Characterization of immune responses associated with ERAP-1 expression in HSV-induced Behçet's disease mouse model.

Behçet's disease (BD) is a chronic multisystem inflammatory disorder. Endoplasmic reticulum aminopeptidase 1 (ERAP1) polymorphism has been reported as a risk factor for BD. However, the immunological role of ERAP1 in BD remains unclear. Therefore, the purpose of this study was to investigate the immunological role of ERAP1 in BD using a mouse model. ERAP1 incomplete expressing mice (ERAP1 hetero, +/-) were generated and inoculated with herpes simplex virus 1 to produce a BD mouse model. In these mice, dendritic cell activation markers and other immune response-related markers were analyzed. Among them, the factor showing a significant difference between ERAP1+/- BD mice and WT BD mice was IL-17. In ERAP1+/-, BD had significantly different expression levels of CD80, CD11b, Ly6G, RORγt, IFNγ, and IL-17 compared to asymptomatic controls. This study demonstrates ERAP1 defective expressions play an important role in BD development through inappropriate regulation of Th17.

CD83 Regulates the Immune Responses in Inflammatory Disorders.

Activating the immune system plays an important role in maintaining physiological homeostasis and defending the body against harmful infections. However, abnormalities in the immune response can lead to various immunopathological responses and severe inflammation. The activation of dendritic cells (DCs) can influence immunological responses by promoting the differentiation of T cells into various functional subtypes crucial for the eradication of pathogens. CD83 is a molecule known to be expressed on mature DCs, activated B cells, and T cells. Two isotypes of CD83, a membrane-bound form and a soluble form, are subjects of extensive scientific research. It has been suggested that CD83 is not only a ubiquitous co-stimulatory molecule but also a crucial player in monitoring and resolving inflammatory reactions. Although CD83 has been involved in immunological responses, its functions in autoimmune diseases and effects on pathogen immune evasion remain unclear. Herein, we outline current immunological findings and the proposed function of CD83 in inflammatory disorders.

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.

The emerging role of xanthine oxidase inhibition for suppression of breast cancer cell migration and metastasis associated with hypercholesterolemia.

Hypercholesterolemia is reported to increase reactive oxygen species (ROS) and to promote breast cancer progression. ROS play an important role in tumor biology, and xanthine oxidase (XO) is an enzyme that generates ROS. The effects of febuxostat (FBX), an XO inhibitor, on breast cancer cell migration under LDL stimulation in vitro and metastasis of breast cancer associated with hypercholesterolemia in vivo were studied. In vitro, FBX significantly inhibited LDL-induced ROS production and cell migration. Treatment of small interfering RNA against XO was consistent with the findings of FBX treatment. In vivo, a significant increase of tumor growth and pulmonary metastasis was observed in a xenograft mouse model with 4T1 cells on a high cholesterol diet (HCD), both of which were markedly inhibited by FBX or allopurinol treatment. Moreover, ERK represented the main target-signaling pathway that was affected by FBX treatment in a xenograft mouse model on an HCD evaluated by NanoString nCounter analysis. Consistently, MEK/ERK inhibitors directly decreased the LDL-induced cell migration in vitro. In conclusion, FBX mitigates breast cancer cell migration and pulmonary metastasis in the hyperlipidemic condition, associated with decreased ROS generation and MAPK phosphorylation. The inhibition of ERK pathways is likely to underlie the XO inhibitor-mediated suppression of breast cancer cell migration.-Oh, S.-H., Choi, S.-Y., Choi, H.-J., Ryu, H.-M., Kim, Y.-J., Jung, H.-Y., Cho, J.-H., Kim, C.-D., Park, S.-H., Kwon, T.-H., Kim, Y.-L. The emerging role of xanthine oxidase inhibition for suppression of breast cancer cell migration and metastasis associated with hypercholesterolemia.

Fimasartan attenuates renal ischemia-reperfusion injury by modulating inflammation-related apoptosis.

