High serum level of methylglyoxal-derived AGE, Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine, independently relates to renal dysfunction.

BACKGROUND: The dicarbonyl methylglyoxal reacts primarily with arginine residues to form advanced glycation end products, including Nδ-(5-hydro-5-methyl-4 -imidazolone-2-yl)-ornithine (MG-H1), which are risk factors for not only diabetic complications but also lifestyle-related disease including renal dysfunction. However, the data on serum level and clinical significance of this substance in chronic kidney disease are limited. METHODS: Serum levels of MG-H1 and Nε-(carboxymethyl) lysine (CML) in 50 patients with renal dysfunction were measured by liquid chromatography/triple-quadruple mass spectrometry. RESULTS: The median serum MG-H1 levels in patients with estimated glomerular filtration rate (eGFR) of ≥30, 15-30, and <15 ml/min/1.73 m2 was 4.16, 12.58, and 14.66 mmol/mol Lys, respectively (p > 0.05). On the other hand, MG-H1 levels in patients with HbA1c of <6 and ≥6 % was 12.85 and 10.45 mmol/mol Lys, respectively, the difference between which is not significant. In logistic regression analysis, decreased renal function (eGFR <15 ml/min/1.73 m2) significantly associated with high serum levels of MG-H1 [odds ratio: 9.39 (95 % confidence interval 1.528-57.76), p = 0.015; Spearman rank correlation: MG-H1 vs. eGFR, r = -0.691, p < 0.01]. In contrast, the serum level of CML did not correlate with eGFR, but correlated with systolic blood pressure [odds ratio 16.17 (95 % confidence interval 1.973-132.5), p = 0.010; Spearman rank correlation coefficient: CML vs. eGFR, r = 0.454, p < 0.01]. CONCLUSION: These results showed that the serum concentration of MG-H1 was strongly related to renal function rather than to DM.

IL-1ß promotes tubulointerstitial injury in MPO-ANCA-associated glomerulonephritis
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BACKGROUND: It is widely accepted that tubulointerstitial injury (TII) is caused by glomerular injury (GI) in glomerular diseases. Glomerular endocapillary inflammation may result in crescent formation and exuded protein leakage, which may induce TII in antineutrophil cytoplasmic antibody-associated glomerulonephritis (ANCAGN). However, some reports have indicated a glomerulonephritis-independent mechanism of TII in ANCAGN. The aim of this study was to determine the principle cytokines correlated with TII severity and to elucidate a characteristic mechanism for TII in ANCAGN. METHODS: 28 myeloperoxidase-ANCA-positive ANCAGN patients were enrolled, and their kidney biopsy specimens were histologically evaluated with regard to GI and TII. The mRNA expression of various cytokines was examined in 28 specimens. RESULTS: Interleukin (IL)-1ß was significantly correlated with the severity of TII. The mRNA expression of Toll-like receptor 4 (TLR4) and Nod-like receptor family pyrin domain-containing-3 (NLRP3) also correlated with TII severity. Immunohistochemical analysis demonstrated that TLR4 protein was positively stained in the tubulointerstitial infiltrating cells. NRLP3 protein was detected in macrophages in the severe infiltrating area but was absent or only very faintly expressed in the glomeruli. These results indicated that NLRP3 inflammasome-dependent processing in macrophages releases the mature active form of IL-1ß, which may lead to the development and deterioration of TII. CONCLUSIONS: Sterile inflammation leads to the formation of ANCA-mediated neutrophil extracellular traps (NETs), which may stimulate macrophages and dendritic cells via TLR4 and induce NF-κB-dependent mRNA expression and translation of pro-IL-1ß. Simultaneously, damage-associated molecular pattern signals resulting from NETs promote NLRP3 inflammasome-dependent processing and release mature active IL-1ß. Sterile inflammation utilizing the NLRP3 inflammasome might be a characteristic reaction limited to the tubulointerstitium. Thus, neutralizing IL-1ß may be a promising strategy to suspend the progress of TII and improve the prognosis of chronic kidney disease resulting from ANCAGN.
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Generation mechanism of RANKL(+) effector memory B cells: relevance to the pathogenesis of rheumatoid arthritis.

BACKGROUND: The efficacy of B cell-depleting therapies for rheumatoid arthritis underscores antibody-independent functions of effector B cells such as cognate T-B interactions and production of pro-inflammatory cytokines. Receptor activator of nuclear factor κB ligand (RANKL) is a key cytokine involved in bone destruction and is highly expressed in synovial fluid B cells in patients with rheumatoid arthritis. In this study we sought to clarify the generation mechanism of RANKL(+) effector B cells and their impacts on osteoclast differentiation. METHODS: Peripheral blood and synovial fluid B cells from healthy controls and patients with rheumatoid arthritis were isolated using cell sorter. mRNA expression of RANKL, osteoprotegerin, tumor necrosis factor (TNF)-α, and Blimp-1 was analyzed by quantitative real-time polymerase chain reaction. Levels of RANKL, CD80, CD86, and CXCR3 were analyzed using flow cytometry. Functional analysis of osteoclastogenesis was carried out in the co-culture system using macrophage RAW264 reporter cells. RESULTS: RANKL expression was accentuated in CD80(+)CD86(+) B cells, a highly activated B-cell subset more abundantly observed in patients with rheumatoid arthritis. Upon activation via B-cell receptor and CD40, switched-memory B cells predominantly expressed RANKL, which was further augmented by interferon-γ (IFN-γ) but suppressed by interleukin-21. Strikingly, IFN-γ also enhanced TNF-α expression, while it strongly suppressed osteoprotegerin expression in B cells. IFN-γ increased the generation of CXCR3(+)RANKL(+) effector B cells, mimicking the synovial B cell phenotype in patients with rheumatoid arthritis. Finally, RANKL(+) effector B cells in concert with TNF-α facilitated osteoclast differentiation in vitro. CONCLUSIONS: Our current findings have shed light on the generation mechanism of pathogenic RANKL(+) effector B cells that would be an ideal therapeutic target for rheumatoid arthritis in the future.

