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The pathogenesis of lupus nephritis is mainly attributable to a complex interaction between the innate and adaptive immune systems, including T and B cell function abnormalities. In addition to autoantibody production and immune complex deposition, Th1 and Th17 cytokines may play key roles in the development and progression of lupus nephritis. Acute onset of severe lupus nephritis remains a challenge in terms of prevention and treatment. In the present study, we evaluated the therapeutic effects of DCB-SLE1, an extract of a mixture of four traditional Chinese medicinal herbs ( Atractylodis macrocephalae Rhizoma, Eucommiae cortex , Lonicerae caulis , and Hedyotidis diffusae Herba), on an accelerated severe lupus nephritis model, characterized by acute onset of proteinuria, azotemia, autoantibody production, and development of severe nephritis, induced by twice weekly injection of New Zealand black/white F1 mice with Salmonella -type lipopolysaccharide. DCB-SLE1 was administered daily by gavage starting 2 days after the first dose of induction of lipopolysaccharide, and the mice were euthanized at week 1 or week 5. The results showed that DCB-SLE1 significantly ameliorated the hematuria, proteinuria, renal dysfunction, and severe renal lesions by 1) suppression of B cell activation and decreased autoantibody production; 2) negative regulation of T cell activation/proliferation and natural killer cell activity; 3) suppression of IL-18, IL-6, and IL-17 production and blocking of NF-κB activation in the kidney; and 4) prevention of lymphoid and renal apoptosis. These results show that DCB-SLE1 can protect the kidney from autoimmune response-mediated acute and severe damage through systemic immune modulation and anti-inflammation pathways.

Patients with lupus nephritis show an impaired oxidative status and increased levels of interleukin (IL)-1β and IL-18, which are closely linked to inflammation and correlated with disease activity. Although epigallocatechin-3-gallate (EGCG), the major bioactive polyphenol present in green tea with antioxidant and free radical scavenging activities, has been reported to have anti-inflammatory effects by inhibiting nuclear factor-kappa B (NF-κB)-mediated inflammatory responses in vivo, its effectiveness for the treatment of lupus nephritis is still unknown. In the present study, 12-week-old New Zealand black/white (NZB/W) F1 lupus-prone mice were treated daily with EGCG by gavage until sacrificed at 34 weeks old for clinical, pathological, and mechanistic evaluation. We found that the administration (1) prevented proteinuria, renal function impairment, and severe renal lesions; (2) increased renal nuclear factor E2-related factor 2 (Nrf2) and glutathione peroxidase activity; (3) reduced renal oxidative stress, NF-κB activation, and NLRP3 mRNA/protein expression and protein levels of mature caspase-1, IL-1β, and IL-18; and (4) enhanced splenic regulatory T (Treg) cell activity. Our data clearly demonstrate that EGCG has prophylactic effects on lupus nephritis in these mice that are highly associated with its effects of enhancing the Nrf2 antioxidant signaling pathway, decreasing renal NLRP3 inflammasome activation, and increasing systemic Treg cell activity.

Aim of the study To evaluate the protective effect of baicalein on myocardial dysfunction caused by endotoxaemia in rats and to explore the possible mechanisms. Materials and methods Baicalein (10 mg/kg, intravenous) was administered to conscious Wistar rats 30 min after lipopolysaccharide (LPS; 10 mg/kg, intravenous) challenge. Six hours after LPS administration, the contractile function of the isolated heart was examined using the Langendorff technique. Cardiac protein expression related to inflammatory responses, superoxide anion production and caspase-3 activity were measured. Results Post-treatment with baicalein significantly attenuated the LPS-induced hypotension with accompanying tachycardia. The contractile function of isolated heart was significantly preserved 6 h after LPS administration, following treatment with baicalein. Furthermore, baicalein induced the expression of heme oxygenase-1 protein and reduced superoxide anion formation in the myocardium of LPS-treated rats. Cardiac levels of inducible nitric oxide synthase, monocyte chemoattractant protein-1, phospho-IκBα and phospho-p65 protein and caspase-3 activity significantly increased 6 h after LPS challenge but baicalein significantly attenuated these LPS-induced changes. Conclusions Baicalein improves myocardial contractility in LPS-induced sepsis, which may be related to reductions in oxidative stress, myocardial inflammatory responses and apoptosis.

Rab23, a novel member of the Rab family of small GTPases, has recently been identified in mesangial cells (MCs). Although Rab23 levels in MCs are associated with glomerular nephropathies, the exact physiological and pathological roles of Rab23 in MCs are unknown. In the present study, its roles in MCs were explored by performing proteomics and systems biology analyses in MCs after knockdown or overexpression of Rab23. Knockdown of Rab23 was achieved by transfecting MCs with a plasmid expressing short hairpin RNA against Rab23, while overexpression of Rab23 was accomplished by transfection with the wild-type, dominant negative, and constitutively active Rab23 gene constructs. The effects of different levels of Rab23 activity on proteome of various biological pathways were investigated. Gel-based proteomic approaches and systems biology tools, respectively, were used to identify the Rab23-regulated proteins and the functional pathways. Proteomic analysis revealed the potential roles for Rab23 in multiple processes, including G-protein signal transduction, transcription modulation, RNA stabilization, protein synthesis and degradation, cytoskeleton reorganization, anti-oxidation and detoxification, circadian rhythm regulation and phagocytosis. Bioinformatics analyses showed that Rab23 impacts on multiple biological networks in MCs. These data may shed light on the roles of Rab23 in mesangiopathy or MC damage.

Multiple organ dysfunction caused by hyperinflammation remains the major cause of mortality during sepsis. Excessive M1-macrophage activation leads to systemic inflammatory responses. Gene related to anergy in lymphocytes (Grail) is regarded as an important regulator of T cells that functions by diminishing cytokine production. However, its role in regulating macrophage activation and organ injury during sepsis remains unclear. Our aim was to examine the effects of Grail on macrophage reactivity and organ injury in endotoxemic animals. Wild-type and Grail knockout mice were injected with vehicle or Escherichia coli lipopolysaccharide and observed for 24 h. Changes in blood pressure, heart rate, blood glucose, and biochemical variables were then examined. Moreover, levels of neutrophil infiltration, MMP-9, and caspase 3 were analyzed in the lungs of animals. The expression of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages stimulated with LPS were also assessed in the presence or absence of Grail. Results indicated that loss of Grail expression enhances the induction of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages treated with LPS. Furthermore, LPS-induced macrophage hyperactivation was alleviated by ectopic Grail overexpression. In vivo studies showed that Grail deficiency exacerbates organ damage in endotoxemic animals. Levels of neutrophil infiltration, MMP-9, and caspase 3 were significantly increased in the lungs of Grail-deficient endotoxemic mice. Thus, these results suggest that Grail contributes to the attenuation of hyperinflammation caused by activated macrophages and prevents organ damage in endotoxemic mice. We suggest that Grail signaling could be a therapeutic target for endotoxemia.

