Ryu ES, Kim MJ, Shin HS, Jang YH, Choi HS, Jo I, Johnson RJ, Kang DH. Uric acid-induced phenotypic transition of renal tubular cells as a novel mechanism of chronic kidney disease.

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.

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-b1 (TGF-b1)-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 a-SMA, a-smooth muscle actin) and cell migration, DEXA inhibited the TGF-b1-induced EMT. RU486, a glucocorticoid receptor (GR) antagonist, blocked the effect of DEXA on the TGF-b1-induced EMT. Importantly, DEXA also induced the mesenchymal-to-epithelial transition of TGF-b1-stimulated HPMCs. The beneficial effect of DEXA on the TGF-b1-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-b1-induced activation of Smad2/3 signaling. DEXA inhibited glycogen synthase kinase-3b (GSK-3b) phosphorylation and the Snail upregulation induced by TGF-b1, 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-b1-stimulated HPMCs by inhibiting MAPK activation, GSK-3b phosphorylation and Snail upregulation.

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 a-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 mg/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. KEYWORDS: apoptosis; chronic kidney disease; epithelial-to-mesenchymal transition; indoxyl sulfate; MAPKinase Indoxyl sulfate (IS) is one of the protein-bound uremic solutes derived from tryptophan, which is converted to indole and subsequently undergoes sulfate conjugation in hepatocytes to form IS.1 It is taken by organic anion transporters (OATs) at the basolateral membrane of proximal tubular cells, and excreted mostly into urine through tubular secretion.2 Therefore, IS accumulates in patients with chronic kidney disease (CKD), 3 demonstrated as an increase in serum level and positive immunostaining area of IS in the kidneys. 4 Although IS is regarded as a marker of renal dysfunction, recent data have revealed that IS also has a substantial role in the progression of CKD.5 Indole, the precursor of IS, caused glomerular sclerosis in uremic rats, 6 and oral administration of IS increased serum creatinine and decreased inulin clearance in uremic rats.3 In a clinical study, patients with urinary IS levels 490 mg/day showed faster progression rate of CKD than did those with urinary IS o30 mg/day.7 Furthermore, the administration of oral adsorbent of IS resulted in a decrease in glomerular sclerosis and interstitial fibrosis in uremic rats associated with a decrease in plasma and urinary IS levels.3 Lowering IS also resulted in an improvement of renal function and a delay in the initiation of dialysis in CKD patients. 8 In a multicenter, randomized clinical study, Schulman et al 9 showed that an administration of AST-120, oral adsorbent of IS, ameliorated uremic symptoms in a dose-dependent manner with a decrease in serum IS and a stable maintenance of serum creatinine. The mechanism of IS-induced renal progression has been expla...

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 a-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.

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