Effects of diesel exhaust particles on cytokine production by splenocytes stimulated with lipopolysaccharide.

It was previously shown that pulmonary exposure of mice to diesel exhaust particles (DEP) enhances inflammatory conditions induced by allergens or bacterial endotoxin (lipopolysaccharide: LPS) via enhanced local expression of cytokines. However, resolution of the underlying mechanisms, in which DEP exaggerate inflammation, remains uncompleted. Investigation of the actions of DEP on mouse-derived mononuclear cells may provide a clue to the mechanisms, because mononuclear cells produce and release several types of cytokines. The present study elucidated the effects of DEP on mononuclear cell reactions stimulated with LPS in vitro. ICR mouse-derived mononuclear cells, isolated from splenocytes, one of the secondary lymphoid tissues, were co-cultured with LPS (1 microg ml(-1)) and DEP (1, 10 or 100 microg ml(-1)). The protein levels of interferon (IFN)-gamma, interleukin (IL)-2, IL-10, and IL-13 in the culture supernatants were measured 72 h after the co-culture. LPS significantly increased the protein levels of IFN-gamma, IL-2 and IL-10. In the presence of LPS, DEP decreased the protein levels in a concentration-dependent manner with an overall trend, whereas DEP (1, 10 microg ml(-1)) moderately elevated the IL-13 level. These results suggest that DEP suppress cytokine production from mononuclear cells stimulated with LPS and provide a possible hint for DEP facilitation on inflammatory conditions, especially related to Th2 response, in vivo.

Effects of diesel exhaust on lung inflammation related to bacterial endotoxin in mice.

We have previously shown that intratracheal instillation of diesel exhaust particles enhances lung inflammation and lung expression of proinflammatory cytokines and chemokines related to bacterial endotoxin (lipopolysaccharide) in mice. The present study was designed to elucidate the effects of inhalation of diesel exhaust on lung inflammation related to lipopolysaccharide. ICR mice were exposed for 12 hr to clean air or diesel exhaust at a soot concentration of 0.3, 1.0, or 3.0 mg/m(3) after intratracheal challenge with 125 microg/kg of lipopolysaccharide. Lung inflammation and lung expression of proinflammatory chemokines such as macrophage chemoattractant protein-1 and keratinocyte chemoattractant were evaluated 24 hr after intratracheal administration. Diesel exhaust inhalation decreased lipopolysaccharide-elicited inflammatory cell recruitment into the bronchoalveolar lavage fluid as compared with clean air inhalation. Histological study demonstrated that exposure to diesel exhaust did not affect lipopolysaccharide-enhanced neutrophil recruitment into the lung parenchyma. Lipopolysaccharide instillation elevated lung expression of macrophage chemoattractant protein-1 and keratinocyte chemoattractant under clean air or diesel exhaust inhalation. However, diesel exhaust exposure did not influence but rather did suppress these levels in the presence of lipopolysaccharide. These results suggest that short-term exposure to diesel exhaust did not exacerbate lung inflammation related to bacterial endotoxin.

Effects of 15-deoxy-delta(12,14)-prostaglandin J2 on nuclear localization of GATA-3 in the murine lung in the presence of lipopolysaccharide.

15-Deoxy-delta(12, 14)-prostaglandin J2 (15d-PG J2) is a regulator of a nuclear transcriptional factor, peroxisome proliferator-activated receptor (PPAR)-gamma. A previous study has demonstrated that 15d-PG J2 enhanced acute lung injury induced by lipopolysaccharide (LPS) in mice. 15d-PG J2 induced mucin-producing cells in the bronchial epithelium, especially in the presence of LPS. The present study investigated the effects of 15d-PG J2 on the activation of GATA-3 and Signal Transducer and Activator of Transcription (STAT) 6, important transcriptional factors in mucus secretion, in the lung in the presence or absence of LPS. ICR mice were divided into 4 experimental groups that intratracheally received vehicle, lipopolysaccharide (LPS: 125 microg/kg), 15d-PG J2 (1 mg/kg), or 15d-PG J2 + LPS. The nuclear localization of GATA-3 and phosphorylated STAT 6 was evaluated 2 h after the intratracheal administration. 15d-PG J2 enhanced the nuclear localization of GATA-3 in the presence of LPS, whereas the nuclear localization of phosphorylated STAT 6 was not altered in the groups. These results suggest that the enhancing effects of 15d-PG J2 on the production of mucin-producing cells might be related, at least in part, to the activation of GATA-3.