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.

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.

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.

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.

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.

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.

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.

Effects of secretome derived from macrophages exposed to calcium oxalate crystals on renal fibroblast activation.

The association between kidney stone disease and renal fibrosis has been widely explored in recent years but its underlying mechanisms remain far from complete understanding. Using label-free quantitative proteomics (nanoLC-ESI-LTQ-Orbitrap MS/MS), this study identified 23 significantly altered secreted proteins from calcium oxalate monohydrate (COM)-exposed macrophages (COM-MP) compared with control macrophages (Ctrl-MP) secretome. Functional annotation and protein-protein interactions network analysis revealed that these altered secreted proteins were involved mainly in inflammatory response and fibroblast activation. BHK-21 renal fibroblasts treated with COM-MP secretome had more spindle-shaped morphology with greater spindle index. Immunofluorescence study and gelatin zymography revealed increased levels of fibroblast activation markers (α-smooth muscle actin and F-actin) and fibrotic factors (fibronectin and matrix metalloproteinase-9 and -2) in the COM-MP secretome-treated fibroblasts. Our findings indicate that proteins secreted from macrophages exposed to COM crystals induce renal fibroblast activation and may play important roles in renal fibrogenesis in kidney stone disease.

Calcium oxalate monohydrate crystal disrupts tight junction via F-actin reorganization.

Tight junction is an intercellular protein complex that regulates paracellular permeability and epithelial cell polarization. This intercellular barrier is associated with actin filament. Calcium oxalate monohydrate (COM), the major crystalline composition in kidney stones, has been shown to disrupt tight junction but with an unclear mechanism. This study aimed to address whether COM crystal disrupts tight junction via actin deregulation. MDCK distal renal tubular epithelial cells were treated with 100 µg/ml COM crystals for 48 h. Western blot analysis revealed that level of a tight junction protein, zonula occludens-1 (ZO-1), significantly decreased, whereas that of ß-actin remained unchanged after exposure to COM crystals. Immunofluorescence study showed discontinuation and dissociation of ZO-1 and filamentous actin (F-actin) expression at the cell border. In addition, clumping of F-actin was found in some cytoplasmic areas of the COM-treated cells. Moreover, transepithelial resistance (TER) was reduced by COM crystals, indicating the defective barrier function of the polarized cells. All of these COM-induced defects could be completely abolished by pretreatment with 20 µM phalloidin, an F-actin stabilizer, 2-h prior to the 48-h crystal exposure. These findings indicate that COM crystal does not reduce the total level of actin but causes tight junction disruption via F-actin reorganization.

ARID1A knockdown in human endothelial cells directly induces angiogenesis by regulating angiopoietin-2 secretion and endothelial cell activity.

AT-rich interactive domain 1A (ARID1A) is a novel tumor suppressor gene found in several human cells and its loss/defect is commonly observed in many cancers. However, its roles in angiogenesis, which is one of the hallmarks for tumor progression, remained unclear. Herein, we demonstrated the direct effects of ARID1A knockdown in human endothelial cells by lentivirus-based short-hairpin RNA (shRNA) (shARID1A) on angiogenesis. Functional assays revealed that shARID1A significantly enhanced cell proliferation and migration/invasion and endothelial tube formation compared with the control cells transfected with scramble shRNA (shControl). Additionally, the shARID1A-transfected cells had significantly increased podosome formation and secretion of angiopoietin-2 (ANG2), a key angiogenic factor. Moreover, neutralization of ANG2 with monoclonal anti-ANG2 antibody strongly reduced cell proliferation and migration/invasion and endothelial tube formation in the shARID1A-transfected cells. These findings indicate that down-regulation of ARID1A in human endothelial cells directly induces angiogenesis by regulating angiopoietin-2 secretion and endothelial cell activity.

Differential bound proteins and adhesive capabilities of calcium oxalate monohydrate crystals with various sizes.

