Eun-Joo Oh

Impact of low glucose degradation product bicarbonate/lactate-buffered dialysis solution on the epithelial-mesenchymal transition of peritoneum.

Background: Epithelial-mesenchymal transition (EMT) is important in the development of peritoneal fibrosis. Glucose degradation products (GDPs) may induce EMT in human peritoneal mesothelial cells (HPMCs). Methods: The effects of individual GDPs and GDPs derived from peritoneal dialysis fluid (PDF) in both HPMCs and peritoneal membranes were evaluated. EMT was assessed with α-smooth muscle actin (α-SMA) and E-cadherin. Results: In vitro, α-SMA protein and mRNA levels increased in the presence of the GDPs (formaldehyde, glyoxal, methylglyoxal, and 3-deoxyglucosone), and E-cadherin decreased. Changes in the EMT markers were most prominent after exposure to 3-deoxyglucosone. Changes in both α-SMA and E-cadherin protein levels were less with low (L)-GDP bicarbonate/lactate-buffered PDF compared to high (H)-GDP PDF. In the rat model after 8 weeks’ PDF infusion, the α-SMA/E-cadherin mRNA ratio increased in the H-GDP group compared with the L-GDP group (p < 0.05). The peritoneum in the H-GDP group tended to be thicker (p = 0.052) and had more blood vessels than that in the L-GDP group (p < 0.05). Tissue staining for TGF-β1 decreased in the L-GDP group. Dual-stained cytokeratin and α-SMA-positive myofibroblasts in the submesothelial layer were more prominent in the H-GDP group. Conclusion: GDPs found in PDF induce EMT of HPMCs, which is associated with peritoneal fibrosis and vascularization. Conversely, L-GDP PDF reduces EMT and peritoneal fibrosis.

Effects of Low Glucose Degradation Products Peritoneal Dialysis Fluid on the Peritoneal Fibrosis and Vascularization in a Chronic Rat Model

Abstract:  In the present study, we examined the effects of a new peritoneal dialysis fluid (PDF) with a low level of low glucose degradation products (GDP) on the functional and structural stability of the peritoneal membrane (PM). Male Sprague‐Dawley rats were divided into three groups: group C (n = 8), without dialysate infusion; group P (n = 12), infused with low‐level GDP solution (4.25% Physioneal, pH 7.0–7.4); and group D (n = 12), infused with conventional solution (4.25% Dianeal, pH 5.2, adjusted to pH 7.0). In groups D and P, animals were infused through a permanent catheter with 25 mL of PDF, twice daily for 8 weeks. Lipopolysaccharide was added into the PDF immediately before infusion on days 8, 9 and 10 in the two dialysis groups. When compared with group P, group D showed a higher glucose mass transfer at weeks 6 and 8, D/P urea at week 8, TGF‐β1 at weeks 4 and 8, and VEGF level at week 8. The submesothelial matrix layer of the parietal peritoneum was significantly thickened in group D and the lectin‐stained blood vessels in this layer were well‐visualized in group D compared with group P. There were significantly more peritoneal blood vessels in group D than group P. The transforming growth factor‐β induced gene‐h3 (βig‐h3) and TGF‐β1 levels in the peritoneal effluent correlated with the submesothelial thickness, which correlated with the dialysate‐to‐plasma ratio (D/P) of protein and, inversely, with the rate of glucose transport (D/D0 glucose, where D is glucose concentration in the dialysate and D0 is glucose concentration in the dialysis solution before it is infused into the peritoneal cavity). The present study showed that low‐GDP PDF effectively attenuated the peritoneal vascularization and fibrosis related to conventional solution.

Aquaporin 3 expression is up-regulated by TGF-β1 in rat peritoneal mesothelial cells and plays a role in wound healing.

