and to classify the potential stone modulatory proteins as either "solid" (modulator score ≥ 50) or "weak" (modulator score < 50) modulators. Furthermore, the updated version has been designed with more user-friendly interfaces and advanced visualization tools. In addition to the monthly scheduled update, the users can directly submit their experimental evidence online anytime. Therefore, StoneMod 2.0 is a powerful database with prediction scores that will be very useful for many future studies on the stone modulatory proteins.

Proteomic insight towards key modulating proteins regulated by the aryl hydrocarbon receptor involved in ovarian carcinogenesis and chemoresistance.

Gynecological malignancies pose a severe threat to female lives. Ovarian cancer (OC), the most lethal gynecological malignancy, is clinically presented with chemoresistance and a higher relapse rate. Several studies have highly correlated the incidence of OC to exposure to environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a process mainly mediated through activating the aryl hydrocarbon receptor (AhR). We have previously reported that exposure of OC cells to TCDD, an AhR activator, significantly modulated the expression of several genes that play roles in stemness and chemoresistance. However, the effect of AhR activation on the whole OC cell proteome aiming at identifying novel druggable targets for both prevention and treatment intervention purposes remains unrevealed. For this purpose, we conducted a comparative proteomic analysis of OC cells A2780 untreated/treated with TCDD for 24 h using a mass spectrometry-based label-free shotgun proteomics approach. The most significantly dysregulated proteins were validated by Western blot analysis. Our results showed that upon AhR activation by TCDD, out of 2598 proteins identified, 795 proteins were upregulated, and 611 were downregulated. STRING interaction analysis and KEGG-Reactome pathway analysis approaches identified several significantly dysregulated proteins that were categorized to be involved in chemoresistance, cancer progression, invasion and metastasis, apoptosis, survival, and prognosis in OC. Importantly, selected dysregulated genes identified by the proteomic study were validated at the protein expression levels by Western blot analysis. In conclusion, this study provides a better understanding of the the cross-talk between AhR and several other molecular signaling pathways and the role and involvement of AhR in ovarian carcinogenesis and chemoresistance. Moreover, the study suggests that AhR is a potential therapeutic target for OC prevention and maintenance. SIGNIFICANCE: To our knowledge, this is the first study that investigates the role and involvement of AhR and its regulated genes in OC by performing a comparative proteomic analysis to identify the critical proteins with a modulated expression upon AhR activation. We found AhR activation to play a tumor-promoting and chemoresistance-inducing role in the pathogenesis of OC. The results of our study help to devise novel therapeutics for better management and prevention and open the doors to finding novel biomarkers for the early detection and prognosis of OC.

The upregulation of lamin A/C as a compensatory mechanism during tight junction disruption in renal tubular cells mediated by calcium oxalate crystals.

Calcium oxalate monohydrate (COM), the most important crystal causing kidney stone disease, upregulates lamin A/C but downregulates zonula occludens-1 (ZO-1) in renal tubular cells. While roles for F-actin and α-tubulin and their association with ZO-1 are known to regulate COM-mediated tight junction (TJ) disruption, roles of lamin A/C and its interplay with ZO-1 in COM kidney stone model remain unclear and are thus the objectives of this study. Lamin A/C was knocked down in MDCK cells by silencing RNA specific for LMNA (siLMNA). Both wild-type (WT) and siLMNA cells were treated with COM for 48-h compared with the untreated (control) cells. Western blotting and immunofluorescence staining revealed upregulated lamin A/C and downregulated ZO-1 in the COM-treated WT cells. siLMNA successfully reduced lamin A/C expression in both control and COM-treated cells. Nonetheless, siLMNA did not reverse the effect of COM on the decreases in ZO-1 and transepithelial resistance, but further reduced their levels in both control and COM-treated cells. Protein-protein interaction analysis demonstrated that two cytoskeletal proteins (actin and tubulin) served as the linkers to connect lamin A/C with ZO-1 and occludin (both of which are the TJ proteins). Altogether, these data implicate that lamin A/C and ZO-1 are indirectly associated to control TJ function, and ZO-1 expression is regulated by lamin A/C. Moreover, COM-induced upregulation of lamin A/C most likely serves as a compensatory mechanism to cope with the downregulation of ZO-1 during COM-mediated TJ disruption.

The protective effect of caffeine against oxalate-induced epithelial-mesenchymal transition in renal tubular cells via mitochondrial preservation.

