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.

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.

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.

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.

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 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.

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.

Characterizations of annexin A1-interacting proteins in apical membrane and cytosolic compartments of renal tubular epithelial cells.

Annexin A1 (ANXA1) is a multifunctional calcium-binding protein that can bind to membrane phospholipids. Under high-calcium condition, ANXA1 expression increases on renal epithelial cell surface, leading to enhanced adhesion of calcium oxalate (CaOx) crystal (stone material) onto the cells. To regulate various cellular processes, ANXA1 interacts with many other intracellular protein partners. However, components of the ANXA1-interacting protein complex remain unclear. Herein, we characterized the interacting complexes of apical membrane (ApANXA1) and cytosolic (cyANXA1) forms of ANXA1 in apical membrane and cytosolic compartments, respectively, of renal epithelial cells under high-calcium condition using proteomic and bioinformatic approaches. After fractionation, the ApANXA1- and CyANXA1-interacting partners were identified by immunoprecipitation followed by nanoLC­ESI­Qq-TOF tandem mass spectrometry (IP-MS/MS). The ANXA1-interacting partners that were common in both apical membrane and cytosolic compartments and those unique in each compartment were then analyzed for their physico-chemical properties (molecular weight, isoelectric point, amino acid contents, instability index, aliphatic index, and grand average of hydropathicity), secondary structure (α-helix, ß-turn, random coil, and extended strand), molecular functions, biological processes, reactome pathways and KEGG pathways. The data demonstrated that each set of these interacting proteins exhibited common and unique characteristics and properties. The knowledge from this study may lead to better understanding of the ApANXA1 and CyAXNA1 biochemistry and functions as well as the pathophysiology of CaOx kidney stone formation induced by high-calcium condition.

OxaBIND: A tool for identifying oxalate-binding domain(s)/motif(s) in protein(s).

High oxalate level in blood and urine may cause oxalate-related disorders, particularly kidney stone disease. To unravel disease mechanisms, investigations of oxalate level and its binding proteins are required. However, the information on oxalate-binding proteins is limited due to a lack of appropriate tool for their investigations. Therefore, we have developed a freely accessible web-based tool, namely OxaBIND (https://www.stonemod.org/oxabind.php), to identify oxalate-binding site(s) in any proteins of interest. The prediction model was generated by recruiting all of the known oxalate-binding proteins with solid experimental evidence (from PubMed and RCSB Protein Data Bank). The potential oxalate-binding domains/motifs were predicted from these oxalate-binding proteins using PRATT tool and used to discriminate these known oxalate-binding proteins from the known non-oxalate-binding proteins. The best one, which provided highest fitness score, sensitivity and specificity, was then implemented to create the OxaBIND tool. After inputting protein identifier or sequence (which can be single or multiple), details of all the identified oxalate-binding site(s), if any, are presented in both textual and graphical formats. OxaBIND also provides theoretical three-dimensional (3D) structure of the protein with oxalate-binding site(s) being highlighted. This tool will be beneficial for future research on the oxalate-binding proteins, which play important roles in the oxalate-related disorders.

Kidney Stone Prevention.

Kidney stone disease (KSD) (alternatively nephrolithiasis or urolithiasis) is a global health care problem that affects people in almost all of developed and developing countries. Its prevalence has been continuously increasing with a high recurrence rate after stone removal. Although effective therapeutic modalities are available, preventive strategies for both new and recurrent stones are required to reduce physical and financial burdens of KSD. To prevent kidney stone formation, its etiology and risk factors should be first considered. Low urine output and dehydration are the common risks of all stone types, whereas hypercalciuria, hyperoxaluria, and hypocitraturia are the major risks of calcium stones. In this article, up-to-date knowledge on strategies (nutrition-based mainly) to prevent KSD is provided. Important roles of fluid intake (2.5-3.0 L/d), diuresis (>2.0-2.5 L/d), lifestyle and habit modifications (for example, maintain normal body mass index, fluid compensation for working in high-temperature environment, and avoid cigarette smoking), and dietary management [for example, sufficient calcium at 1000-1200 mg/d, limit sodium at 2 or 3-5 g/d of sodium chloride (NaCl), limit oxalate-rich foods, avoid vitamin C and vitamin D supplements, limit animal proteins to 0.8-1.0 g/kg body weight/d but increase plant proteins in patients with calcium and uric acid stone and those with hyperuricosuria, increase proportion of citrus fruits, and consider lime powder supplementation] are summarized. Moreover, uses of natural bioactive products (for example, caffeine, epigallocatechin gallate, and diosmin), medications (for example, thiazides, alkaline citrate, other alkalinizing agents, and allopurinol), bacterial eradication, and probiotics are also discussed.

Protein network analysis and functional enrichment via computational biotechnology unravel molecular and pathogenic mechanisms of kidney stone disease.