Objective. Intra-renal T cells and macrophages play a key pathogenic role in the development and progression of glomerular crescents. We aimed to establish (S)-armepavine [(S)-ARM], a major bioactive compound of a Chinese medicinal plant, Nelumbo nucifera, as a potential therapeutic agent in the treatment of autoimmune crescentic glomerulonephritis (ACGN). Methods. A mouse ACGN model associated with T-cell dysregulation, was used to evaluate the therapeutic effects of (S)-ARM on the rapidly progressive glomerular disorder. Results. The results showed that (S)-ARM administered in the established phase of ACGN is capable of dramatically decreasing glomerular crescents in the kidney and improving proteinuria and renal dysfunction. These effects were associated with greatly inhibited infiltration of T cells/macrophages and suppressed nuclear factor (NF)-kB activation in the kidney, lowered serum levels of autoantibodies and both serum and intra-renal levels of pro-inflammatory cytokines, suppressed T/B-cell activation and T-cell proliferation of the spleen, reduced glomerular immune deposits and apoptosis in both the spleen and kidney in (S)-ARM-treated ACGN mice, compared with the vehicle-treated (disease control) group of ACGN mice. Conclusion. We demonstrated therapeutic effects of (S)-ARM on ACGN as a result of: (i) early systemic negative modulation of T/B cells; (ii) intra-renal regulation of combined NF-kB activation and mononuclear leucocytic infiltration, thereby preventing glomerular crescent formation; and (iii) protection from apoptosis in both the spleen and kidney. Key word: Crescentic glomerulonephritis, Crescent, (S)-armepavine, Nelumbo nucifera, T cell, Macrophage, Nuclear factor-kB, Apoptosis.

Focal segmental glomerulosclerosis (FSGS) is characterized by typical sclerosis but also shows other non-sclerotic lesions that provide prognostic informations. The glomerular epithelial hyperplasia lesion (EPHL) that develops earlier than the sclerotic lesions is a key determinant of progression of FSGS. However, the relationship among EPHL, glomeular sclerosis, and macrophage infiltration in FSGS is unclear, and the EPHL-associated markers helpful for prognosis of FSGS have still not been completely identified. Here, we performed clinicopathologic, immunochemical, and molecular analyses to examine whether osteopontin (OPN), a macrophage chemokine, is an injury marker of EPHLs correlating with glomerular sclerosis and macrophage mobilization. First, the FSGS model was induced in Balb/c mice by a single injection of adriamycin, and consecutive sclerosis changes were evaluated. In parallel, we used reverse transcription-polymerase chain reaction and Western blot analyses to determine levels of OPN in isolated glomeruli and urine, respectively. Immunohistochemistry was applied to assess the OPN expression in EPHLs and macrophage infiltration around the glomeruli. Our results showed that, within glomeruli, OPN expressed restrictedly within EPHL; the OPN mRNA and protein of glomeruli increased on day 11, correlating well with the early EPHL, and following sclerosis and macrophage infiltration. In addition, immunohistochemistry (IHC) staining of OPN greatly highlighted early glomerular EPHLs, helping microscopic identification of EPHLs. We propose that the OPN expression in EPHLs could contribute to the progression of FSGS by recruiting macrophage toward the compromised glomeruli. Detection of OPN in glomeruli and urine could be helpful in prognosis of FSGS.

Autoimmune crescentic glomerulonephritis (ACGN) is a variant of crescentic glomerulonephritis. The outcome of treatment of crescentic glomerulonephritis is poor. Binding of decoy receptor 3 (DCR3) to its ligand is capable of downregulating the alloresponsiveness of T cells. DCR3 has also been shown to benefit an experimental autoimmune model of diabetes. This study tested the hypothesis that a potential immune regulator, DCR3, could prevent the evolution of ACGN. With the use of an established ACGN model in mice, mice were treated with 100 microg/10 g body wt human DCR3 by hydrodynamics-based gene delivery at 14-d intervals. The results showed that the gene therapy resulted in (1) suppression of T and B cell activation and T cell proliferation; (2) a reduction in serum levels of proinflammatory cytokines; (3) improvement of proteinuria and renal dysfunction; (4) prevention of glomerular crescent formation, renal interstitial inflammation, and glomerulosclerosis; (5) a reduction in serum levels of autoantibodies and glomerular immune deposits; (6) inhibition of apoptosis in the spleen and kidney; (7) prevention of T cell and macrophage infiltration of the kidney; and (8) suppression of fibrosis-related gene expression in the kidney compared with empty vector-treated (disease control) ACGN mice. On the basis of these findings, it is proposed that human DCR3 exerts its preventive and protective effects on ACGN through modulation of T cell activation/proliferation, B cell activation, protection against apoptosis, and suppression of mononuclear leukocyte infiltration in the kidney.

Background Focal segmental glomerulosclerosis (FSGS) is a chronic nephropathy showing characteristic glomerular sclerosis. So far, the diagnosis and prognosis of FSGS rely mainly on the invasive biopsy. Searching for potential FSGS-associated urinary biomarkers representing pre-sclerotic and serial sclerotic stages of FSGS could be helpful to the non-invasive diagnosis and prognosis of FSGS. Methods In the present study, we used a 2D gel-based proteomic approach to identify urinary proteins at pre-sclerotic and different sclerotic stages of an FSGS mouse model in order to find FSGS-related urinary proteins. The FSGS mouse model was established in Balb/c mice by a single injection of adriamycin, and disease severity was monitored by renal biological parameters and histopathological features. Urine was collected on days 0, 4, 7, 11, 15 and 20, and subjected to two-dimensional electrophoresis (2-DE) analysis. Proteins were identified by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) and a protein database search. Some of the identified proteins were confirmed by western blot analysis. Results We identified 37 urinary proteins showing characteristic patterns of dynamic changes along the disease course of FSGS. Early urinary proteins appearing before glomerular sclerosis were noticed. Importantly, 11 urine proteins are novel to FSGS and have known functions highly associated with different pathogenetic steps of the disease, including haemodynamic disturbance, podocyte apoptosis, ECM-protein deposition and glomerular sclerosis. Conclusions Some urinary proteins appearing earlier than glomerular sclerosis could serve as potential early diagnostic biomarkers. The proteins with the pathogenic roles could serve as potential non-invasive prognostic markers of FSGS, and give an insight into pathogenic mechanisms of this sclerosis disease.

Objectives Systemic lupus erythematosus is occasionally accompanied with bacterial infection. Lipopolysaccharide (LPS) from bacteria can accelerate and exacerbate lupus nephritis (LN) in animal models, but some mechanisms underlying the LPS-induced acceleration are still unclear. First, it is not known whether LPS can stimulate mesangial cells (MCs) to secrete the pro-inflammatory cytokine, interleukin (IL)-18. Second, it is also unclear whether LPS and/or IL-18 can induce MC apoptosis. Here, we attempted to clarify the cause-and-effect relationships between LPS stimulation, IL-18 production and MC apoptosis to address the above questions. Methods LPS was used to induce accelerated LN in LN-prone mice. LPS and IL-18 were also used to treat cultured MCs isolated from the mice. IL-18 expression and MC apoptosis were investigated by in situ hybridization, the TUNEL method, reverse transcription- polymerase chain reaction (RT-PCR), western blotting, DNA electrophoresis and flow cytometry. NFkappaB was detected by immunofluorescent staining. Results In the LPS-accelerated LN mice, we observed co-existence of IL-18 expression, hyperplasia, apoptosis, and activated apoptotic signal transduction in MCs, as well as marked neutrophil infiltration in the glomerulus, especially around the mesangial region. In cultured MCs, LPS greatly enhanced IL-18 expression, but did not induce apoptosis, while mouse IL-18 did not induce apoptosis or activate apoptotic signal transduction in MCs. Conclusions We conclude that LPS can evoke IL-18 production in MCs, but neither LPS nor IL-18 directly induces apoptosis or activates apoptotic signal transduction in the cells. We infer that LPS-induced IL-18 production by MCs could be a mediator by which LPS accelerates and exacerbates LN.

