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. FASEB J. 33, 13334–13345 (2019). www.fasebj.org

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 TGFβ-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.

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-1β 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-β1-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-1β and IL-18, which was ameliorated by small interfering (si)NLRP3, siASC, caspase inhibitors, or neutralizing antibodies for IL-1β and IL-18. TGF-β1 enhanced reactive oxygen species generation with an increase in NADPH oxidase (NOX) activity and mitochondrial NOX4 production. Paricalcitol alleviated TGF-β1-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-β1-induced phenotype transition of human peritoneal mesothelial cells (HPMCs) via modulation of oxidative stress and NLRP3 inflammasome.

// Dal-Ah Kim 1, 2 , Hack Sun Choi 1, 2 , Eun-Sun Ryu 1, 2 , Jiyeon Ko 1, 2 , Heesung Chung 3 , Eun-Sung Jun 4 , Eok-Soo Oh 3 and Duk-Hee Kang 1, 2 1 The Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul 07985, Republic of Korea 2 Ewha Medical Research Center, Ewha Womans University School of Medicine, Seoul 07985, Republic of Korea 3 Department of Life Science, The Research Center Homeostasis, Ewha Womans University, Seoul 03760, Republic of Korea 4 Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, South Korea Correspondence to: Duk-Hee Kang, email: dhkang@ewha.ac.kr Keywords: cancer stem cells; tannic acid; mammosphere; NF-kB signaling; p65 Received: October 16, 2017 Accepted: January 03, 2018 Epub: January 02, 2018 ABSTRACT Cancer stem cells (CSCs) are known to mediate metastasis and recurrence, and are therefore a promising therapeutic target. Likewise, aberrant NF-κB signaling has been identified in many cancers, and shown to promote CSC formation via several mechanisms, including induction of epithelial-to-mesenchymal transition (EMT). Tannic acid (C76H52O46, TA) is reported to inhibit the proliferation of cancer cells, however there is no data regarding the effect of TA on CSCs. The present study investigated the effects of TA on CSC formation, NF-κB signaling, and EMT in breast cancer cells. TA inhibited the formation and growth of mammosphere in MCF7 cells expressed as a decrease in mammosphere formation efficiency (MFE) and ALDH1 activity. An activation of NF-κB pathway was observed in MCF7-derived 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) and IKK inhibitor (Bay11-7082) alleviated MFE. Other markers of CSCs such as an increase in ALDH1 and the number of CD44 high /CD24 low cells were ameliorated by interfering p65. TA alleviated the markers of NF-κB activation in MCF7-derived mammosphere. TGFβ-induced EMT, increase in MFE and NF-κB activation was alleviated by TA. In in-vivo murine xenograft model, tumor volume was decreased by TA with a decrease in CD44 and IKK phosphorylation. Taken together, these results strongly suggest TA as a promising potential therapeutic agent capable of inhibiting NF-κB signaling in breast CSCs, and thereby preventing cancer cells from undergoing EMT and subsequent metastasis.

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-β1-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-3β phosphorylation, nuclear translocalization of β-catenin and Snail in HPMCs. Effect of metformin on TGF-β1-induced EMT was ameliorated by either AMPK inhibitor or AMPK gene silencing. Another AMPK agonist, 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide partially blocked TGF-β1-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.

Objective: Recent data suggested a role of uric acid (UA) in the pathogenesis of cardiovascular and renal disease. Endothelial dysfunction, which is characterized by a decrease in nitric oxide, is regarded as the key mechanism of UA-induced vascular disease. Endo-MT is an early process of endothelial dysfunction, and is also known to play a role in the progression of renal fibrosis. Glycocalyx is a structure covering endothelium composed of membrane-bound proteoglycans and glycoproteins associated with adsorbed plasma components, which may lead to endothelial dysfunction via intraluminal shedding. Design and Method: Endo-MT was evaluated by cell morphology and the expression of the endothelial markers, VE-cadherin or CD31 and the mesenchymal marker, α-SMA by real time PCR, western blotting (WB) and immunocytochemistry in HUVECs and animal model of hyperuricemia (Sprague-Dawley rats fed with 2% oxonic acid for 6 weeks). NAPDH oxidase (NOX) activity, reactive oxygen species (ROS) production, endothelial permeability and glycocalyx shedding were evaluated by WB and ELISA. Results: Stimulation of HUVEC with UA resulted in an alteration of cell morphology into fibroblastoid cells associated with a decrease in CD31 and VE-cadherin and de-novo α-SMA expression from 24 hours. UA increased ROS production via NOX (15 min) and mitochondrial activation (6 hours) with an increase in glycocalyx shedding (6 hours), which were blocked by an inhibitor of organic anion transporter, probenecid (5 μM). Anti-oxidant treatment [NAC (5mM), apocynin (100 μM), and mitotempo (10 μM)] ameliorated endo-MT and glycocalyx shedding in HUVEC. Matrix metalloproteinase inhibitor, GM6001, also alleviated UA-induced endoMT. In the kidney of hyperuricemic rats, endothelial staining in peritubular capillaries was decreased with de-novo staining of α-SMA, which was ameliorated allopurinol treatment. Conclusions: UA per se induced a phenotypic transition of endothelial cells via oxidative stress and glycocalyx shedding, which could be one of the mechanisms of UA-induced vascular disease.