Adhesion of calcium oxalate (CaOx) crystals onto renal tubular epithelial cells is one of the critical steps in kidney stone formation. However, effects of crystal size on the crystal adhesive capability remained unclear. This study compared the adhesive capabilities of CaOx monohydrate (COM) crystals with various sizes (<10 µm, 20-30 µm, 50-60 µm, and > 80 µm). Crystal-cell adhesion assay showed size-dependent increase of COM crystal adhesion onto epithelial cell surface using the larger crystals. Identification of apical membrane proteins that could bind to COM crystals by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) demonstrated size-specific sets of the COM crystal-binding proteins. Among these, numbers of known oxalate-binding proteins and COM crystal receptors were greatest in the set of the largest size (>80 µm). Atomic force microscopy (AFM) revealed that adhesive forces between carboxylic-immobilized AFM tip and COM crystal surface and between COM-mounted AFM tip and renal epithelial cell surface were size-dependent (greater for the larger crystals). In summary, the adhesive capability of COM crystals is size-dependent - the larger the greater adhesive capability. These data may help better understanding of the pathogenic mechanisms of kidney stone formation at an initial stage when renal tubular cells are exposed to various sizes of COM crystals.

Protective roles of trigonelline against oxalate-induced epithelial-to-mesenchymal transition in renal tubular epithelial cells: An in vitro study.

Fibrogenesis is a common feature for all types of chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the main processes involving renal fibrosis and its inhibition is considered as a preventive/therapeutic strategy for CKD. Trigonelline (TRIG), a plant alkaloid commonly found in herbs, coffee bean, soy bean and other edible food plants, has several beneficial effects on human health and has been proposed to reduce renal fibrosis but with unclear mechanisms. This study thus addressed cellular mechanism underlying the anti-fibrogenic effects of TRIG in renal tubular epithelial cells grown in vitro. EMT was successfully induced by oxalate treatment as indicated by morphological changes into spindle-shape cells, increased expression of mesenchymal proteins (fibronectin, vimentin and α-smooth muscle actin (α-SMA)), decreased expression of epithelial proteins (E-cadherin and zonula occludens-1 (ZO-1)) and increased activity of a profibrotic factor (matrix metalloproteinase-9 (MMP-9)). Interestingly, these oxalate-induced EMT features could be attenuated by TRIG pretreatment. Moreover, TRIG also prevented oxalate-induced cell migration, reactive oxygen species (ROS) overproduction, and down-regulation of Nrf-2 signaling molecule. These data indicated that TRIG could attenuate the effects of oxalate-induced EMT and thus may serve as the anti-fibrotic compound for prevention and/or treatment of CKD.

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.

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.

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.

Protein Network Analysis and Functional Studies of Calcium Oxalate Crystal-Induced Cytotoxicity in Renal Tubular Epithelial Cells.

Our previous expression study has reported a set of proteins with altered levels in renal tubular cells after exposure to calcium oxalate monohydrate (COM) crystals, which are the main composition of kidney stones. However, their functional significance remained largely unknown. In this study, protein network analysis revealed that the significantly altered proteins induced by COM crystals were involved mainly in three main functional networks, including i) cell proliferation and wound healing; ii) oxidative stress and mitochondrial function; and iii) cellular junction complex and integrity. Cell proliferation and wound healing assays showed that the COM-treated cells had defective proliferation and tissue healing capability, respectively. Oxyblot analysis demonstrated accumulation of the oxidized proteins, whereas intracellular ATP level was significantly increased in the COM-treated cells. Additionally, level of zonula occludens-1 (ZO-1), a tight junction protein, was significantly decreased, consistent with the significant declines in transepithelial resistance (TER) and level of RhoA signaling molecule in the COM-treated cells. These findings indicate significant perturbations in mitochondrial and oxidative stress axis that cause defective cell proliferation, tissue healing capability, junctional protein complex, and cellular integrity of renal tubular epithelial cells exposed to COM crystals that may play important roles in kidney stone pathogenesis.

