Uric acid induced the phenotype transition of vascular endothelial cells via induction of oxidative stress and glycocalyx shedding.

Recent data suggested a causative role of uric acid (UA) in the development of renal disease, in which endothelial dysfunction is regarded as the key mechanism. Endothelial-to-mesenchymal transition (EndoMT) and shedding of the glycocalyx are early changes of endothelial dysfunction. We investigated whether UA induced EndoMT in HUVECs and an animal model of hyperuricemia fed with 2% oxonic acid for 4 wk. UA induced EndoMT in HUVECs with a generation of reactive oxygen species via the activation of membranous NADPH oxidase (from 15 min) and mitochondria (from 6 h) along with glycocalyx shedding (from 6 h), which were blocked by probenecid. GM6001, an inhibitor of matrix metalloproteinase, alleviated UA-induced glycocalyx shedding and EndoMT. Antioxidants including N-acetyl cysteine, apocynin, and mitotempo ameliorated EndoMT; however, they did not change glycocalyx shedding in HUVECs. In the kidney of hyperuricemic rats, endothelial staining in peritubular capillaries (PTCs) was substantially decreased with a de novo expression of α-smooth muscle actin in PTCs. Plasma level of syndecan-1 was increased in hyperuricemic rats, which was ameliorated by allopurinol. UA caused a phenotypic transition of endothelial cells via induction of oxidative stress with glycocalyx shedding, which could be one of the mechanisms of UA-induced endothelial dysfunction and kidney disease.-Ko, J., Kang, H.-J., Kim, D.-A., Kim, M.-J., Ryu, E.-S., Lee, S., Ryu, J.-H., Roncal, C., Johnson, R. J., Kang, D.-H. Uric acid induced the phenotype transition of vascular endothelial cells via induction of oxidative stress and glycocalyx shedding.

Paricalcitol attenuates TGF-ß1-induced phenotype transition of human peritoneal mesothelial cells (HPMCs) via modulation of oxidative stress and NLRP3 inflammasome.

Phenotype transition of mesothelial cells, such as epithelial-to-mesenchymal transition (EMT), is one of the early mechanisms of peritoneal fibrosis, which is mediated by oxidative stress and inflammation. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein oligomer that promotes the maturation of IL-1ß and IL-18. Paricalcitol is reported to exert an anti-inflammatory effect; however, there are no studies as to whether paricalcitol modulates the activation of NLRP3 inflammasome. We investigated the role of NLRP3 inflammasome in peritoneal EMT with an exploration of the effect of paricalcitol on oxidative stress, NLRP3 inflammasome, and EMT of mesothelial cells. TGF-ß1-induced EMT in human peritoneal mesothelial cells (HPMCs) was associated with an up-regulation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and procaspase-1, with an increased production of IL-1ß and IL-18, which was ameliorated by small interfering (si)NLRP3, siASC, caspase inhibitors, or neutralizing antibodies for IL-1ß and IL-18. TGF-ß1 enhanced reactive oxygen species generation with an increase in NADPH oxidase (NOX) activity and mitochondrial NOX4 production. Paricalcitol alleviated TGF-ß1-induced EMT and the NLRP3 inflammasome, which was associated with a down-regulation of NOX activity by interfering with p47phox and p22phox interaction and mitochondrial NOX4 production in HPMCs. Taken together, paricalcitol ameliorated EMT of HPMCs via modulating an NOX-dependent increase in the activity of NLRP3 inflammasome. Paricalcitol could be a novel approach to protect the peritoneum from the development of EMT and peritoneal fibrosis.-Ko, J., Kang, H.-J., Kim, D.-A., Ryu, E.-S., Yu, M., Lee, H., Lee, H. K., Ryu, H.-M., Park, S.-H., Kim, Y.-L., Kang, D.-H. Paricalcitol attenuates TGF-ß1-induced phenotype transition of human peritoneal mesothelial cells (HPMCs) via modulation of oxidative stress and NLRP3 inflammasome.

Endoplasmic reticulum stress as a novel target to ameliorate epithelial-to-mesenchymal transition and apoptosis of human peritoneal mesothelial cells.