Mitochondrial dysfunction is one of the key mechanisms for developing chronic kidney disease (CKD). Hyperoxaluria and nephrolithiasis are also associated with mitochondrial dysfunction. Increasing evidence has shown that caffeine, the main bioactive compound in coffee, exerts both anti-fibrotic and anti-lithogenic properties but with unclear mechanisms. Herein, we address the protective effect of caffeine against mitochondrial dysfunction during oxalate-induced epithelial-mesenchymal transition (EMT) in renal cells. Analyses revealed that oxalate successfully induced EMT in MDCK renal cells as evidenced by the increased expression of several EMT-related genes (i.e., Snai1, Fn1 and Acta2). Oxalate also suppressed cellular metabolic activity and intracellular ATP level, but increased reactive oxygen species (ROS). Additionally, oxalate reduced abundance of active mitochondria and induced mitochondrial fragmentation (fission). Furthermore, oxalate decreased mitochondrial biogenesis and content as evidenced by decreased expression of sirtuin-1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), cytochrome c oxidase subunit 4 (COX4), and total mitochondrial proteins. Nonetheless, these oxalate-induced deteriorations in MDCK cells and their mitochondria were successfully hampered by caffeine. Knockdown of Snai1 gene by small interfering RNA (siRNA) completely abolished the effects of oxalate on suppression of cellular metabolic activity, intracellular ATP and abundance of active mitochondria, indicating that these oxalate-induced renal cell deteriorations were mediated through the Snai1 EMT-related gene. These data, at least in part, unveil the anti-fibrotic mechanism of caffeine during oxalate-induced EMT in renal cells by preserving mitochondrial biogenesis and function.

Tight junction and kidney stone disease.

Defects of tight junction (TJ) are involved in many diseases related to epithelial cell functions, including kidney stone disease (KSD), which is a common disease affecting humans for over a thousand years. This review provides brief overviews of KSD and TJ, and summarizes the knowledge on crystal-induced defects of TJ in renal tubular epithelial cells (RTECs) in KSD. Calcium oxalate (CaOx) crystals, particularly COM, disrupt TJ via p38 MAPK and ROS/Akt/p38 MAPK signaling pathways, filamentous actin (F-actin) reorganization and α-tubulin relocalization. Stabilizing p38 MAPK signaling, reactive oxygen species (ROS) production, F-actin and α-tubulin by using SB239063, N-acetyl-L-cysteine (NAC), phalloidin and docetaxel, respectively, successfully prevent the COM-induced TJ disruption and malfunction. Additionally, genetic disorders of renal TJ, including mutations and single nucleotide polymorphisms (SNPs) of CLDN2, CLDN10b, CLDN14, CLDN16 and CLDN19, also affect KSD. Finally, the role of TJ as a potential target for KSD therapeutics and prevention is also discussed.

The inhibitory effects of epigallocatechin-3-gallate on calcium oxalate monohydrate crystal growth, aggregation and crystal-cell adhesion.

Epigallocatechin-3-gallate (EGCG), a predominant phytochemical in tea plant, has been reported to prevent kidney stone formation but with vague mechanism. We investigated modulatory effects of EGCG (at 0.1-100 µM) on calcium oxalate monohydrate (COM) crystals at various stages of kidney stone development. EGCG significantly increased crystal size (at 1-100 µM), but decreased crystal number (at 10-100 µM), resulting in unchanged crystal mass and volume. Interestingly, EGCG at 10-100 µM caused morphological change of the crystals from typical monoclinic prismatic to coffee-bean-like shape, which represented atypical/aberrant form of COM as confirmed by attenuated total reflection - Fourier transform infrared (ATR-FTIR) spectroscopy. EGCG at all concentrations significantly inhibited crystal growth in a concentration-dependent manner. However, only 100 µM and 10-100 µM of EGCG significantly inhibited crystal aggregation and crystal-cell adhesion, respectively. Immunofluorescence staining (without permeabilization) revealed that surface expression of heat shock protein 90 (HSP90) (a COM crystal receptor) on MDCK renal cells was significantly decreased by 10 µM EGCG, whereas other surface COM receptors (annexin A1, annexin A2, enolase 1 and ezrin) remained unchanged. Immunoblotting showed that 10 µM EGCG did not alter total level of HSP90 in MDCK cells, implicating that its decreased surface expression was due to translocation. Our data provide a piece of evidence explaining mechanism underlying the anti-lithiatic property of EGCG by inhibition of COM crystal growth, aggregation and crystal-cell adhesion via reduced surface expression of HSP90, which is an important COM crystal receptor.

