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.

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.

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.

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.

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.

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.

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.

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.

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.

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-ß1-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-3ß phosphorylation, nuclear translocalization of ß-catenin and Snail in HPMCs. Effect of metformin on TGF-ß1-induced EMT was ameliorated by either AMPK inhibitor or AMPK gene silencing. Another AMPK agonist, 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide partially blocked TGF-ß1-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.

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.

The role of Toll-like receptor 4 in high-glucose-induced inflammatory and fibrosis markers in human peritoneal mesothelial cells.

PURPOSE: High glucose stimulates peritoneal inflammation and extracellular matrix accumulation in human peritoneal mesothelial cells (HPMCs). However, the roles of Toll-like receptor 4 (TLR4) and TLR2 in high-glucose-induced inflammation and fibrosis in peritoneal dialysis (PD) remain unclear. This study aimed to evaluate the effect of high glucose on TLR2 and TLR4 expression in HPMCs and to assess their impact on peritoneal inflammatory and fibrosis markers. METHODS: Using cultured HPMCs, TLRs expression by high-glucose (50 mM) stimulation was assessed by quantitative real-time PCR. The association of reactive oxygen species (ROS) in high-glucose-induced TLR2 and TLR4 expression was measured by 2',7'-dichlorodihydrofluorescein diacetate staining with or without ROS inhibitor. In addition, the role of TLR2 and TLR4 on high-glucose-induced inflammatory and fibrosis markers including chemoattractant protein-1 (MCP-1), NF-κB, alpha-smooth muscle actin (α-SMA), transforming growth factor-ß (TGF-ß), and fibronectin was evaluated after inhibition of TLR2 and TLR4 by small-interfering RNA (siRNA) or anti-TLR4/TLR2 antibodies, respectively. RESULTS: High glucose induced TLR1, TLR2, and TLR4 mRNAs expressions. High-glucose-induced TLR4 and TLR2 mRNAs were associated partly with the generation of ROS. Inhibition of TLR4 attenuated the high-glucose-induced expression of MCP-1 mRNA and protein, MyD88 mRNA, nuclear NF-κB p65 protein, TGF-ß, fibronectin, and α-SMA mRNA and protein. However, inhibition of TLR2 did not change the expression of MCP-1 mRNA and protein. CONCLUSIONS: High glucose induces inflammatory and fibrosis markers in HPMCs partly through the TLR4/MyD88/NF-κB signaling pathway rather than TLR2. Therefore, TLR4 might be a therapeutic target for ameliorating peritoneal inflammation and fibrosis in PD.

Alpha1-Antitrypsin Attenuates Renal Fibrosis by Inhibiting TGF-ß1-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)-ß1 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-ß1 (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-ß1 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-ß1 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-ß/Smad3 signaling.

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.

Changes in serum metabolites with the stage of chronic kidney disease: Comparison of diabetes and non-diabetes.

BACKGROUND: The renal dysfunction of chronic kidney disease (CKD) alters serum metabolite levels, but it is not clear how diabetes mellitus (DM) affects the metabolic changes in CKD. METHODS: Serum metabolites from pre-dialysis CKD patients (n=291) with or without DM and from healthy controls (n=56) was measured using nuclear magnetic resonance. RESULTS: Initial principal components analysis and partial least squares-discriminant analysis score plots segregated the CKD patients according to CKD stage and separated DM from non-DM patients. In the CKD patients, associations were seen with clinical characteristics, hyperglycemia, altered amino acid metabolism, accumulated uremic toxins, and dyslipidemia. Of interest, diabetes more strongly affected the metabolic signature during early stage CKD. Furthermore, serum metabolite profiles were successfully applied to the PLS regression model to predict the estimated glomerular filtration rate. The R(2) values from the PLS models for CKD patients with DM were higher than those for CKD without DM. CONCLUSIONS: Metabolomics is useful clinically for providing a metabolic signature that is associated with the CKD phenotype and diabetes more seriously affects patients with early stage CKD compared to those with advanced CKD.

Effect of DNA demethylation in experimental encapsulating peritoneal sclerosis.

Encapsulating peritoneal sclerosis (EPS) involves excessive peritoneal fibrosis in patients on peritoneal dialysis, eventually leading to visceral constriction and bowel obstruction. Few studies have investigated epigenetic mechanisms relating to EPS. Here we evaluated the therapeutic effects of DNA demethylation in experimental EPS. Experimental EPS was induced by intraperitoneal injection of 0.1% chlorhexidine gluconate (CG) and 15% ethanol in non-uremic male Sprague-Dawley (SD) rats. Rats were divided into three groups: group C (N=5) with saline injection only, group CG (N=7) with EPS induction for 4 weeks, and chlorhexidine gluconate and azacytidine (CGA) treated group (N=7) with EPS induction for 4 weeks and 5'-azacytidine injection for the last 2 weeks. Morphometric analysis of peritoneum and immunohistochemical staining for type 1 collagen and α-smooth muscle actin (α-SMA) were performed. Expressions of transforming growth factor-ß (TGF-ß), fibroblast-specific protein 1 (FSP1), and DNA methyltransferase 1 (DNMT1) were analyzed by Western blot. Methylation-specific polymerase chain reaction (PCR) for Ras GTPase activating-like protein 1 (RASAL1) was performed with measurement of RASAL1 protein expression. Parietal peritoneal thickness and the number of vessels in omental tissue were significantly decreased in group CGA compared to group CG, as were the expressions of type 1 collagen, α-SMA, TGF-ß, and FSP1. DNMT1 was significantly increased in group CG, and reduced in group CGA. RASAL1 hypermethylation was associated with decreased RASAL1 protein expression in group CG, which was reversed in group CGA. DNA demethylation by 5'-azacytidine treatment improved pathologic changes of the peritoneum in experimental EPS, and was associated with reversal of increased DNMT1 expression and RASAL1 hypermethylation.

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.

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.