Mass spectrometry-based proteomics has been extensively applied to current biomedical research. From such large-scale identification of proteins, several computational tools have been developed for determining protein-protein interactions (PPI) network and functional significance of the identified proteins and their complex. Analyses of PPI network and functional enrichment have been widely applied to various fields of biomedical research. Herein, we summarize commonly used tools for PPI network analysis and functional enrichment in kidney stone research and discuss their applications to kidney stone disease (KSD). Such computational approach has been used mainly to investigate PPI networks and functional significance of the proteins derived from urine of patients with kidney stone (stone formers), stone matrix, Randall's plaque, renal papilla, renal tubular cells, mitochondria and immune cells. The data obtained from computational biotechnology leads to experimental validation and investigations that offer new knowledge on kidney stone formation processes. Moreover, the computational approach may also lead to defining new therapeutic targets and preventive strategies for better outcome in KSD management.

Quantitative proteomics reveals common and unique molecular mechanisms underlying beneficial effects of caffeine and trigonelline on human hepatocytes.

Caffeine and trigonelline are the major bioactive compounds in coffee. Caffeine alone or combined with other coffee compounds shows hepatoprotective effects. However, molecular mechanisms underlying such hepatoprotective effects remain unclear. We therefore addressed molecular effects of caffeine and trigonelline on human hepatocytes using quantitative proteomics followed by bioinformatic analyses to obtain topological and functional significance. HepG2 cells were treated with 100 µM caffeine or trigonelline for 24-h and evaluated by quantitative proteomics using nanoLC-ESI-LTQ-Orbitrap MS/MS. A total of 26 and 25 significantly altered proteins were identified in caffeine-treated and trigonelline-treated cells, respectively, compared with control cells. Topological analyses revealed that ribosomal and translation regulatory proteins predominantly served as the hub proteins associated with protein clusters. Functional analyses also revealed that these two bioactive compounds shared some molecular mechanisms via induction of translational processes. There were also other unique molecular functions and biological processes triggered or suppressed by either caffeine or trigonelline. These data highlight common and unique molecular mechanisms underlying the hepatoprotective effects of caffeine and trigonelline that may be useful for future clinical applications.

Alpha-tubulin relocalization is involved in calcium oxalate-induced tight junction disruption in renal epithelial cells.

Microtubule (MT) is associated with tight junction (TJ) structure and function. While calcium oxalate monohydrate (COM) commonly causes TJ disruption, its effects on MT remain unknown. This study thus addressed the involvement of a major MT protein, α-tubulin, in COM-induced TJ disruption. Protein-protein interactions analysis demonstrated that α-tubulin directly interacted with a TJ protein, zonula occludens-1 (ZO-1). MDCK renal cells were polarized and incubated with COM crystals for 48 h. Western blotting showed that COM reduced ZO-1, but not α-tubulin, level. Immunofluorescence staining revealed COM-induced relocalization of α-tubulin from apical membranes to cytoplasm and ZO-1 disruption at cell borders. COM also mediated progressive fall of epithelial barrier function, represented by transepithelial resistance (TER), which reached the lowest at 12-h till the end of crystal exposure. Pretreatment of the cells with docetaxel, the MT/tubulin stabilizer, completely prevented such α-tubulin relocalization, ZO-1 disruption/down-regulation, and TER reduction. These data indicate that α-tubulin relocalization is involved in COM-induced TJ disruption in renal epithelial cells.

Calcium oxalate crystals trigger epithelial-mesenchymal transition and carcinogenic features in renal cells: a crossroad between kidney stone disease and renal cancer.

Increasing evidence of association between kidney stone disease (KSD) and renal cell carcinoma (RCC) has been reported. Nevertheless, mechanism underlying such association remained unknown. Herein, we investigated the effects of calcium oxalate monohydrate (COM), a major crystalline component causing KSD, on induction of carcinogenic features in non-cancerous renal cells. COM crystals induced morphological changes from epithelial to fibroblast-like spindle shape. Additionally, COM increased spindle index and mesenchymal markers (fibronectin and vimentin) but declined epithelial markers (E-cadherin and zonula occludens-1). Moreover, COM down-regulated ARID1A, a tumor suppressor gene recently reported to be reversely associated with RCC, at both mRNA and protein levels. COM also down-regulated other RCC-related tumor suppressor genes, PTEN and VHL, but up-regulated oncogene TPX2. Finally, COM enhanced invading capability, cell-aggregate formation, chemoresistance to cisplatin, and secretion of an angiogenic factor (VEGF). These data indicate that COM crystals trigger epithelial-mesenchymal transition (EMT) and several carcinogenic features in the non-cancerous renal cells. These mechanisms may explain and strengthen the association between KSD and RCC.