Uncontrolled inflammation is a leading cause of various chronic diseases. Cinnamaldehyde (CA) is a major bioactive compound isolated from the essential oil of the leaves of Cinnamomum osmophloeum kaneh that exhibits anti-inflammatory activity; however, the use of CA is limited by its cytotoxicity. Here, we synthesized three CA derivatives and identified 4-hydroxycinnamaldehyde-galactosamine (HCAG) as a low toxicity anti-inflammatory compound in vitro (HCAG IC50 ≫ 1600 µM; CA IC50=40 µM) and in vivo. HCAG reduced pro-inflammatory mediator expression in LPS-activated macrophages by inhibiting MAPK and PKC-α/δ phosphorylation, decreasing ROS generation and reducing NF-κB activation. HCAG also reduced NLRP3 inflammasome-derived IL-1β secretion by inhibiting the ATP-mediated phosphorylation of AKT and PKC-α/δ. In a mouse model of LPS-induced renal inflammation, we observed reduced albuminuria and a mild degree of glomerular proliferation, glomerular sclerosis and periglomerular inflammation in the HCAG-treated mice compared with the vehicle-treated mice. The underlying mechanisms for these renoprotective effects involved: (1) inhibited NLRP3 inflammasome activation; (2) decreased superoxide anion levels and apoptosis; and (3) suppressed activation of NF-κB and related downstream inflammatory mediators.

Myofibroblasts are a main cell-type of collagen-producing cells during tissue fibrosis, but their origins remains controversial. While bone marrow-derived myofibroblasts in renal fibrosis has been reported, the cell origin and mechanisms regulating their transition into myofibroblasts remain undefined. In the present study, cell lineage tracing studies by adoptive transfer of GFP+ or dye-labelled macrophages identified that monocyte/macrophages from bone marrow can give rise to myofibroblasts via the process of macrophage-myofibroblast transition (MMT) in a mouse model of unilateral ureteric obstruction. The MMT cells were a major source of collagen-producing fibroblasts in the fibrosing kidney, accounting for more than 60% of α-SMA+ myofibroblasts. The MMT process occurred predominantly within M2-type macrophages and was regulated by TGF-β/Smad3 signalling as deletion of Smad3 in the bone marrow compartment of GFP+ chimeric mice prevented the M2 macrophage transition into the MMT cells and progressive renal fibrosis. In vitro studies in Smad3 null bone marrow macrophages also showed that Smad3 was required for TGF-β1-induced MMT and collagen production. In conclusion, we have demonstrated that bone marrow-derived fibroblasts originate from the monocyte/macrophage population via a process of MMT. This process contributes to progressive renal tissue fibrosis and is regulated by TGF-β/Smad3 signalling.

Background Nanoparticles can be used for targeted drug delivery, in particular for brain cancer therapy. However, this requires a detailed analysis of nanoparticles from the associated microvasculature to the tumor, not easy because of the required high spatial resolution. The objective of this study is to demonstrate an experimental solution of this problem, based in vivo and post-mortem whole organ imaging plus nanoscale 3-dimensional (3D) X-ray microscopy. Results The use of gold nanoparticles (AuNPs) as contrast agents paved the way to a detailed high-resolution three dimensional (3D) X-ray and fluorescence imaging analysis of the relation between xenografted glioma cells and the tumor-induced angiogenic microvasculature. The images of the angiogenic microvessels revealed nanoparticle leakage. Complementary tests showed that after endocytotic internalization fluorescent AuNPs allow the visible-light detection of cells. Conclusions AuNP-loading of cells could be extended from the case presented here to other imaging techniques. In our study, they enabled us to (1) identify primary glioma cells at inoculation sites in mice brains; (2) follow the subsequent development of gliomas. (3) Detect the full details of the tumor-related microvasculature; (4) Finding leakage of AuNPs from the tumor-related vasculature, in contrast to no leakage from normal vasculature. Electronic supplementary material The online version of this article (doi:10.1186/s12951-015-0140-2) contains supplementary material, which is available to authorized users.

BACKGROUND Focal segmental glomerulosclerosis (FSGS) is caused by gradual deposition of extracellular matrix proteins, one of which, fibronectin (FN) is critical for sclerosis development. The origin of the FN deposited at an early stage of FSGS is still unclear. METHODS For investigating the origin of FN, the onset of increases in FN levels in the serum, glomeruli and urine were studied in a mouse model induced by adriamycin and compared with the time-course of development of glomerulosclerosis and expression of FN mRNA. RESULTS In the FSGS mice, serum FN levels were significantly increased as early as the onset of proteinuria on day 4 (7.26 +/- 0.37 mg/ml compared with 5.58 +/- 0.76 mg/ml in normal controls, P < 0.05). This was followed by an increase in glomerular deposition of FN protein on day 7 (FN/actin ratio, 0.216 +/- 0.003 compared with 0.039 +/- 0.009 in normal controls, P < 0.05). Glomerular m-RNA expression was also significantly elevated on day 7, but the locally synthesized FN did not show any increase until day 15. A significant increase in urinary FN protein and focal glomerulosclerosis was seen on day 11. CONCLUSIONS We infer that FN in blood acts as an initiator of the development of FSGS in this mouse model. In addition, serum and urine FN proteins could serve as useful biomarkers for monitoring the progression of FSGS.

Calcium-binding proteins annexin A2 and S100A6 are sensors of tubular injury and recovery in acute renal failure. Background Rise in cellular calcium is associated with acute tubular necrosis, the most common cause of acute renal failure (ARF). The mechanisms that calcium signaling induce in the quiescent tubular cells to proliferate and differentiate during acute tubular necrosis have not been elucidated. Methods Acute tubular necrosis induced in mice by single intravenous injection of uranyl nitrate and examined after 1, 3, 7, and 14 days. Renal function was monitored and kidneys were evaluated by histology, immunohistochemistry, Western blotting, in situ hybridization, and real-time reverse transcription-polymerase chain reaction (RT-PCR). Models of folic acid induced-ARF and ischemic/reperfusion (I/R) injury were similarly investigated. Results Analysis of mRNA expression of intracellular calcium and phospholipid-binding proteins demonstrated selective expression of S100A6 and Annexin A2 (Anxa2) in the renal cortex with marked elevation on day 3, and gradually decline on day 7 and further attenuation on day 14. Similarly, the expression of both proteins, as demonstrated by immunohistochemistry and Western blot analysis, was increased and reached the peak level on day 7 and then gradually declined by day 14. Vimentin, a marker of dedifferentiated cells, was highly expressed during the recovery phase. Combined in situ hybridization immunohistochemistry revealed colocalization of both S100A6 and Anxa2 with proliferating cell nuclear antigen (PCNA). The universality of this phenomenon was confirmed in two other mouse acute tubular necrosis models, the ischemic-reperfusion injury and folic acid-induced ARF. Conclusion Collectively, these findings demonstrate that S100A6 and Anxa2 expression, initiated in response to tubular injury, persist in parallel throughout the recovery process of tubular cells in acute renal failure.