Epithelial-to-mesenchymal transition (EMT) and apoptosis of peritoneal mesothelial cells are known to be the earliest mechanisms of peritoneal fibrosis in peritoneal dialysis (PD). Endoplasmic reticulum (ER) stress with an unfolded protein response is regarded to have a role in the development of organ fibrosis. To investigate the potential role of ER stress as a target to prevent and/or delay the development of peritoneal fibrosis, we examined the effect of ER stress on EMT or apoptosis of human peritoneal mesothelial cells (HPMCs) and elucidated the mechanisms underlying the protective effect of ER stress preconditioning on TGF-ß1-induced EMT. ER stress inducers, tunicamycin (TM) and thapsigargin (TG), induced EMT with Smad2/3 phosphorylation, an increased nuclear translocation of ß-catenin and Snail expression. Low concentrations of TM and TG did not induce apoptosis within 48 h; however, high concentrations of TM- (>1 ng/ml) and TG- (>1 nM) induced apoptosis at 12 h with a persistent increase in C/EBP homologous protein. TGF-ß1 induced EMT and apoptosis in HPMCs, which was ameliorated by taurine-conjugated ursodeoxycholic acid, an ER stress blocker. Interestingly, pre-treatment with TM or TG for 4 h also protected the cells from TGF-ß1-induced EMT and apoptosis, demonstrating the role of ER stress as an adaptive response to protect HPMCs from EMT and apoptosis. Peritoneal mesothelial cells isolated from PD patients displayed an increase in GRP78/94, which was correlated with the degree of EMT. These findings suggest that the modulation of ER stress in HPMCs could serve as a novel approach to ameliorate peritoneal damage in PD patients.

Uric acid-induced phenotypic transition of renal tubular cells as a novel mechanism of chronic kidney disease.

Recent experimental and clinical studies suggest a causal role of uric acid in the development of chronic kidney disease. Most studies have focused on uric acid-induced endothelial dysfunction, oxidative stress, and inflammation in the kidney. The direct effects of uric acid on tubular cells have not been studied in detail, and whether uric acid can mediate phenotypic transition of renal tubular cells such as epithelial-to-mesenchymal transition (EMT) is not known. We therefore investigated whether uric acid could alter E-cadherin expression and EMT in the kidney of hyperuricemic rats and in cultured renal tubular cells (NRK cells). Experimental hyperuricemia was associated with evidence of EMT before the development of significant tubulointerstitial fibrosis at 4 wk, as shown by decreased E-cadherin expression and an increased α-smooth muscle actin (α-SMA). Allopurinol significantly inhibited uric acid-induced changes in E-cadherin and α-SMA with an amelioration of renal fibrosis at 6 wk. In cultured NRK cells, uric acid induced EMT, which was blocked by the organic anion transport inhibitor probenecid. Uric acid increased expression of transcriptional factors associated with decreased synthesis of E-cadherin (Snail and Slug). Uric acid also increased the degradation of E-cadherin via ubiquitination, which is of importance since downregulation of E-cadherin is considered to be a triggering mechanism for EMT. In conclusion, uric acid induces EMT of renal tubular cells decreasing E-cadherin synthesis via an activation of Snail and Slug as well as increasing the degradation of E-cadherin.

Effects of dexamethasone on the TGF-ß1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells.

The epithelial-to-mesenchymal transition (EMT) is known to have a role in appropriate embryonic development, the physiological response to injury and pathological events such as organ fibrosis and cancer progression. Glucocorticoid (GC), one of the most commonly used anti-inflammatory drugs, inhibits the deposition of extracellular matrix independent of its anti-inflammatory effect. The EMT of human peritoneal mesothelial cells (HPMCs) is a key mechanism of peritoneal fibrosis; however, it has not yet been investigated whether GC imposes any effect on the EMT of HPMCs. To investigate the therapeutic potential of GC on preserving peritoneal membrane function, we studied the effect of dexamethasone (DEXA), a synthetic GC, on the transforming growth factor-ß1 (TGF-ß1)-induced EMT in HPMCs. As assessed by changes in cell morphology, the expression of epithelial and mesenchymal cell markers (such as E-cadherin, ZO-1 and α-SMA, α-smooth muscle actin) and cell migration, DEXA inhibited the TGF-ß1-induced EMT. RU486, a glucocorticoid receptor (GR) antagonist, blocked the effect of DEXA on the TGF-ß1-induced EMT. Importantly, DEXA also induced the mesenchymal-to-epithelial transition of TGF-ß1-stimulated HPMCs. The beneficial effect of DEXA on the TGF-ß1-induced EMT was mediated through the amelioration of ERK and p38 mitogen-activated protein kinase (MAPK) phosphorylation; however, this effect was not related to the TGF-ß1-induced activation of Smad2/3 signaling. DEXA inhibited glycogen synthase kinase-3ß (GSK-3ß) phosphorylation and the Snail upregulation induced by TGF-ß1, which were also ameliorated by inhibitors of MAPK. In conclusion, this is the first study demonstrating the protective effect of DEXA on the EMT in TGF-ß1-stimulated HPMCs by inhibiting MAPK activation, GSK-3ß phosphorylation and Snail upregulation.