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.

Comparative analyses of various IgE-mediated and non-IgE-mediated inducers of mast cell degranulation for in vitro study.

In vitro investigations of mast cell (MC) degranulation are essential for studying many diseases, particularly allergy and urticaria. Many MC-degranulation inducers are currently available. However, there is no previous systematic comparative analysis of these available inducers in term of their efficacies to induce MC degranulation. Herein, we performed systematic comparisons of efficacies of five well-known and commonly used MC-degranulation inducers. RBL-2H3 cells were sensitized with 50 ng/ml anti-DNP IgE or biotinylated IgE followed by stimulation with 100 ng/ml DNP-BSA or streptavidin, respectively. For non-IgE-mediated inducers, the cells were treated with 5 µg/ml substance P, compound 48/80, or A23187. At 15-, 30-, 45- and 60-min post-induction, several common MC-degranulation markers (including intracellular [Ca2+], ß-hexosaminidase release, tryptase expression by immunofluorescence staining, cellular tryptase level by immunoblotting, secretory tryptase level by immunoblotting, CD63 expression by immunofluorescence staining, and CD63 expression by flow cytometry) were evaluated. The data showed that all these markers significantly increased after activation by all inducers. Among them, A23187 provided the greatest degrees of increases in intracellular [Ca2+] and ß-hexosaminidase release at all time-points and upregulation of CD63 at one time-point. These data indicate that all these IgE-mediated (anti-DNP IgE/DNP-BSA and biotinylated IgE/streptavidin) and non-IgE-mediated (substance P, compound 48/80, and A23187) inducers effectively induce MC degranulation, while A23187 seems to be the most effective inducer for MC degranulation.

Quercetin inhibits calcium oxalate crystallization and growth but promotes crystal aggregation and invasion.

Recent evidence has shown an association between kidney stone pathogenesis and oxidative stress. Many anti-oxidants have been studied with an aim for stone prevention. Quercetin, a natural flavonol, is one among those eminent anti-oxidants with satisfactory anti-inflammatory property to cope with renal tissue injury in kidney stone disease. Nevertheless, its direct effect (if any) on calcium oxalate (CaOx) crystals and the stone formation mechanism had not been previously explored. This study has addressed the ability of quercetin at various concentrations (2.5, 5, 10, 20, 40, 80 and 160 µM) to directly modulate CaOx crystallization, growth, aggregation, adhesion on kidney cells, and invasion through the matrix. The data have shown that quercetin significantly inhibits CaOx crystallization and crystal growth but promotes crystal aggregation in concentration-dependent manner. However, quercetin at all these concentrations do not affect CaOx adhesion on kidney cells. For the invasion, quercetin at all concentrations constantly promotes CaOx invasion through the matrix without concentration-dependent pattern. These discoveries have demonstrated for the first time that quercetin has direct but dual modulatory effects on CaOx crystals. While quercetin inhibits CaOx crystallization and growth, on the other hand, it promotes CaOx crystal aggregation and invasion through the matrix. These data highlight the role for quercetin in direct modulation of the CaOx crystals that may intervene the stone pathogenesis.

Proteomic and computational analyses followed by functional validation of protective effects of trigonelline against calcium oxalate-induced renal cell deteriorations.

Trigonelline is a phytoalkaloid commonly found in green and roasted coffee beans. It is also found in decaffeinated coffee. Previous report has shown that extract from trigonelline-rich plant exhibits anti-lithiatic effects in a nephrolithiatic rat model. Nevertheless, cellular mechanisms underlying the anti-lithiatic properties of trigonelline remain hazy. Herein, we used nanoLC-ESI-Qq-TOF MS/MS and MaxQuant-based quantitative proteomics to identify trigonelline-induced changes in protein expression in MDCK renal cells. From a total of 1006 and 1011 proteins identified from control and trigonelline-treated cells, respectively, levels of 62 (23 upregulated and 39 downregulated) proteins were significantly changed by trigonelline. Functional enrichment and reactome pathway analyses suggested that these 62 altered proteins were related to stress response, cell cycle and cell polarity. Functional validation by corresponding experimental assays revealed that trigonelline prevented calcium oxalate monohydrate crystal-induced renal cell deteriorations by inhibiting crystal-induced overproduction of intracellular reactive oxygen species, G0/G1 to G2/M cell cycle shift, tight junction disruption, and epithelial-mesenchymal transition. These findings provide cellular mechanisms and convincing evidence for the renoprotective effects of trigonelline, particularly in kidney stone prevention.