Fimasartan, a new angiotensin II receptor antagonist, reduces myocyte damage and stabilizes atherosclerotic plaque through its anti-inflammatory effect in animal studies. We investigated the protective effects of pretreatment with fimasartan on ischemia-reperfusion injury (IRI) in a mouse model of ischemic renal damage. C57BL/6 mice were pretreated with or without 5 (IR-F5) or 10 (IR-F10) mg/kg/day fimasartan for 3 days. Renal ischemia was induced by clamping bilateral renal vascular pedicles for 30 min. Histology, pro-inflammatory cytokines, and apoptosis assays were evaluated 24 h after IRI. Compared to the untreated group, blood urea nitrogen and serum creatinine levels were significantly lower in the IR-F10 group. IR-F10 kidneys showed less tubular necrosis and interstitial fibrosis than untreated kidneys. The expression of F4/80, a macrophage infiltration marker, and tumor necrosis factor (TNF)-α, decreased in the IR-F10 group. High-dose fimasartan treatment attenuated the upregulation of TNF-α, interleukin (IL)-1ß, and IL-6 in ischemic kidneys. Fewer TUNEL positive cells were observed in IR-F10 compared to control mice. Fimasartan caused a significant decrease in caspase-3 activity and the level of Bax, and increased the Bcl-2 level. Fimasartan preserved renal function and tubular architecture from IRI in a mouse ischemic renal injury model. Fimasartan also attenuated upregulation of inflammatory cytokines and decreased apoptosis of renal tubular cells. Our results suggest that fimasartan inhibited the process of tubular injury by preventing apoptosis induced by the inflammatory pathway.

Hypoxanthine causes endothelial dysfunction through oxidative stress-induced apoptosis.

Endothelial cell injury and dysfunction caused by reactive oxygen species (ROS) are implicated in the pathogenesis of vascular diseases. ROS are generated and hypoxanthine is degraded by xanthine oxidase. Smoking and alcohol consumption are associated with an increased level of hypoxanthine. We aimed to study the direct role of hypoxanthine in endothelial dysfunction in human umbilical vascular endothelial cells (HUVECs). Hypoxanthine induced cell death and production of ROS. Furthermore, hypoxanthine induced apoptosis through regulation of protein expression related to apoptosis. When cells were pretreated with N-acetylcysteine or a pancaspase inhibitor (Z-VAD-fmk) and stimulated with hypoxanthine, Z-VAD-fmk and N-acetylcysteine prevented hypoxanthine-induced apoptosis by inhibiting the ROS production and caspase pathway. Thus, an increased extracellular concentration of hypoxanthine induces endothelial dysfunction through ROS production and regulates expression of apoptosis-related proteins in HUVECs. These effects are expected to be associated with some vascular diseases.

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, H2 O2 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.

Aquaporin 3 expression is up-regulated by TGF-ß1 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-ß1 (TGF-ß1) 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-ß1 and AQP3 expression in a model of long-term peritoneal dialysis. In vitro, TGF-ß1 induced AQP3 expression in HPMCs. AQP3 knockdown by small-interfering RNA inhibited TGF-ß1-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-ß1-induced AQP3 expression in HPMCs. These data suggest that TGF-ß1 induces AQP3 and that AQP3 has a critical role in TGF-ß-induced HPMC migration. These findings provide evidence of a novel role for AQP3 in peritoneal fibrosis and wound healing. The effect of TGF-ß1 on AQP3 expression in HPMCs is mediated, at least in part, by ERK and p38 signaling.

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-ß-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.

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.

Tetragenococcus halophilus Alleviates Intestinal Inflammation in Mice by Altering Gut Microbiota and Regulating Dendritic Cell Activation via CD83.