Aquaporin 3 (AQP3) is expressed in many tissues including the peritoneum and kidney. In cultured mesothelial cells, glucose up-regulates AQP3, which may be important for water transport through the peritoneal membrane. However, there has been no research into the role of AQP3 in human peritoneal mesothelial cell (HPMC) migration or peritoneal fibrosis. We investigated the effects of transforming growth factor-β1 (TGF-β1) on AQP3 expression in HPMCs. We also investigated the role of AQP3 in the peritoneal wound healing process in rats. Chronic exposure to glucose-containing solution increased peritoneal myofibroblasts, with TGF-β1 and AQP3 expression in a model of long-term peritoneal dialysis. In vitro, TGF-β1 induced AQP3 expression in HPMCs. AQP3 knockdown by small-interfering RNA inhibited TGF-β1-induced AQP3 and α-smooth muscle actin expression and also slowed HPMC migration. AQP3 overexpression induced faster migration of HPMCs. Treatment with an extracellular signal-regulated kinase inhibitor and p38 kinase inhibitor attenuated TGF-β1-induced AQP3 expression in HPMCs. These data suggest that TGF-β1 induces AQP3 and that AQP3 has a critical role in TGF-β-induced HPMC migration. These findings provide evidence of a novel role for AQP3 in peritoneal fibrosis and wound healing. The effect of TGF-β1 on AQP3 expression in HPMCs is mediated, at least in part, by ERK and p38 signaling.

Alpha1-Antitrypsin Attenuates Renal Fibrosis by Inhibiting TGF-β1-Induced Epithelial Mesenchymal Transition.

Alpha1-antitrypsin (AAT) exerts its anti-inflammatory effect through regulating the activity of serine proteinases. This study evaluated the inhibitory effects of AAT against the transforming growth factor (TGF)-β1 induced epithelial-to-mesenchymal transition (EMT) in unilateral ureter obstruction (UUO) mice and Madin-Darby canine kidney (MDCK) cells. C57BL/6 mice with induced UUO were injected intraperitoneally with AAT (80 mg/Kg) or vehicle for 7 days. MDCK cells were treated with TGF-β1 (2 ng/mL) for 48 hours to induce EMT, and co-treated with AAT (10 mg/mL) to inhibit the EMT. Masson’s trichrome and Sirius red staining was used to estimate the extent of renal fibrosis in UUO mice. The expression of alpha-smooth muscle actin (α-SMA), vimentin, fibronectin, collagen I, and E-cadherin in MDCK cells and kidney tissue were evaluated. Masson’s and Sirius red staining revealed that the area of renal fibrosis was significantly smaller in AAT treated UUO group compared with that of UUO and vehicle treated UUO groups. AAT treatment attenuated upregulation of Smad2/3 phosphorylation in UUO mouse model. Co-treatment of MDCK cells with TGF-β1 and AAT significantly attenuated the changes in the expression of α-SMA, vimentin, fibronectin, collagen I, and E-cadherin. AAT also decreased the phosphorylated Smad3 expression and the phosphorylated Smad3/Smad3 ratio in MDCK cells. AAT treatment inhibited EMT induced by TGF-β1 in MDCK cells and attenuated renal fibrosis in the UUO mouse model. The results of this work suggest that AAT could inhibit the process of EMT through the suppression of TGF-β/Smad3 signaling.

Are ex vivo mesothelial cells representative of the in vivo transition from epithelial-to-mesenchymal cells in peritoneal membrane?