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.

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.

Trigonelline prevents kidney stone formation processes by inhibiting calcium oxalate crystallization, growth and crystal-cell adhesion, and downregulating crystal receptors.

Trigonelline is the second most abundant bioactive alkaloid found in coffee. It is classified as a phytoestrogen with similar structure as of estradiol and exhibits an estrogenic effect. A previous study has reported that fenugreek seed extract rich with trigonelline can reduce renal crystal deposition in ethylene glycol-induced nephrolithiatic rats. However, direct evidence of such anti-lithogenic effects of trigonelline and underlying mechanisms have not previously been reported. Our study therefore addressed the protective effects and mechanisms of trigonelline against kidney stone-forming processes using crystallization, crystal growth, aggregation and crystal-cell adhesion assays. Also, proteomics was applied to identify changes in receptors for calcium oxalate monohydrate (COM), the most common stone-forming crystal, on apical membranes of trigonelline-treated renal tubular cells. The analyses revealed that trigonelline significantly reduced COM crystal size, number and mass during crystallization. Additionally, trigonelline dose-dependently inhibited crystal growth and crystal-cell adhesion, but did not affect crystal aggregation. Mass spectrometric protein identification showed the smaller number of COM crystal receptors on apical membranes of the trigonelline-treated cells. Western blotting confirmed the decreased levels of some of these crystal receptors by trigonelline. These data highlight the protective mechanisms of trigonelline against kidney stone development by inhibiting COM crystallization, crystal growth and crystal-cell adhesion via downregulation of the crystal receptors on apical membranes of renal tubular cells.

Systematic analysis of modulating activities of native human urinary Tamm-Horsfall protein on calcium oxalate crystallization, growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix.

Functions of Tamm-Horsfall protein (THP), the most abundant human urinary protein, have been studied for decades. However, its precise roles in kidney stone formation remain controversial. In this study, we aimed to clarify the roles of native human urinary THP in calcium oxalate monohydrate (COM) kidney stone formation. THP was purified from the human urine by adsorption method using diatomaceous earth (DE). Its effects on stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix (ECM), were examined. SDS-PAGE and Western blotting confirmed that DE adsorption yielded 84.9% purity of the native THP isolated from the human urine. Systematic analyses revealed that THP (at 0.4-40 µg/ml) concentration-dependently reduced COM crystal size but did not affect the crystal mass during initial crystallization. At later steps, THP concentration-dependently inhibited COM crystal growth and aggregation, and prevented crystal-cell adhesion only at 40 µg/ml. However, THP did not affect crystal invasion through the ECM. Sequence analysis revealed two large calcium-binding domains (residues 65-107 and 108-149) and three small oxalate-binding domains (residues 199-207, 361-368 and 601-609) in human THP. Immunofluorescence study confirmed the binding of THP to COM crystals. Analyses for calcium-affinity and/or oxalate-affinity demonstrated that THP exerted a high affinity with only calcium, not oxalate. Functional validation revealed that saturation of THP with calcium, not with oxalate, could abolish the inhibitory effects of THP on COM crystal growth, aggregation and crystal-cell adhesion. These data highlight the inhibitory roles of the native human urinary THP in COM crystal growth, aggregation and crystal-cell adhesion, which are the important processes for kidney stone formation. Such inhibitory effects of THP are most likely mediated via its high affinity with calcium ions.

ARID1A knockdown enhances carcinogenesis features and aggressiveness of Caco-2 colon cancer cells: An in vitro cellular mechanism study.

Loss of ARID1A, a tumor suppressor gene, is associated with the higher grade of colorectal cancer (CRC). However, molecular and cellular mechanisms underlying the progression and aggressiveness of CRC induced by the loss of ARID1A remain poorly understood. Herein, we evaluated cellular mechanisms underlying the effects of ARID1A knockdown on the carcinogenesis features and aggressiveness of CRC cells. A human CRC cell line (Caco-2) was transfected with small interfering RNA (siRNA) specific to ARID1A (siARID1A) or scrambled (non-specific) siRNA (siControl). Cell death, proliferation, senescence, chemoresistance and invasion were then evaluated. In addition, formation of polyploid giant cancer cells (PGCCs), self-aggregation (multicellular spheroid) and secretion of an angiogenic factor, vascular endothelial growth factor (VEGF), were examined. The results showed that ARID1A knockdown led to significant decreases in cell death and senescence. On the other hand, ARID1A knockdown enhanced cell proliferation, chemoresistance and invasion. The siARID1A-transfected cells also had greater number of PGCCs and larger spheroid size and secreted greater level of VEGF compared with the siControl-transfected cells. These data, at least in part, explain the cellular mechanisms of ARID1A deficiency in carcinogenesis and aggressiveness features of CRC.