Ulcerative colitis (UC) is one of the major subtypes of inflammatory bowel disease with unknown etiology. Probiotics have recently been introduced as a treatment for UC. Tetragenococcus halophilus (T. halophilus) is a lactic acid-producing bacterium that survives in environments with high salt concentrations, though little is known about its immunomodulatory function as a probiotic. The purpose of this study is to determine whether T. halophilus exerts an anti-inflammatory effect on intestinal inflammation in mice. Colitis was induced in C57BL/6J mice by feeding 4% DSS in drinking water for 7 days. T. halophilus was orally administered with DSS. Anti-inflammatory functions were subsequently evaluated by flow cytometry, qRT-PCT, and ELISA. Gut microbial composition was analyzed by 16S rRNA metagenomic analysis. DSS-induced colitis mice treated with T. halophilus showed less weight loss and significantly suppressed colonic shortening compared to DSS-induced colitis mice. T. halophilus significantly reduced the frequency of the dendritic cell activation molecule CD83 in peripheral blood leukocytes and intestinal epithelial lymphocytes. Frequencies of CD8+NK1.1+ cells decreased in mice with colitis after T. halophilus treatment and IL-1ß levels were also reduced. Alteration of gut microbiota was observed in mice with colitis after administration of T. halophilus. These results suggest T. halophilus is effective in alleviating DSS-induced colitis in mice by altering immune regulation and gut microbiome compositions.

Phosphate-induced apoptosis in human peritoneal mesothelial cells in vitro.

BACKGROUND: It has been demonstrated that phosphate uptake through the type III sodium-dependent phosphate co-transporter, Pit-1, induced apoptosis of aortic vascular smooth muscle cells and endothelial cells in vitro. However, the apoptotic effects of high phosphate (HP) level in human peritoneal mesothelial cells (HPMCs) are not known. METHODS: To examine whether Pit-1 is expressed in HPMCs, we checked the Western blot assay of immunoreactive Pit-1 and the transcription of Pit-1 by reverse transcriptase PCR. We treated several different phosphate concentrations (1-4 mM) and calcium concentrations (1.8 and 2.8 mM) on HPMCs to assess the effects of concentration. MTT, TUNEL assays, and flow cytometry analysis using Annexin V and propidium iodide were performed to identify cell death and apoptosis. Bax and Bcl-2 by Western blot and caspase-3 activity were evaluated by colorimetric assay. In addition, phosphonoformic acid (PFA) and pan-caspase inhibitor, Z-VAD-FMK, were given to prevent phosphate-induced apoptosis. RESULTS: Pit-1 expression on HPMCs was demonstrated. Apoptosis in HPMCs significantly increased with a high concentration of phosphate in a dose- and time-dependent manner, and was enhanced in the presence of 2.8 mM calcium. HP concentrations significantly decreased the anti-apoptotic Bcl-2/Bax ratio and increased caspase-3 activity. The treatment with PFA and Z-VAD-FMK prevented cell death by HP. CONCLUSION: Phosphate uptake through Pit-1 induces apoptosis in HPMCs by a caspase-related mechanism.

Interrelationship of Stress, Environment, and Herpes Simplex Virus Type-1 on Behçet's Disease: Using a Mouse Model.

The purpose of this study was to investigate effects of stress and environment factors on the induction of Behçet's disease (BD) using HSV-1 infected mouse model. BD is a chronic multisystemic inflammatory disease of unknown etiology. Environmental factors, immune dysfunction, and herpes simplex virus type-1 (HSV) infection might be triggers of BD. To investigate effects of environmental factors on the incidence of BD, HSV was inoculated into mice. Mice were then maintained in conventional facility or SPF facility to compare BD incidence rates. The incidence of BD was also tracked by adding stressors such as substance P (anxiety stress), 4°C (cold stress), xanthine sodium salt (oxidative stress), or 77 dB noise (noise stress). To clarify immune mechanisms involved in the difference in BD incidence caused by various stresses, dendritic cell activation markers were analyzed using flow cytometry. The combination of conventional environment, noise stress, and HSV had the highest rate of BD (38.1%) among all groups. However, HSV inoculated group in a SPF environment had the lowest incidence (2.2%). Frequencies of dendritic cell activation markers such as CD40, CD83, CD80, and CD86 were expressed differently under various stresses. Noise stress increased frequencies of CD83 positive cells. Noise stress also upregulated transcription factors T-bet and ROR-γt. Different gut microbiota compositions were observed between SPF and conventional environment by 16S rRNA sequence analysis. Environment and stress influenced the incidence of HSV-induced BD. Microbial diversity due to environmental differences might be one explanation for regional differences in the incidence of BD.