BACKGROUND We investigated whether ex vivo mesothelial cells found in peritoneal dialysis (PD) effluents were representative of the in vivo epithelial-to-mesenchymal transition (EMT) in peritoneal membrane. METHODS Thirty-six male Sprague-Dawley rats were equally divided into three groups: Group C (control), no PD; Group D, infused with 4.25% Dianeal and Group P, infused with 4.25% Physioneal. PD infusions (25 mL) were given twice daily for 8 weeks. The in vivo study included morphometric analyses performed on the peritoneal membranes of tissue specimens obtained at the end of the study. The ex vivo study included peritoneal mesothelial cells collected from PD effluent and cultured to confluence. Cells were scored with light microscopy. RESULTS PD for 8 weeks induced significant EMT. The in vivo expression of EMT markers (α-smooth muscle actin:E-cadherin ratio, matrix metalloproteinase-2 and Snail) was higher in Group D than in Group P. However, ex vivo EMT marker expression was similar in cells derived from Groups D and P. A significant correlation was observed among in vivo EMT markers. Moreover, the ex vivo cell score increased with time on PD. However, changes in the ex vivo cell score did not correlated with changes in the in vivo EMT marker expression. Furthermore, we found no correlation between ex vivo and in vivo cells in the expression of EMT markers. CONCLUSIONS In this animal study, ex vivo findings did not reflect the in vivo EMT changes in the peritoneum. It may be necessary to improve the current methodology for ex vivo studies.

Hypoxanthine induces cholesterol accumulation and incites atherosclerosis in apolipoprotein E-deficient mice and cells.

Reactive oxygen species (ROS) generation during purine metabolism is associated with xanthine oxidase and uric acid. However, the direct effect of hypoxanthine on ROS generation and atherosclerosis has not been evaluated. Smoking and heavy drinking are associated with elevated levels of hypoxanthine. In this study, we investigated the role of hypoxanthine on cholesterol synthesis and atherosclerosis development, particularly in apolipoprotein E (APOE)‐deficient mice. The effect of hypoxanthine on the regulation of cholesterol synthesis and atherosclerosis were evaluated in Apoe knockout (KO) mice and cultured HepG2 cells. Hypoxanthine markedly increased serum cholesterol levels and the atherosclerotic plaque area in Apoe KO mice. In HepG2 cells, hypoxanthine increased intracellular ROS production. Hypoxanthine increased cholesterol accumulation and decreased APOE and ATP‐binding cassette transporter A1 (ABCA1) mRNA and protein expression in HepG2 cells. Furthermore, H2O2 also increased cholesterol accumulation and decreased APOE and ABCA1 expression. This effect was partially reversible by treatment with the antioxidant N‐acetyl cysteine and allopurinol. Hypoxanthine and APOE knockdown using APOE‐siRNA synergistically induced cholesterol accumulation and reduced APOE and ABCA1 expression. Hypoxanthine induces cholesterol accumulation in hepatic cells through alterations in enzymes that control lipid transport and induces atherosclerosis in APOE‐deficient cells and mice. These effects are partially mediated through ROS produced in response to hypoxanthine.

Oxidative stress caused by activation of NADPH oxidase 4 promotes contrast-induced acute kidney injury

Contrast-induced acute kidney injury (CIAKI) is a leading cause of acute kidney injury following radiographic procedures. Intrarenal oxidative stress plays a critical role in CIAKI. Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidases (Noxs) are important sources of reactive oxygen species (ROS). Among the various types of Noxs, Nox4 is expressed predominantly in the kidney in rodents. Here, we evaluated the role of Nox4 and benefit of Nox4 inhibition on CIAKI using in vivo and in vitro models. HK-2 cells were treated with iohexol, with or without Nox4 knockdown, or the most specific Nox1/4 inhibitor (GKT137831). Effects of Nox4 inhibition on CIAKI mice were examined. Expression of Nox4 in HK-2 cells was significantly increased following iohexol exposure. Silencing of Nox4 rescued the production of ROS, downregulated pro-inflammatory markers (particularly phospho-p38) implicated in CIAKI, and reduced Bax and caspase 3/7 activity, which resulted in increased cellular survival in iohexol-treated HK-2 cells. Pretreatment with GKT137831 replicated these effects by decreasing levels of phospho-p38. In a CIAKI mouse model, even though the improvement of plasma blood urea nitrogen was unclear, pretreatment with GKT137831 resulted in preserved structure, reduced expression of 8-hydroxy-2’-deoxyguanosine (8OHdG) and kidney injury molecule-1 (KIM-1), and reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cells. These results suggest Nox4 as a key source of reactive oxygen species responsible for CIAKI and provide a novel potential option for prevention of CIAKI.