Hyperglycemia and mannitol activate protein kinase C (PKC) and induce mesangial cell hypocontractility that subsequently may modulate renal function. Since focal adhesion kinase (FAK) activation is known to be linked with PKC activity, FAK may also be involved in mesangial cell contraction. To facilitate our understanding of the PKC- and FAK-modulating mechanism, we developed an in vitro model of mouse mesangial cell hypocontractility induced by hyperglycemia or mannitol. Mouse mesangial cells (CRL-1927) were exposed to: normal D-glucose (group N), high D-glucose (group H), and control groups at the same osmolality as H plus L-glucose (group L) and mannitol (group M). Changes in the planar surface area of cells in response to 1 microM phorbol 12-myristate 13-acetate (PMA) were determined. Western blot analyses for PKC, phosphorylated (p)-PKC, tyrosine phosphorylation, FAK, and p-FAK were done on each of these four groups. The effects of mannitol in various doses on cell contraction and activation of PKC and FAK were also assayed. The planar surface areas of groups H and M both showed an attenuated change in response to PMA stimulation. Before PMA stimulation, the baseline PKC expression of groups H and M showed a higher expression of p-PKC alpha and delta than that seen in group N (p0.05). Results of tyrosine phosphorylation and immunoprecipitation showed that FAK may be involved in this contraction process. The total amount of FAK showed no significant difference among the four experimental groups; however, p-FAK was found to have significantly increased in group M (p0.05). The use of PKC and tyrosine kinase inhibitors reduced PMA-induced mesangial cell contraction in all four groups. Activation of PKC alpha, delta, and FAK with the resultant inhibition of mesangial cell contraction by mannitol was found to be dose-dependent. These results may provide a correlation between increased expression of several PKC isoforms and, in particular, increased phosphorylation levels of PKC alpha and delta and hypocontractility induced by high glucose and mannitol treatment. Furthermore, the mannitol-induced hypocontractility involving PKC and FAK occurred in a dose-dependent manner.

Introduction The aim of this study was to investigate the effects of levosimendan on rodent septic shock induced by cecal ligation and puncture (CLP). Methods Three hours after peritonitis-induced sepsis, male Wistar rats were randomly assigned to receive an intravenous infusion of levosimendan (1.2 μg/kg/min for 10 min and then 0.3 μg/kg/min for 6 h) or an equivalent volume of saline and vehicle (5% dextrose) solution. Results The levosimendan-treated CLP animals had significantly higher arterial pressure and lower biochemical indices of liver and kidney dysfunction compared to the CLP animals (P

Sepsis and its associated multiple organ failure are related to high mortality in critical patients. Several studies have reported that gabexate mesilate, a synthetic inhibitor of trypsin-like serine protease, protects tissues/organs against injury in the models of endotoxemia. The aim of this study was to examine whether gabexate mesilate could attenuate coagulopathy and organ dysfunction in lipopolysaccharide (LPS)-induced sepsis model by using thrombelastography (TEG). LPS (7.5 mg/kg/h, intravenouly for 4 h) was administered to male adult Wistar rats. Some of the LPS rats received a continuous infusion of gabexate mesilate (10 mg/kg/h, intravenously for 8.5 h) for 30 min before the LPS administration. Variable parameters of hemodynamics, biochemistry, hemostasis and inflammatory response were measured for 6 h after the LPS infusion. TEG variables (R-time, K-time, α-angle, and maximal amplitude) were also measured. The pretreatment of LPS rats with gabexate mesilate significantly attenuated the lung, liver and kidney dysfunction, consumptive coagulopathy, the increases in serum tumor necrosis factor-α, interleukin-6, plasma thrombin-antithrombin complex and plasminogen activator inhibitor-1, and neutrophils infiltration score in lung, liver and kidney, compared with the LPS alone group. In addition, TEG parameters correlated with tissue and liver injury in the late phase of endotoxemia. In particular, a strong negative correlation between maximal amplitude at 4 h and Ln (lactate dehydrogenase) at 6 h after LPS infusion was noted (r = -0.752, P 0.001, R = 0.566). These results indicate that beneficial effects of anticoagulants (e.g. gabexate mesilate) in endotoxemia could be monitored by TEG per se.

Selegiline, an anti-Parkinson drug, has antioxidant and anti-apoptotic effects. To explore the effect of selegiline on sepsis, we used a clinically relevant animal model of polymicrobial sepsis. Cecal ligation and puncture (CLP) or sham operation was performed in male rats under anesthesia. Three hours after surgery, animals were randomized to receive intravenously selegiline (3 mg/kg) or an equivalent volume of saline. The administration of CLP rats with selegiline (i) increased arterial blood pressure and vascular responsiveness to norepinephrine, (ii) reduced plasma liver and kidney dysfunction, (iii) attenuated metabolic acidosis, (iv) decreased neutrophil infiltration in liver and lung, and (v) improved survival rate (from 44% to 65%), compared to those in the CLP alone rats. The CLP-induced increases of plasma interleukin-6, organ superoxide levels, and liver inducible nitric oxide synthase and caspase-3 expressions were ameliorated by selegiline treatment. In addition, the histological changes in liver and lung were significantly attenuated in the selegiline -treated CLP group compared to those in the CLP group. The improvement of organ dysfunction and survival through reducing inflammation, oxidative stress and apoptosis in peritonitis-induced sepsis by selegiline has potential as an adjuvant agent for critical ill.

Image registration of biological data is challenging as complex deformation problems are common. Possible deformation effects can be caused in individual data preparation processes, involving morphological deformations, stain variations, stain artifacts, rotation, translation, and missing tissues. The combining deformation effects tend to make existing automatic registration methods perform poor. In our experiments on serial histopathological images, the six state of the art image registration techniques, including TrakEM2, SURF + affine transformation, UnwarpJ, bUnwarpJ, CLAHE + bUnwarpJ and BrainAligner, achieve no greater than 70% averaged accuracies, while the proposed method achieves 91.49% averaged accuracy. The proposed method has also been demonstrated to be significantly better in alignment of laser scanning microscope brain images and serial ssTEM images than the benchmark automatic approaches (p < 0.001). The contribution of this study is to introduce a fully automatic, robust and fast image registration method for 2D image registration.

The NLRP3 inflammasome is a caspase-1-containing multi-protein complex that controls the release of IL-1β and plays important roles in the development of inflammatory disease. Here, we report that resveratrol, a polyphenolic compound naturally produced by plants, inhibits NLRP3 inflammasome-derived IL-1β secretion and pyroptosis in macrophages. Resveratrol inhibits the activation step of the NLRP3 inflammasome by suppressing mitochondrial damage. Resveratrol also induces autophagy by activating p38, and macrophages treated with an autophagy inhibitor are resistant to the suppressive effects of resveratrol. In addition, resveratrol administration mitigates glomerular proliferation, glomerular sclerosis, and glomerular inflammation in a mouse model of progressive IgA nephropathy. These findings were associated with decreased renal mononuclear leukocyte infiltration, reduced renal superoxide anion levels, and inhibited renal NLRP3 inflammasome activation. Our data indicate that resveratrol suppresses NLRP3 inflammasome activation by preserving mitochondrial integrity and by augmenting autophagy.