Eubacterium rectale Attenuates HSV-1 Induced Systemic Inflammation in Mice by Inhibiting CD83.

The purpose of this study was to determine whether administration of the microorganism Eubacterium rectale (E. rectale) could regulate dendritic cell (DC) activation and systemic inflammation in herpes simplex virus type 1-induced Behçet's disease (BD). E. rectale, butyrate-producing bacteria, was administered to BD mice. Peripheral blood leukocytes (PBL) and lymph node cells were isolated and analyzed by flow cytometry. 16S rRNA metagenomic analysis was performed in the feces of mice to determine the differences in the composition of the microbial population between normal and BD mice. Serum cytokine levels were measured by enzyme-linked immunosorbent assay. The frequency of DC activation marker CD83 positive cells was significantly increased in PBL of BD mice. Frequencies of CD83+ cells were also significantly increased in patients with active BD. 16S rRNA metagenomic analysis revealed different gut microbiota composition between normal and BD mice. The administration of E. rectale to BD mice reduced the frequency of CD83+ cells and significantly increased the frequency of NK1.1+ cells with the improvement of symptoms. The co-administration of colchicine and E. rectale also significantly reduced the frequency of CD83+ cells. Differences in gut microbiota were observed between normal mice and BD mice, and the administration of E. rectale downregulated the frequency of CD83, which was associated with BD deterioration. These data indicate that E. rectale could be a new therapeutic adjuvant for BD management.

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 alpha-smooth muscle actin (alpha-SMA) and E-cadherin. RESULTS: In vitro, alpha-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 alpha-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 alpha-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-beta1 decreased in the L-GDP group. Dual-stained cytokeratin and alpha-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.

Impact of systemic and local peritoneal inflammation on peritoneal solute transport rate in new peritoneal dialysis patients: a 1-year prospective study.

BACKGROUND: The association between peritoneal solute transport rates (PSTRs) and inflammatory markers in patients on peritoneal dialysis (PD) is still under investigation. We aimed to elucidate their relationship during the first year on PD. METHODS: We performed a prospective observational study with 187 incident PD patients who were treated with either biocompatible solution (BCS) or conventional solution (CS). Peritoneal dialysate effluent (PDE) and blood samples for the markers and the calculation of mass transfer area coefficient of creatinine (MTAC) were performed at 1, 6 and 12 months after commencing PD. RESULTS: Of the 187 enrolled patients, 110 completed a 1-year study protocol. All PDE markers [interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta), TGF-beta-induced gene-h3 (beta ig-h3), vascular endothelial growth factor (VEGF)] except CA125 increased over time, whereas PSTRs, high-sensitivity C-reactive protein (hs-CRP) and serum IL-6 levels did not change. Serum albumin and log PDE appearance rates (ARs) of IL-6, TGF-beta and CA125 predicted MTAC. The Delta value (12-month minus 1-month) of PDE AR of IL-6 was correlated with those of all other PDE markers. Both 12-month IL-6 and Delta IL-6 ARs in PDE were highest in the upper Delta MTAC tertile. PSTRs in the CS group, unlike BCS, had a tendency to increase over time, demonstrating a time-by-group interaction. Solution type and MTAC were not associated with patient and technique survival. CONCLUSIONS: The change in PSTR during the first year of PD is related to PDE IL-6 AR, which may represent intraperitoneal inflammation; however, there does not seem to be a close association between PSTR and the degree of systemic inflammation.