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-β1-induced EMT. ER stress inducers, tunicamycin (TM) and thapsigargin (TG), induced EMT with Smad2/3 phosphorylation, an increased nuclear translocation of β-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-β1 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-β1-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.

Introduction and Aims: Management of Renal failure in the setting of HIV infection has been scarcelystudied particularly in low resource settings where accessto renal replacement therapy is not widelyavailable. This study aims to evaluate the outcomes of Continuous Ambulatory Peritoneal Dialysis (CAPD) in the management of renal failure associated with HIV infection. Methods: This is an ongoing prospective cohort study following-up 70 HIV positive and 70 HIV negative renal failure patients newly inserted atenckhoff catheter in our unit between September 2012 and February 2015, monthly followed up data of the first 365 days was used to assess outcomes of catheter failure and mortality Results: Both the HIV positive cohort and the HIV negative controls had statistically similar catheter failure rates 0.252 vs 0.270 per-person-years, RR 0.93 (95% CI 0.38 - 2.21 p=0.873) with severe or non-resolving peritonitis being the leading cause for catheter removal (84.6% vs 80.0%, respectively). However, the HIV positive cohort had a lower proportion of patients with a patent catheter reaching 1 year (45.7% vs 58.6%, p=0.128) due to an increased all-cause mortality rate (0.550 vs 0.251 per-person-years, RR 2.20 (CI 1.07- 4.69, P=0.0207), with sudden unspecified home death accounting for the majorityof mortalities (50% vs 62%, respectively) whereas infective causes were associated with 37.5% and 30.8% of deaths, respectively.Baseline factors associated with the increased mortality risk were CD4 count of less than 200/µl, unsuppressed viral load, and ARV duration of lessthan 6 months, linked to COX hazard ratios of 5.39 (95% CI 2.20 to 13.21, p<0.001), 2.79 (CI 1.34 to 5.80, p=0.006), and 2.65 (CI 1.26 to 5.58, p=0.010), respectively Conclusions: This study suggeststhatHIV infection in patients on CAPD does not adversely influence catheter failure rates at one year, however, uncontrolled HIV infection may be associated with increased mortality relative risk.

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.

Eun-Sun Ryu, Mi Jin Kim, Hyun-Soo Shin, Yang-Hee Jang, Hack Sun Choi, Inho Jo, Richard J. Johnson, and Duk-Hee Kang Division of Nephrology, Department of Internal Medicine, Ewha Womans University School of Medicine, Ewha Medical Research Center, Seoul, Korea; Department of Molecular Medicine, Ewha Womans University School of Medicine, Ewha Medical Research Center, Seoul, Korea; and Division of Renal Diseases and Hypertension, University of Colorado-Denver, Aurora, Colorado

Eun-Sun Ryu, Mi Jin Kim, Hyun-Soo Shin, Yang-Hee Jang, Hack 6 Sun Choi, Inho Jo, Richard J. Johnson, and Duk-Hee Kang 7 8 9 10 The Division of Nephrology, Department of Internal Medicine,Department of 11 Molecular Medicine, Ewha Womans University School of Medicine, Ewha Medical 12 Research Center, Seoul, Korea; The Division of Renal Diseases and Hypertension, 13 University of Colorado Denver, Aurora, Colorado, U. S. A. 14 15 16 17 18 Address for reprint requests and other correspondence: Duk-Hee Kang, M.D., Ph.D., 19 Division of Nephrology, Ewha University College of Medicine, 911 Mok-dong 20 Yangcheon-Ku, Seoul 158-710, Korea (e-mail : dhkang@ewha.ac.kr). 21 22 Articles in PresS. Am J Physiol Renal Physiol (January 2, 2013). doi:10.1152/ajprenal.00560.2012