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is a reactive oxygen species-sensitive multiprotein complex that regulates IL-1β maturation via caspase-1. It also plays an important role in the pathogenesis of inflammation-related disease. Cyclooxygenase-2 (COX-2) is induced by inflammatory stimuli and contributes to the pathogenesis of inflammation-related diseases. However, there is currently little known about the relationship between COX-2 and the NLRP3 inflammasome. Here, we describe a novel role for COX-2 in regulating the activation of the NLRP3 inflammasome. NLRP3 inflammasome-derived IL-1β secretion and pyroptosis in macrophages were reduced by pharmaceutical inhibition or genetic knockdown of COX-2. COX-2 catalyzes the synthesis of prostaglandin E2 and increases IL-1β secretion. Conversely, pharmaceutical inhibition or genetic knockdown of prostaglandin E2 receptor 3 reduced IL-1β secretion. The underlying mechanisms for the COX-2-mediated increase in NLRP3 inflammasome activation were determined to be the following: (1) enhancement of lipopolysaccharide-induced proIL-1β and NLRP3 expression by increasing NF-κB activation and (2) enhancement of the caspase-1 activation by increasing damaged mitochondria, mitochondrial reactive oxygen species production and release of mitochondrial DNA into cytosol. Furthermore, inhibition of COX-2 in mice in vivo with celecoxib reduced serum levels of IL-1β and caspase-1 activity in the spleen and liver in response to lipopolysaccharide (LPS) challenge. These findings provide new insights into how COX-2 regulates the activation of the NLRP3 inflammasome and suggest that it may be a new potential therapeutic target in NLRP3 inflammasome-related diseases.

The pathogenesis of focal segmental glomerulosclerosis (FSGS) is considered to be associated with oxidative stress, mononuclear leukocyte recruitment and infiltration, and matrix production and/or matrix degradation, although the exact etiology and pathogenic pathways remain to be determined. Establishment of a pathogenesis-based therapeutic strategy for the disease is clinically warranted. Citral (3,7-dimethyl-2,6-octadienal), a major active compound in Litsea cubeba , a traditional Chinese herbal medicine, can inhibit oxidant activity, macrophage and NF-κB activation. In the present study, first, we used a mouse model of FSGS with the features of glomerular epithelial hyperplasia lesions (EPHLs), a key histopathology index of progression of FSGS, peri-glomerular inflammation, and progressive glomerular hyalinosis/sclerosis. When treated with citral for 28 consecutive days at a daily dose of 200 mg/kg of body weight by gavage, the FSGS mice showed greatly reduced EPHLs, glomerular hyalinosis/sclerosis and peri-glomerular mononuclear leukocyte infiltration, suggesting that citral may be renoprotective for FSGS and act by inhibiting oxidative stress and apoptosis and early activating the Nrf2 pathway. Meanwhile, a macrophage model involved in anti-oxidative and anti-inflammatory activities was employed and confirmed the beneficial effects of citral on the FSGS model.

Renal reactive oxygen species (ROS) and mononuclear leukocyte infiltration are involved in the progressive stage (exacerbation) of IgA nephropathy (IgAN), which is characterized by glomerular proliferation and renal inflammation. The identification of the mechanism responsible for this critical stage of IgAN and the development of a therapeutic strategy remain a challenge. Osthole is a pure compound isolated from Cnidiummonnieri (L.) Cusson seeds, which are used as a traditional Chinese medicine, and is anti-inflammatory, anti-apoptotic, and anti-fibrotic both in vitro and in vivo. Recently, we showed that osthole acts as an anti-inflammatory agent by reducing nuclear factor-kappa B (NF-κB) activation in and ROS release by activated macrophages. In this study, we examined whether osthole could prevent the progression of IgAN using a progressive IgAN (Prg-IgAN) model in mice. Our results showed that osthole administration resulted in prevention of albuminuria, improved renal function, and blocking of renal progressive lesions, including glomerular proliferation, glomerular sclerosis, and periglomerular mononuclear leukocyte infiltration. These findings were associated with (1) reduced renal superoxide anion levels and increased Nrf2 nuclear translocation, (2) inhibited renal activation of NF-κB and the NLRP3 inflammasome, (3) decreased renal MCP-1 expression and mononuclear leukocyte infiltration, (4) inhibited ROS production and NLRP3 inflammasome activation in cultured, activated macrophages, and (5) inhibited ROS production and MCP-1 protein levels in cultured, activated mesangial cells. The results suggest that osthole exerts its reno-protective effects on the progression of IgAN by inhibiting ROS production and activation of NF-κB and the NLRP3 inflammasome in the kidney. Our data also confirm that ROS generation and activation of NF-κB and the NLRP3 inflammasome are crucial mechanistic events involved in the progression of the renal disorder.

Overt systemic inflammatory response is a predisposing mechanism for infection-induced skeletal muscle damage and rhabdomyolysis. Aberrant DNA methylation plays a crucial role in the pathophysiology of excessive inflammatory response. The antiarrhythmic drug procainamide is a non-nucleoside inhibitor of DNA methyltransferase 1 (DNMT1) used to alleviate DNA hypermethylation. Therefore, we evaluated the effects of procainamide on the syndromes and complications of rhabdomyolysis rats induced by lipopolysaccharide (LPS). Rhabdomyolysis animal model was established by intravenous infusion of LPS (5 mg/kg) accompanied by procainamide therapy (50 mg/kg). During the experimental period, the changes of hemodynamics, muscle injury index, kidney function, blood gas, blood electrolytes, blood glucose, and plasma interleukin-6 (IL-6) levels were examined. Kidneys and lungs were exercised to analyze superoxide production, neutrophil infiltration, and DNMTs expression. The rats in this model showed similar clinical syndromes and complications of rhabdomyolysis including high levels of plasma creatine kinase, acute kidney injury, hyperkalemia, hypocalcemia, metabolic acidosis, hypotension, tachycardia, and hypoglycemia. The increases of lung DNMT1 expression and plasma IL-6 concentration were also observed in rhabdomyolysis animals induced by LPS. Treatment with procainamide not only inhibited the overexpression of DNMT1 but also diminished the overproduction of IL-6 in rhabdomyolysis rats. In addition, procainamide improved muscle damage, renal dysfunction, electrolytes disturbance, metabolic acidosis, hypotension, and hypoglycemia in the rats with rhabdomyolysis. Moreover, another DNMT inhibitor hydralazine mitigated hypoglycemia, muscle damage, and renal dysfunction in rhabdomyolysis rats. These findings reveal that therapeutic effects of procainamide could be based on the suppression of DNMT1 and pro-inflammatory cytokine in endotoxin-induced rhabdomyolysis.