Systematic evaluation for effects of urine pH on calcium oxalate crystallization, crystal-cell adhesion and internalization into renal tubular cells.

Urine pH has been thought to be an important factor that can modulate kidney stone formation. Nevertheless, there was no systematic evaluation of such pH effect. Our present study thus addressed effects of differential urine pH (4.0-8.0) on calcium oxalate (CaOx) crystallization, crystal-cell adhesion, crystal internalization into renal tubular cells, and binding of apical membrane proteins to the crystals. Microscopic examination revealed that CaOx monohydrate (COM), the pathogenic form, was crystallized with greatest size, number and total mass at pH 4.0 and least crystallized at pH 8.0, whereas COD was crystallized with the vice versa order. Fourier-transform infrared (FT-IR) spectroscopy confirmed such morphological study. Crystal-cell adhesion assay showed the greatest degree of crystal-cell adhesion at the most acidic pH and least at the most basic pH. Crystal internalization assay using fluorescein isothiocyanate (FITC)-labelled crystals and flow cytometry demonstrated that crystal internalization into renal tubular cells was maximal at the neutral pH (7.0). Finally, there were no significant differences in binding capacity of the crystals to apical membrane proteins at different pH. We concluded that the acidic urine pH may promote CaOx kidney stone formation, whereas the basic urine pH (i.e. by alkalinization) may help to prevent CaOx kidney stone disease.

Chromosome-centric Human Proteome Project (C-HPP): Chromosome 12.

Following an official announcement of the Chromosome-centric Human Proteome Project (C-HPP), the Chromosome 12 (Ch12) Consortium has been established by five representative teams from five Asian countries including Thailand (Siriraj Hospital, Mahidol University), Singapore (National University of Singapore), Taiwan (Academia Sinica), Hong Kong (The Chinese University of Hong Kong), and India (Institute of Bioinformatics). We have worked closely together to extensively and systematically analyze all missing and known proteins encoded by Ch12 for their tissue/cellular/subcellular localizations. The target organs/tissues/cells include kidney, brain, gastrointestinal tissues, blood/immune cells, and stem cells. In the later phase, post-translational modifications and functional significance of Ch12-encoded proteins as well as their associations with human diseases (i.e., immune diseases, metabolic disorders, and cancers) will be defined. We have collaborated with other chromosome teams, Human Kidney and Urine Proteome Project (HKUPP), AOHUPO Membrane Proteomics Initiative, and other existing HUPO initiatives in the Biology/Disease-Based Human Proteome Project (B/D-HPP) to delineate functional roles and medical implications of Ch12-encoded proteins. The data set to be obtained from this multicountry consortium will be an important piece of the jigsaw puzzle to fulfill the missions and goals of the C-HPP and the global Human Proteome Project (HPP).

p38 MAPK mediates calcium oxalate crystal-induced tight junction disruption in distal renal tubular epithelial cells.

We examined whether p38 MAPK plays role in calcium oxalate monohydrate (COM) crystal-induced tight junction disruption. Polarized MDCK cells were pretreated with or without 20 µM SB239063 (p38 MAPK inhibitor) for 2-h, and then incubated with 100 µg/ml COM crystals for up to 48-h. Western blotting showed increased level of phospho-p38, not total p38, in COM-treated cells, whereas SB239063 pretreatment successfully maintained phospho-p38 at its basal level. COM crystals also caused decreased levels of two tight junction proteins, zonula occludens-1 (ZO-1) and occludin. Immunofluorescence study revealed disruption of tight junction, redistribution, and dissociation of ZO-1 and occludin. Moreover, transepithelial resistance (TER) showed defective barrier function, whereas Western blotting for Na(+)/K(+)-ATPase-α1 revealed defective fence function of tight junction in COM-treated cells. All these expression and functional defects were successfully prevented by SB239063 pretreatment. These findings indicate that COM crystals cause tight junction disruption in distal renal tubular epithelial cells through p38 MAPK activation.