3,4-dideoxyglucosone-3-ene induces apoptosis in human peritoneal mesothelial cells.

OBJECTIVE: Glucose degradation products (GDPs) are formed during heat sterilization and storage of peritoneal dialysis (PD) fluids. 3,4-dideoxyglucosone-3-ene (3,4-DGE) has been identified as the most bioreactive GDP. 3,4-DGE induces apoptosis in leukocytes and renal tubular epithelial cells. Our aim was to evaluate the apoptotic effects of 3,4-DGE on human peritoneal mesothelial cells (HPMCs). METHODS: Primary cultured HPMCs were treated with 25 or 50 micromol/L 3,4-DGE. MTT assay was used to determine cell viability. Apoptosis was measured using TUNEL assay and flow cytometry. Expressions of procaspase-3, Bax, and Bcl-2 were estimated by Western blot. Activity of caspase-3 was measured and the effect of the caspase inhibitor zVAD-fmk (Z-Val-Ala-DL-Asp-fluoromethylketone) was evaluated by TUNEL assay. RESULTS: 3,4-DGE treatment accelerated cell death in HPMCs in a dose- and time-dependent manner. Treatment with 3,4-DGE (25 and 50 micromol/L) significantly increased apoptosis compared to control (p<0.05 and p<0.01 respectively) by TUNEL assay. Flow cytometry showed treatment with 50 micromol/L 3,4-DGE significantly increased apoptosis compared to control (p<0.05). Decreased expression of procaspase-3 and increased activity of caspase-3 were observed in the presence of 50 micromol/L 3,4-DGE compared to control and 25 micromol/L 3,4-DGE (p<0.05). 3,4-DGE-induced HPMC apoptosis was decreased after pretreatment with the pan-caspase inhibitor zVAD-fmk in the 50 micromol/L 3,4-DGE-treated group (p<0.001). The ratio of Bcl-2 to Bax expression was decreased in the 25 micromol/L and the 50 micromol/L 3,4-DGE-treated groups compared to control (p<0.05). CONCLUSIONS: 3,4-DGE promotes apoptosis in HPMCs by a caspase-related mechanism.

Network-based integrated analysis of omics data reveal novel players of TGF-ß1-induced EMT in human peritoneal mesothelial cells.

Long-term peritoneal dialysis is associated with progressive fibrosis of the peritoneum. Epithelial-mesenchymal transition (EMT) of mesothelial cells is an important mechanism involved in peritoneal fibrosis, and TGF-ß1 is considered central in this process. However, targeting currently known TGF-ß1-associated pathways has not proven effective to date. Therefore, there are still gaps in understanding the mechanisms underlying TGF-ß1-associated EMT and peritoneal fibrosis. We conducted network-based integrated analysis of transcriptomic and proteomic data to systemically characterize the molecular signature of TGF-ß1-stimulated human peritoneal mesothelial cells (HPMCs). To increase the power of the data, multiple expression datasets of TGF-ß1-stimulated human cells were employed, and extended based on a human functional gene network. Dense network sub-modules enriched with differentially expressed genes by TGF-ß1 stimulation were prioritized and genes of interest were selected for functional analysis in HPMCs. Through integrated analysis, ECM constituents and oxidative stress-related genes were shown to be the top-ranked genes as expected. Among top-ranked sub-modules, TNFAIP6, ZC3H12A, and NNT were validated in HPMCs to be involved in regulation of E-cadherin, ZO-1, fibronectin, and αSMA expression. The present data shows the validity of network-based integrated analysis in discovery of novel players in TGF-ß1-induced EMT in peritoneal mesothelial cells, which may serve as new prognostic markers and therapeutic targets for peritoneal dialysis patients.

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.

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.