High levels of reactive oxygen species (ROS), systemic T cell activation, and macrophage infiltration in the kidney are implicated in the acceleration and progression of IgA nephropathy (IgAN), the most frequent type of primary glomerulonephritis. However, the pathogenic mechanism of IgAN is still little understood, and it remains a challenge to establish a specific therapeutic strategy for this type of glomerular disorder. Recently, we showed that antroquinonol (Antroq), a pure active compound from Antrodia camphorata mycelium, inhibits renal inflammation and reduces oxidative stress in a mouse model of renal fibrosis. But the anti-inflammatory and immune-regulatory effects of Antroq on the acceleration and progression of primary glomerular disorders have not been determined. In this study, we show that Antroq administration substantially impeded the development of severe renal lesions, such as intense glomerular proliferation, crescents, sclerosis, and periglomerular interstitial inflammation, in mice with induced accelerated and progressive IgAN (AcP-IgAN). Further mechanistic analysis in AcP-IgAN mice showed that, early in the developmental stage of the AcP-IgAN model, Antroq promoted the Nrf2 antioxidant pathway and inhibited the activation of T cells and NLRP3 inflammasome. Significantly improved proteinuria/renal function and histopathology in AcP-IgAN mice of an established stage supported potential therapeutic effects of Antroq on the disease. In addition, Antroq was shown to inhibit activation of NLRP3 inflammasome in vitro by an IgA immune complex (IC) partly involving a reduced ROS production in IgA-IC-primed macrophages, and this finding may be helpful in the understanding of the mode of action of Antroq in the treated AcP-IgAN mice.

Reactive oxygen species (ROS) plays a critical role in the regulation of NLRP3 inflammasome activation. However, the ROS-mediated signaling pathways controlling NLRP3 inflammasome activation are not well defined. Using lipopolysaccharide (LPS) and adenosine triphosphate (ATP) activated murine macrophages as the testing model, cytokine release and protein expression were quantified by enzyme-linked immunosorbent assay and Western blot, respectively. ROS was scavenged by N-acetyl cysteine; NADPH oxidase, the major source of ROS, was inhibited by diphenyliodonium, apocynin or gp91-phox siRNA transfection; and protein kinase was inhibited by its specific inhibitor. LPS-induced NLRP3 protein expression was regulated through the NADPH oxidase/ROS/NF-κB-dependent, JAK2/PI3-kinase/AKT/NF-κB-dependent, and MAPK-dependent pathways, while ATP-induced caspase-1 activation was regulated through the NADPH oxidase/ROS-dependent pathway. These results demonstrate that ROS regulates not only the priming stage, but also the activation stage, of NLRP3 inflammasome activation in LPS + ATP-activated macrophages.

Background Hyperoncotic albumin may be a therapeutic option to improve tissue perfusion and organ injury in sepsis. To clarify the hypothesis and its mechanism, hyperoncotic albumin was administered to the rats in a polymicrobial sepsis–peritonitis model. Materials and methods Peritonitis was induced by a surgery of cecal ligation and puncture (CLP) in 27 male Wistar rats. For control purposes, sham operations without ligating and puncturing the cecum were performed in 20 rats. Three hours later, rats were randomized to receive intravenously 3 mL/kg of 5% albumin, 25% albumin, or normal saline. All the hemodynamic and biochemical parameters were measured during the 18-h observation. Results In septic rats, 25% albumin attenuated hypotension, vascular hyporeactivity to norepinephrine, and the elevated serum levels of lactate dehydrogenase and blood urea nitrogen. However, these improvements were not noted in CLP rats after 5% albumin treatment. In addition, 25% albumin decreased metabolic acidosis and improved the CLP-induced hypoperfusion in the intestine and kidney. Superoxide levels in the aorta and lung and the protein expression of inducible nitric oxide synthase in the lung were also attenuated by 25% albumin in CLP rats. Microscopic findings confirmed that 25% albumin attenuated the substantial swelling and cell infiltration in the intestine and lung caused by CLP. Conclusions In this sepsis rat model, 25% albumin reduced macro- and microhemodynamic changes and attenuated intestine and lung injuries in peritonitis-induced sepsis.

Liver injury developing in patients with sepsis may lead to an increased risk of mortality. Thrombelastography (TEG) is generally applied to evaluate hemostatic disturbance in patients undergoing liver transplantation or cardiopulmonary bypass. The aim of this study was to investigate the development of liver injury and coagulopathy in a lipopolysaccharide (LPS)-induced animal model and to assess the relationship between TEG variables and liver injury. Male Wistar rats received LPS (30 mg/kg over a 4-h intravenous infusion) to induce experimental liver injury or isotonic saline as a control. Variables of hemodynamics and liver biochemistry were measured during the subsequent 6 h after the start of infusion. TEG variables (R-time, K-time, α-angle and maximal amplitude), thrombin-antithrombin complex and plasminogen activator inhibitor-1 were also measured. After LPS infusion, liver injury [examined by biochemical variables (e.g. alanine aminotransferase, ALT) and histological studies] was developed and inflammatory cytokines (tumor necrosis factor-α and interleukin-6) were raised. At the initial period of LPS infusion, R-time was shortened and α-angle was increased. Thereafter, α-angle and maximal amplitude were decreased progressively, demonstrating that endotoxin induced coagulation disturbances. Furthermore, there were strong positive correlation between K-time and natural log (Ln)(ALT) (r = 0.823, P = 0.001); also, there were strong negative correlations between α-angle and Ln(ALT) (r = -0.762, P = 0.002) as well as maximal amplitude and Ln(ALT) (r = -0.732, P = 0.004) at 6 h after LPS infusion. These results demonstrated that TEG could be a potential tool to evaluate the development of liver injury in endotoxemia.

Ultrasound (US) irradiation has been found to facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, its transfection efficiency is generally low, and the expression duration of transfected gene is short. Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. The purpose of this study is to determine whether PEI can also prolong the expression duration after US-mediated transfection. A mixture of pCMViLUC and 22-kDa linear PEI was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulsed US. The duration of expression was assessed periodically following US treatment. As expected, strong expression of luciferase could be found 30days after the treatment of DNA-PEI complex with US exposure, both in vitro and in vivo. However, without US, only very low transfection level could be obtained in vivo. The DNA/PEI complex showed protective effect against digestion of DNase I enzymes as compared with groups without PEI or to which PEI was added following the mixing of plasmid DNA with DNase I. PEI enhanced the US transfection efficiency by increasing both the intracellular uptake of plasmid DNA and the percentage of transfected cells. Most of the DNA uptake occurred at 3h after US exposure, suggesting that endocytosis took place. Moreover, the PEI-facilitated US gene transfection depended on the ratio of PEI and DNA (N/P ratio), which was different for in-vitro and in-vivo conditions. This system could be applied in future human gene therapies.

Oxidative stress, inflammation, and fibrosis are involved in the development and progression of focal segmental glomerulosclerosis (FSGS), a common form of idiopathic nephrotic syndrome that represents a therapeutic challenge because it has a poor response to steroids. Antroquinonol (Antroq), a purified compound, is a major active component of a mushroom, namely Antrodia camphorata, that grows in the camphor tree in Taiwan, and it has inhibitory effects on nitric oxide production and inflammatory reactions. We hypothesized that Antroq might ameliorate FSGS renal lesions by modulating the pathogenic pathways of oxidative stress, inflammation, and glomerular sclerosis in the kidney. We demonstrate that Antroq significantly (1) attenuates proteinuria, renal dysfunction, and glomerulopathy, including epithelial hyperplasia lesions and podocyte injury; (2) reduces oxidative stress, leukocyte infiltration, and expression of fibrosis-related proteins in the kidney; (3) increases renal nuclear factor E2-related factor 2 (Nrf2) and glutathione peroxidase activity; and (4) inhibits renal nuclear factor-κB (NF-κB) activation and decreases levels of transforming growth factor (TGF)-β1 in serum and kidney tissue in a mouse FSGS model. Our data suggest that Antroq might be a potential therapeutic agent for FSGS, acting by boosting Nrf2 activation and suppressing NF-κB-dependent inflammatory and TGF-β1-mediated fibrosis pathways in the kidney.