Effects of Losartan and Pentoxifylline on Renal Dimethylarginine Dimethylaminohydrolase-1 Expression in Proteinuric Nephropathy

Background/Aims: Circulatory asymmetric dimethylarginine (ADMA) is correlated with proteinuria and endothelial dysfunction in patients with proteinuric renal diseases. However, it is not known whether proteinuria itself affects expression of dimethylarginine dimethylaminohydrolase (DDAH), a degrading enzyme of ADMA, in kidney. The aim of this study is to evaluate the direct effects of losartan and/or pentoxifylline on expression of renal DDAH-1 and its relation to oxidative stress in the setting of albuminuria. Methods: Using NRK52E cells, DDAH-1 mRNA and protein were determined after exposure to albumin with losartan and/or pentoxifylline. Reactive oxygen species (ROS), PKC activity, and NOX-4 mRNA were also measured. In addition, the effect of losartan and/or pentoxifylline on renal expression of DDAH-1 and serum ADMA were evaluated in a rat model of proteinuric nephropathy. Results: Exposure to albumin resulted in increased release of N-acetyl-β-D-glucosaminidase along with an increase of TNF-α, 8-hydroxy-2′-deoxyguanosine, and angiotensin II in NRK52E cells. Losartan and pentoxifylline reversed albumin-induced decrease of DDAH-1 mRNA and protein expression and DDAH-1 activity. The effects of losartan and pentoxifylline on DDAH-1 mRNA were associated with reduction of ROS. In addition, treatment with losartan and pentoxifylline resulted in an attenuated change of renal DDAH-1 protein expression and serum ADMA levels in vivo. Conclusion: DDAH-1 was positively regulated by losartan and pentoxifylline with its antioxidative effect in albumin-exposed renal proximal tubular cells. Combined treatment with losartan and pentoxifylline has a direct beneficial effect on expression of renal DDAH-1, and, thus, at least in part, modulates the circulatory levels of ADMA in proteinuric nephropathy.

Dipeptidyl peptidase-4 inhibitor gemigliptin protects against vascular calcification in an experimental chronic kidney disease and vascular smooth muscle cells.

Although dipeptidyl peptidase-4 inhibitors, a class of antidiabetic drugs, have various pleiotropic effects, it remains undetermined whether gemigliptin has a beneficial effect on vascular calcification. Therefore, this study was performed to evaluate the effect of gemigliptin on vascular calcification in a rat model of adenine-induced chronic kidney disease and in cultured vascular smooth muscle cells. Gemigliptin attenuated calcification of abdominal aorta and expression of RUNX2 in adenine-induced chronic kidney disease rats. In cultured vascular smooth muscle cells, phosphate-induced increase in calcium content was reduced by gemigliptin. Gemigliptin reduced phosphate-induced PiT-1 mRNA expression, reactive oxygen species generation, and NADPH oxidase mRNA expression (p22phox and NOX4). The reduction of oxidative stress by gemigliptin was associated with the downregulation of phospho-PI3K/AKT expression. High phosphate increased the expression of frizzled-3 (FDZ3) and decreased the expression of dickkopf-related protein-1 (DKK-1) in the Wnt pathway. These changes were attenuated by gemigliptin treatment. Gemigliptin restored the decreased expression of vascular smooth muscle cells markers (α-SMA and SM22α) and increased expression of osteogenic makers (CBFA1, OSX, E11, and SOST) induced by phosphate. In conclusion, gemigliptin attenuated vascular calcification and osteogenic trans-differentiation in vascular smooth muscle cells via multiple steps including downregulation of PiT-1 expression and suppression of reactive oxygen species generation, phospho-PI3K/AKT, and the Wnt signaling pathway.