Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism.

Antibody responses, involving B cells, CD4 + T cells, and macrophages, are implicated in autoimmune diseases and organ transplant rejection. We have previously shown that inhibiting FROUNT with disulfiram (DSF) suppresses macrophage activation and migration, effectively treating inflammatory diseases. In this study, we investigated the effectiveness of DSF in antibody-producing reactions. Using a heart transplantation mouse model with antibody-mediated rejection, we administered anti-CD8 antibody to exclude cellular rejection. DSF directly inhibited B cell responses in vitro and significantly reduced plasma donor-specific antibodies and graft antibody deposition in vivo, resulting in prolonged survival of the heart graft. DSF also mediated various effects, including decreased macrophage infiltration and increased Foxp3+ regulatory T-cells in the grafts. Additionally, DSF inhibited pyrimidine metabolism-related gene expression induced by B-cell stimulation. These findings demonstrate that DSF modulates antibody production in the immune response complexity by regulating B-cell and macrophage responses.

Rare case of exostosin 1/exostosin 2-related membranous lupus nephritis concomitant with dual ANCA- and anti-GBM antibody-associated crescentic glomerulonephritis effectively diagnosed by mass spectrometry: a case report.

BACKGROUND: Recent developments in mass spectrometry (MS) have revealed target antigens for membranous nephropathy (MN), including phospholipase A2 receptor and exostosin 1/exostosin 2 (EXT1/2). EXT1/2 are known antigens of autoimmune disease-related MN, especially membranous lupus nephritis. We describe the case of an elderly man who developed nephrotic syndrome followed by progressive renal dysfunction. CASE PRESENTATION: A 78-year-old man presented with rapidly progressive renal dysfunction with proteinuria and hematuria. Three years previously, he had developed leg edema but did not receive any treatment. Laboratory tests showed elevated anti-nuclear antibody (Ab), anti-dsDNA Ab titer, and hypocomplementemia, indicating systemic lupus erythematous. Myeloperoxidase anti-neutrophil cytoplasmic Ab (ANCA) and anti-glomerular basement membrane (GBM) Ab were also detected. The renal pathologic findings were compatible with crescentic glomerulonephritis (GN), whereas non-crescentic glomeruli exhibited MN without remarkable endocapillary or mesangial proliferative change. Immunofluorescence microscopy revealed glomerular IgG, C3, and C1q deposition. All IgG subclasses were positive in glomeruli. Anti-PLA2R Ab in serum was negative. MS analysis was performed to detect the antigens of MN, and EXT1/2 was detected in glomeruli. Therefore, we reached a diagnosis of membranous lupus nephritis concurrent with both ANCA-associated vasculitis and anti-GBM-GN. The simultaneous occurrence of these three diseases is extremely rare. CONCLUSIONS: This is the first report of EXT1/2-related membranous lupus nephritis concurrent with ANCA-associated vasculitis and anti-GBM-GN. This case demonstrates the usefulness of MS in diagnosing complicated cases of MN.

Inhibition of the chemokine signal regulator FROUNT by disulfiram ameliorates crescentic glomerulonephritis.

Activated monocytes/macrophages promote glomerular injury, including crescent formation, in anti-glomerular basement membrane (GBM) glomerulonephritis. Disulfiram, an alcohol-aversion drug, inhibits monocyte/macrophage migration by inhibiting FROUNT, a cytosolic protein that enhances chemokine receptor signaling. Our study found that disulfiram at a human equivalent dose successfully blocked albuminuria and crescent formation with podocyte loss, and later stage kidney fibrotic lesions, in a rat model of anti-GBM glomerulonephritis. A disulfiram derivative, DSF-41, with more potent FROUNT inhibition activity, inhibited glomerulonephritis at a lower dose than disulfiram. Disulfiram markedly reduced the number of monocytes or macrophages at the early stage of glomerulonephritis and that of CD3+ and CD8+ lymphocytes at the established stage. Impaired pseudopodia formation was observed in the glomerular monocytes/macrophages of the disulfiram group; consistent with the in vitro observation that disulfiram blocked chemokine-dependent pseudopodia formation and chemotaxis of bone marrow-derived monocytes/macrophages. Furthermore, disulfiram suppressed macrophage activation as revealed by reduced expression of inflammatory cytokines and chemokines (TNF-α, CCL2, and CXCL9) and reduced CD86 and MHC class II expressions in monocytes/macrophages during glomerulonephritis. The dramatic reduction in monocyte/macrophage number might have resulted from disulfiram suppression of both the chemotactic response of monocytes/macrophages and their subsequent activation to produce cytokines and chemokines, which further recruit monocytes. Additionally, FROUNT was expressed in CD68+ monocytes/macrophages infiltrating the crescentic glomeruli in human anti-GBM glomerulonephritis. Thus, disulfiram can be a highly effective and safe drug for the treatment of glomerulonephritis by blocking the chemotactic responses of monocytes/macrophages and their activation status in the glomerulus.