Background Rab23, a member of the Rab family of small GTPase, has a function in antagonizing sonic hedgehog signal transduction. Both Rab-family and hedgehog-related proteins are involved in sclerosis and fibrosis in certain pathological states, but their roles in focal segmental glomerulosclerosis (FSGS) remain unclear. Methods The FSGS model was established in Balb/c mice by a single injection of adriamycin. Serum, urine and mice kidneys were collected on Days 0, 7, 15 and 20. Western blot analysis was performed to detect the levels of Rab23 in the samples. Immunohistochemistry was used to examine the expressional profiles of Rab23 in kidneys. The expressions of transcripts of Rab23, extracellular matrix (ECM) proteins, and various hedgehog signalling pathway genes in kidneys or mesangial cells were evaluated by real-time RT-PCR. The effect of Rab23 on ECM protein expressions was evaluated by the knockdown or overexpression of Rab23 in mesangial cells. Results Our results show that elevations of Rab23 were observed in the urine, but not in the serum, of the FSGS mice. Rab23 and hedgehog signalling pathway genes were constitutively expressed in normal kidneys and were significantly up-regulated in the kidneys of FSGS mice. The basal expression of Rab23 was identified in glomeruli, and mesangial cells displayed obvious elevation of Rab23 in the FSGS state. The knockdown or overexpression of Rab23 affected the collagen expression in cultured mesangial cells. Conclusions An autocrine loop of hedgehog signalling could be activated in mesangial cells in the FSGS state, and Rab23 may be elevated to suppress hedgehog signalling and/or influence collagen synthesis. Importantly, Rab23 could serve as a biomarker that indicates the severity of FSGS.

Gene transfer of Fas ligand (FasL) to tumor cells has been demonstrated to inhibit tumor growth in vivo, and neutrophils are primarily responsible for this immunoprotection. The granulocyte– macrophage colony stimulating factor (GM-CSF) secreted by tumor vaccine can recruit dendritic cells (DCs) for efficient antigen presentation to T cells that generate the tumor-specific response. To investigate whether the combination of FasL and GM-CSF can efficiently suppress tumor growth, we have established Lewis lung carcinoma (LLC-1) cells that are transduced with GM-CSF (LLC/ GM-CSF), FasL (LLC/FasL) or both genes (LLC/FasL/GM-CSF) to test their tumorigenic potential in vivo. Mice inoculated with LLC/GM-CSF display high survival rates along with reduction of tumor growth. In contrast, none of the mice injected with LLC/ FasL or LLC/FasL/GM-CSF develop tumors. Specific memory immune response and delayed LLC-1 tumor growth are found in mice immunized with LLC-1/FasL or LLC-1/FasL/GM-CSF. Furthermore, therapeutic effects are observed only when LLC-1/ FasL/GM-CSF tumor vaccine is employed to retard growth of preexisting LLC-1 tumors. Tumor growth is also completely suppressed in mice injected with a mixture of LLC-1 and LLC-1/ FasL/GM-CSF. In addition, IL-12 production, cytotoxic T-cell activity and IgG against LLC-1 are manifested in mice injected with LLC/FasL/GM-CSF. Our data show that FasL-induced pathway triggers expression of proinflammatory cytokines, including IL1b, IL-6, MIP-2 and MCP-1, while GM-CSF-dependent pathway promotes functional maturation and activation of DCs. Taken together, the results indicate that dual gene-based delivery with FasL and GM-CSF may serve as a more effective tumor vaccine to suppress lung cancer cell growth in vivo. ' 2008 Wiley-Liss, Inc.

Background. Acute tubular necrosis (ATN) is characterized by an initiation phase, followed by an extension phase, and a maintenance and recovery phase, the latter of which involves increased regeneration of tubular cells. Nephronectin (NPNT), a ligand for a8b1 integrin, is expressed in the ureteric bud epithelium during kidney morphogenesis. However, little is known about the potential involvement of NPNT in the regeneration phase of ATN. Methods. cDNA microarray, real-time polymerase chain reaction, in situ hybridization, immunohistochemistry, immuno-electron microscopy and immunoassay (for urine) were used to identify the timecourse NPNT expression in a murine model of ATN. Results. The gene transcript of NPNT was examined during a 14-day course of ATN by a cursory cDNA microarray analysis. Although NPNT was observed focally in normal renal tubular epithelium, it was greatly expressed in regenerating tubular cells during the maintenance and recovery phases of ATN. As early as day 1 following onset of ATN, NPNT was already present in the urine. Importantly, NPNT expression preceded proliferating cell nuclear antigen protein expression in regenerating renal tubular epithelial cells, as demonstrated by double immunohistochemistry. Conclusion. The present study was the first to identify an enhanced expression of NPNT in regenerating tubular epithelium in an experimental model of ATN. NPNT may play a crucial role in the regenerating process of nephrotoxic ATN. Our data also suggest that NPNT may provide a useful tissue and urine biomarker for both the development and evolution of nephrotoxic acute renal injury.

Autoimmune crescentic glomerulonephritis is characterized by severe immune response with glomerular crescentic formation and fibrosis in the kidney. Recent studies indicate that overexpression of renal Smad7 attenuates both renal fibrosis and inflammation in rat remnant kidney. However, little attention has been paid to the potential role of TGF-beta/Smad signaling in autoimmune kidney disease. This study tested the hypothesis that blocking TGF-beta signaling by overexpression of Smad7 may have a therapeutic effect in a mouse model of autoimmune crescentic glomerulonephritis that was induced in C57BL/6 x DBA/2J F1 hybrid mice by giving DBA/2J donor lymphocytes. Smad7 gene was transfected into the kidney using the ultrasound-microbubble-mediated system. Results showed that overexpression of Smad7 blocked both renal fibrosis and inflammatory pathways in terms of Smad2/3 and NF-kappaB activation (P < 0.01), thereby inhibiting alpha-smooth muscle actin; collagen I, III, and IV accumulation; and expression of inflammatory cytokines (IL-1beta and IL-6), adhesion molecule/chemokine (intercellular adhesion molecule-1, monocyte chemoattractant protein-1), and inducible nitric oxide synthase (all P < 0.01). Leukocyte infiltration (CD4(+) cells and macrophages) was also suppressed (P < 0.005). Severe histologic damage (glomerular crescent formation and tubulointerstitial injury) and functional injury including proteinuria were significantly improved (all P < 0.05). This study provides important evidence that overexpression of Smad7 may have therapeutic potential for autoimmune kidney disease.