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.

Caffeine causes cell cycle arrest at G0/G1 and increases of ubiquitinated proteins, ATP and mitochondrial membrane potential in renal cells.

Caffeine is a well-known purine alkaloid commonly found in coffee. Several lines of previous and recent evidence have shown that habitual coffee drinking is associated with lower risks for chronic kidney disease (CKD) and nephrolithiasis. However, cellular and molecular mechanisms underlying its renoprotective effects remain largely unknown due to a lack of knowledge on cellular adaptive response to caffeine. This study investigated cellular adaptive response of renal tubular cells to caffeine at the protein level. Cellular proteome of MDCK cells treated with caffeine at a physiologic concentration (100 µM) for 24 h was analyzed comparing with that of untreated cells by label-free quantitative proteomics. From a total of 936 proteins identified, comparative analysis revealed significant changes in levels of 148 proteins induced by caffeine. These significantly altered proteins were involved mainly in proteasome, ribosome, tricarboxylic acid (TCA) (or Krebs) cycle, DNA replication, spliceosome, biosynthesis of amino acid, carbon metabolism, nucleocytoplasmic transport, cell cycle, cytoplasmic translation, translation initiation, and mRNA metabolic process. Functional validation by various assays confirmed that caffeine decreased cell population at G2/M, increased cell population at G0/G1, increased level of ubiquitinated proteins, increased intracellular ATP and enhanced mitochondrial membrane potential in MDCK cells. These data may help unravelling molecular mechanisms underlying the biological effects of caffeine on renal tubular cells at cellular and protein levels.

Epigenetic regulation of epithelial-mesenchymal transition during cancer development.

Epithelial-mesenchymal transition (EMT) plays essential roles in promoting malignant transformation of epithelial cells, leading to cancer progression and metastasis. During EMT-induced cancer development, a wide variety of genes are dramatically modified, especially down-regulation of epithelial-related genes and up-regulation of mesenchymal-related genes. Expression of other EMT-related genes is also modified during the carcinogenic process. Especially, epigenetic modifications are observed in the EMT-related genes, indicating their involvement in cancer development. Mechanically, epigenetic modifications of histone, DNA, mRNA and non-coding RNA stably change the EMT-related gene expression at transcription and translation levels. Herein, we summarize current knowledge on epigenetic regulatory mechanisms observed in EMT process relate to cancer development in humans. The better understanding of epigenetic regulation of EMT during cancer development may lead to improvement of drug design and preventive strategies in cancer therapy.

Calcineurin B inhibits calcium oxalate crystallization, growth and aggregation via its high calcium-affinity property.

Calcineurin inhibitors (CNIs) are widely used in organ transplantation to suppress immunity and prevent allograft rejection. However, some transplant patients receiving CNIs have hypocitraturia, hyperoxaluria and kidney stone with unclear mechanism. We hypothesized that CNIs suppress activities of urinary calcineurin, which may serve as the stone inhibitor. This study aimed to investigate effects of calcineurin B (CNB) on calcium oxalate monohydrate (COM) stone formation. Sequence and structural analyses revealed that CNB contained four EF-hand (Ca2+-binding) domains, which are known to regulate Ca2+ homeostasis and likely to affect COM crystals. Various crystal assays revealed that CNB dramatically inhibited COM crystallization, crystal growth and crystal aggregation. At an equal amount, degrees of its inhibition against crystallization and crystal growth were slightly inferior to total urinary proteins (TUPs) from healthy subjects that are known to strongly inhibit COM stone formation. Surprisingly, its inhibitory effect against crystal aggregation was slightly superior to TUPs. While TUPs dramatically inhibited crystal-cell adhesion, CNB had no effect on this process. Ca2+-affinity assay revealed that CNB strongly bound Ca2+ at a comparable degree as of TUPs. These findings indicate that CNB serves as a novel inhibitor of COM crystallization, growth and aggregation via its high Ca2+-affinity property.