Eun-Sun Ryu

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-β1-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-β1 (TGF-β1)-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-β1-induced EMT. RU486, a glucocorticoid receptor (GR) antagonist, blocked the effect of DEXA on the TGF-β1-induced EMT. Importantly, DEXA also induced the mesenchymal-to-epithelial transition of TGF-β1-stimulated HPMCs. The beneficial effect of DEXA on the TGF-β1-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-β1-induced activation of Smad2/3 signaling. DEXA inhibited glycogen synthase kinase-3β (GSK-3β) phosphorylation and the Snail upregulation induced by TGF-β1, 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-β1-stimulated HPMCs by inhibiting MAPK activation, GSK-3β phosphorylation and Snail upregulation.

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-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.

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-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.

Paricalcitol attenuates TGF-β1–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‐lβ and IL‐18. Paricalcitol is reported to exert an antiinflammatory 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‐βl‐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 ρrocasρase‐1, with an increased production of IL‐lβ and IL‐18, which was ameliorated by small interfering (si)NLRP3, siASC, caspase inhibitors, or neutralizing antibodies for IL‐lβ and IL‐18. TGF‐βl enhanced reactive oxygen species generation with an increase in NADPH oxidase (NOX) activity and mitochondrial NOX4 production. Paricalcitol alleviated TGF‐βl‐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‐βl–induced phenotype transition of human peritoneal mesothelial cells (HPMCs) via modulation of oxidative stress and NLRP3 inflammasome. FASEB J. 33, 3035–3050 (2019). www.fasebj.org

OS 17-09 URIC ACID-INDUCED ENDOTHELIAL-TO-MESENCHYMAL TRANSITION (Endo-MT) VIA OXIDATIVE STRESS AND GLYCOCALYX SHEDDING

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, &agr;-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 &agr;-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 &mgr;M). Anti-oxidant treatment [NAC (5mM), apocynin (100 &mgr;M), and mitotempo (10 &mgr;M)] 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 &agr;-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.