In animal models of IgA nephropathy, the inevitable endogenous immune response to passively administered antigens alone or in complex with specific IgA mask the exact role each might play in pathogenesis. To delineate the role the immune response might play, we have developed a passive model with exclusive IgA-immune complex-mediated nephropathy in B-cell-deficient (BCD) mice. Glomerular IgA immune deposits were induced by administration of purified IgA antiphosphorylcholine and the specific pneumococcal C-polysaccharide (PnC) antigen daily for 2 weeks into BCD and wild-type (WT) mice. In BCD mice IgA+PnC deposits induced severe glomerular injury and renal dysfunction. In contrast, WT mice developed intense glomerular IgG and IgM and C3 co-deposits of the IgA+PnC with significantly less renal injury. Cytofluorometric analysis revealed that PnC induced in BCD, but not in WT, a rapid and dramatic increase in number of activated CD3(+)/CD69(+) T-cell population. The nuclear factor-kappa B (NF-kappaB) transcription factor was activated early and progressively increased in response to glomerular IgA+PnC deposits. These results suggest that nephritogenic IgA+PnC immune deposits induce glomerular and renal dysfunction through activation of the NF-kappaB. This inflammatory pathway is modulated by the endogenous cellular and antibody response to the antigen affecting the course of IgA nephropathy progression.

Background We examined the alterations in gene expression associated with the development of crescentic glomerulonephritis in murine chronic graft-versus-host disease, a model for human systemic lupus erythematosus. Methods The disease was induced in (C57BL/6 x DBA/2) F(1) hybrids by injection of DBA/2 lymphocytes leading to deposition of auto-antibodies in the glomeruli, and a lupus type of nephritis morphologically. After extensive crescent formation at week 9 of disease, cDNA microarray analysis was performed and highly expressed genes were evaluated as molecular markers by real-time reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunohistochemistry and immunoassay of urine proteins. Results Six genes, secreted acidic cysteine-rich glycoprotein (Sparc), thymosin beta 10 (Tmsb10), S100 calcium-binding protein A6 (S100a6), annexin A2 (Anxa2), osteopontin (OPN) and lipocalin 2 (Lcn2), were quantified by real-time RT-PCR in laser microdissected glomeruli in a time course manner. Sparc was detected early before the onset of proteinuria and continued to increase throughout the course of the disease. The expression of Tmsb10, S100a6 and Anxa2 coincided with heavy proteinuria. By week 9, OPN and Lcn2 were highly expressed. The expression of proteins encoded by these genes was predominant in the glomerular crescent. The protein levels of Sparc, OPN and Lcn2 in urine were significantly elevated. Conclusions These findings implicate these six genes in the development of glomerular crescents. More importantly, detection of Sparc, OPN and Lcn2 in urine may mean that these molecules could serve as important biomarkers for non-invasive diagnosis of glomerular crescents.

The effects of IgA immune complex (IgA-IC) on the contractile function of cultured mesangial cells were measured by the changes in planar surface area in response to treatment with agonists. Incubation of mesangial cells with IgA-IC for 24 hours significantly decreased the contractile responses to angiotensin II (10(-6) M) and phorbol 12-myristate 13-acetate (PMA, 10(-6) M). Pretreatment of mesangial cells with the protein kinase C (PKC) inhibitor, chelerythrine (10(-6) M), eliminated the difference in contractile responses to angiotensin II or PMA between the control and IgA-IC groups indicating IgA-IC may inhibit the activity of PKC. The contractile responses to ionomycin were not significantly different between IgA-IC treated and control mesangial cells, suggesting that the contractile machinery is not impaired by IgA-IC. Intracellular calcium, [Ca2+]i measured by changes in fura-2 level in response to ATP or bradykinin, was significantly inhibited in IgA-IC treated mesangial cells, compared to control cells. In contrast, treatment with thapsigargin did not result in significant differences in [Ca2+]i between IgA-IC and control mesangial cells, suggesting that a negligible role of endoplasmic reticulum in the effects of IgA-IC. Using PKC specific antibodies, IgA-IC significantly increased the particulate fraction of PKC-iota of mesangial cells to 141+/-13% of control, without significantly changing the protein content of PKC-alpha, -delta and -lambda in the cytosolic and particulate fractions. In summary, IgA-IC inhibits the contractile responses of cultured mesangial cells to agonists by inhibiting the activation of PKC and [Ca2+]i.

Background: Antroquinonol (Antroq) is an active component of Antrodia camphorate. The present study was to validate the preventive effects of Antroq in an atherosclerosis model. Materials and Methods: We examined Antroq inhibitory effect on rat aortic smooth muscle cell proliferation and migration and evaluated its effect on neointima formation and inflammation in mouse carotid artery ligation (CAL). Results: Our data show that Antroq [1] inhibited the proliferation (Antroq [3.0 μg/ml] + PDGF 41.7 ± 7.3%, vehicle + PDGF 134.5 ± 7.3%) (p

Two polyenylpyrroles from a soil ascomycete Gymnoascus reessii were previously identified as hit compounds in screening for cytotoxicity against lung cancer cells. These compounds and various analogs, which have been previously synthesized and tested for anti-lung cancer cell activity, were tested for anti-inflammatory activity. After preliminary screening for cytotoxicity for RAW 264.7 murine macrophage cells, the non-toxic compounds were tested for anti-inflammatory activity using lipopolysaccharide (LPS)-activated RAW 264.7 cells. Compounds 1h, 1i, and 1n reduced LPS-induced nitric oxide (NO) production, with respective ED50 values of 15 ± 2, 16 ± 2, and 17 ± 2 µM. They also reduced expression of inducible NO synthase and interleukin-6 (IL-6) without affecting cyclooxygenase-2 expression. Compound 1h also reduced secretion of IL-6 and tumor necrosis factor-α by LPS-activated J774A.1 murine macrophage cells, primary mice peritoneal macrophages, and JAWSII murine bone marrow-derived dendritic cells and reduced NLRP3 inflammasome-mediated interleukin-1β (IL-1β) secretion by LPS + adenosine triphosphate-activated J774A.1 and JAWSII cells. The underlying mechanisms for the anti-inflammatory activity of compound 1h were found to be a decrease in LPS-induced reactive oxygen species (ROS) production, mitogen-activated protein kinase phosphorylation, and NF-κB activation and a decrease in ATP-induced ROS production and PKC-α phosphorylation. These results provide promising insights into the anti-inflammatory activity of these conjugated polyenes and a molecular rationale for future therapeutic intervention in inflammation-related diseases. They also show how compound 1h regulates inflammation and suggest it may be a new source for the development of anti-inflammatory agents to ameliorate inflammation- and NLRP3 inflammasome-related diseases.

Bamboo vinegar (BV), a natural liquid derived from the condensation produced during bamboo charcoal production, has been used in agriculture and as a food additive, but its application to immune modulation has not been reported. Here, we demonstrated that BV has anti-inflammatory activities both in vitro and in vivo. BV reduced inducible nitric oxide synthase expression and nitric oxide levels in, and interleukin-6 secretion by, lipopolysaccharide-activated macrophages without affecting tumor necrosis factor-α secretion and cyclooxygenase-2 expression. The mechanism for the anti-inflammatory effect of BV involved decreased reactive oxygen species production and protein kinase C-α/δ activation. Furthermore, creosol (2-methoxy-4-methylphenol) was indentified as the major anti-inflammatory compound in BV. Impaired cytokine expression and NLR family, pyrin domain-containing 3 (NLRP3) inflammasome activation was seen in mice treated with creosol. These findings provide insights into how BV regulates inflammation and suggest that it may be a new source for the development of anti-inflammatory agents or a healthy supplement for preventing and ameliorating inflammation- and NLRP3 inflammasome-related diseases, including metabolic syndrome.