Indoxyl sulfate-induced epithelial-to-mesenchymal transition and apoptosis of renal tubular cells as novel mechanisms of progression of renal disease.

Indoxyl sulfate (IS), one of the uremic toxins, is regarded to have a substantial role in the progression of chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) and apoptosis of renal tubular cells are known to be the critical mechanisms of the development and aggravation of CKD. We investigated the effect of IS on EMT and apoptosis in renal proximal tubular cells, NRK-52E cells. IS significantly inhibited cell proliferation and induced cell migration with a morphological transition from cuboidal epithelial cells to spindle-shaped scattered fibroblast-like cells. IS downregulated the expressions of zonula occluden-1 and E-cadherin, whereas upregulated α-SMA expression at 48 h, which was blocked by a pretreatment of the organic anion transporter, probenecid. IS also induced apoptosis of NRK cells from a concentration of 25 µg/ml with an activation of ERK1/2 and p38 MAP kinase (MAPK). Pretreatment of ERK1/2 or p38 MAPK inhibitors, PD98059 or SB203580, resulted in no significant effect on IS-induced EMT, whereas it ameliorated IS-induced apoptosis of NRK cells. These findings suggested phenotypic transition and apoptosis as potential mechanisms of IS-induced renal damage and the differential role of MAPK activation in IS-induced EMT and apoptosis of renal tubular cells.

Tannic acid attenuates the formation of cancer stem cells by inhibiting NF-κB-mediated phenotype transition of breast cancer cells.

Cancer stem cells (CSCs) are innately resistant to standard therapies, which positions CSCs in the focus of anti-cancer research. In this study, we investigated the potential inhibitory effect of tannic acid (TA) on CSCs. Our data demonstrated that TA (10 µM), at the concentration not inhibiting the proliferation of normal mammary cells (MCF10A), inhibited the formation and growth of mammosphere in MCF7, T47D, MDA-MB-231 cells shown as a decrease in mammosphere formation efficiency (MFE), cell number, diameter of mammosphere, and ALDH1 activity. NF-κB pathway was activated in the mammosphere indicated by an up-regulation of p65, a degradation of IκBα, and an increased IL-6. The inhibition of NF-κB pathway via gene silencing of p65 (sip65), NF-κB inhibitor (PDTC), or IKK inhibitor (Bay11-7082) alleviated MFE. Other CSCs markers such as an increase in ALDH1 and CD44high/CD24low ratio were ameliorated by sip65. TA also alleviated TGFß-induced EMT, increase in MFE, and NF-κB activation. In murine xenograft model, TA reduced tumor volume which was associated with a decrease in CD44high/CD24low expression and IKK phosphorylation. These results suggest that TA negatively regulates CSCs by inhibiting NF-κB activation and thereby prevents cancer cells from undergoing EMT and CSCs formation, and may thus be a promising therapy targeting CSCs.

Metformin ameliorates the Phenotype Transition of Peritoneal Mesothelial Cells and Peritoneal Fibrosis via a modulation of Oxidative Stress.

Phenotype transition of peritoneum is an early mechanism of peritoneal fibrosis. Metformin, 5'-adenosine monophosphate-activated protein kinase (AMPK) activator, has recently received a new attention due to its preventive effect on organ fibrosis and cancer metastasis by inhibiting epithelial-to-mesenchymal transition (EMT). We investigated the effect of metformin on EMT of human peritoneal mesothelial cells (HPMC) and animal model of peritoneal dialysis (PD). TGF-ß1-induced EMT in HPMC was ameliorated by metformin. Metformin alleviated NAPDH oxidase- and mitochondria-mediated ROS production with an increase in superoxide dismutase (SOD) activity and SOD2 expression. Metformin inhibited the activation of Smad2/3 and MAPK, GSK-3ß phosphorylation, nuclear translocalization of ß-catenin and Snail in HPMCs. Effect of metformin on TGF-ß1-induced EMT was ameliorated by either AMPK inhibitor or AMPK gene silencing. Another AMPK agonist, 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide partially blocked TGF-ß1-induced EMT. In animal model of PD, intraperitoneal metformin decreased the peritoneal thickness and EMT with an increase in ratio of reduced to oxidized glutathione and the expression of SOD whereas it decreased the expression of nitrotyrosine and 8-hydroxy-2'-deoxyguanosine. Therefore, a modulation of AMPK in peritoneum can be a novel tool to prevent peritoneal fibrosis by providing a favorable oxidant/anti-oxidant milieu in peritoneal cavity and ameliorating phenotype transition of peritoneal mesothelial cells.