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 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.

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.

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.

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.