ASCL1 regulates super-enhancer-associated miRNAs to define molecular subtypes of small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor with dismal prognosis. Recently, molecular subtypes of SCLC have been defined by the expression status of ASCL1, NEUROD1, YAP1, and POU2F3 transcription regulators. ASCL1 is essential for neuroendocrine differentiation and is expressed in the majority of SCLC. Although previous studies investigated ASCL1 target genes in SCLC cells, ASCL1-mediated regulation of miRNAs and its relationship to molecular subtypes remain poorly explored. Here, we performed genome-wide profiling of chromatin modifications (H3K27me3, H3K4me3, and H3K27ac) by CUT&Tag assay and ASCL1 knockdown followed by RNA sequencing and miRNA array analyses in SCLC cells. ASCL1 could preferentially regulate genes associated with super-enhancers (SEs) defined by enrichment of H3K27ac marking. Moreover, ASCL1 positively regulated several SE-associated miRNAs, such as miR-7, miR-375, miR-200b-3p, and miR-429, leading to repression of their targets, whereas ASCL1 suppressed miR-455-3p, an abundant miRNA in other molecular subtypes. We further elucidated unique patterns of SE-associated miRNAs in different SCLC molecular subtypes, highlighting subtype-specific miRNA networks with functional relevance. Notably, we found apparent de-repression of common target genes of different miRNAs following ASCL1 knockdown, suggesting combinatorial action of multiple miRNAs underlying molecular heterogeneity of SCLC (e.g., co-targeting of YAP1 by miR-9 and miR-375). Our comprehensive analyses provide novel insights into SCLC pathogenesis and a clue to understanding subtype-dependent phenotypic differences.

Uterine leiomyosarcomas with osteoclast-like giant cells associated with high expression of RUNX2 and RANKL.

Uterine leiomyosarcoma (ULMS) with osteoclast-like giant cells (OLGCs) has been reported as a rare phenomenon in ULMS, and its clinico-pathological features and tumorigenesis remain unclear. We recently reported high expression of receptor activator of nuclear factor κB ligand (RANKL) in ULMS with OLGCs. As osteoblasts produce RANKL, in this study, we analyzed the expression of Runt-related transcription factor 2 (RUNX2), a critical transcription factor for osteoblasts, and osteoclast-related proteins in three cases of ULMS with OLGCs as well as five conventional ULMSs and nine leiomyomas. Immunohistochemistry and real-time reverse transcription quantitative polymerase chain reaction analyses showed high expression of RUNX2 and RANKL in ULMS with OLGCs. In these cases, macrophages expressed receptor activator of nuclear factor κB (RANK), and OLGCs expressed osteoclast-related proteins (nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and cathepsin K). Accumulation sites of cathepsin K-positive OLGCs showed hemorrhagic appearance and degraded type IV collagen. We reviewed reported cases of ULMS with OLGCs, including ours, and found that they presented an aggressive course even at stage I. Furthermore, metastatic lesions showed similar histological features to those of OLGC association in ULMS. Here, we show that tumor cells in ULMS with OLGCs highly express RUNX2 and RANKL and that osteoclastic differentiation of macrophages occurs in the tumor tissue.

IgM monoclonal gammopathy with heavy-and-light-chain amyloidosis resembling fibrillary glomerulonephritis determined by tandem mass spectrometry: a case report.

BACKGROUND: Fibrillary glomerulonephritis (FGN) is distinguished from amyloidosis by thicker fibrils and the lack of staining with histochemical dyes typically reactive with amyloid. However, congophilic FGN has been proposed recently and adding laser microdissection followed by mass spectrometry (LMD/MS) to conventional pathological methods would be helpful to diagnose FGN. Here, we report a patient initially diagnosed with FGN whose final pathological diagnosis was changed to immunoglobulin heavy-and-light-chain amyloidosis (AHL) after LMD/MS. CASE PRESENTATION: A 75-year-old male developed nephrotic syndrome. Protein electrophoresis showed IgM κ type M proteinemia and he was diagnosed with IgM monoclonal gammopathy. A renal biopsy was performed and pathological examination showed marked periodic acid-Schiff-positive enlargement of the mesangial region and silver stain positivity, but weak direct fast scarlet staining. Immunofluorescence analysis showed monoclonal deposition of IgM-κ chain in the glomerulus. Under electron microscopy, the fibrils were about 20 nm in diameter, which was thicker than typical amyloid fibrils. Based on these findings, the patient was diagnosed with FGN. Although cyclophosphamide and prednisolone were administered, his renal function deteriorated and progressed to end stage renal disease requiring maintenance hemodialysis. As congophilic FGN has been recognized since 2018, Congo red staining and LMD/MS were performed. The Congo red staining was positive and LMD/MS results indicated that this was a case of AHL. CONCLUSIONS: We reported a case of µ and κ chain AHL resembling FGN requiring LMD/MS for definitive diagnosis. Since FGN and amyloidosis exhibit pathological findings, even if Congo red staining is positive, LMD/MS needs to be considered in cases atypical pathological findings, such as silver stain positivity or thicker fibrils.

Molecular hydrogen attenuates gefitinib-induced exacerbation of naphthalene-evoked acute lung injury through a reduction in oxidative stress and inflammation.

Although inhibition of epidermal growth factor receptor (EGFR)-mediated cell signaling by the EGFR tyrosine kinase inhibitor gefitinib is highly effective against advanced non-small cell lung cancer, this drug might promote severe acute interstitial pneumonia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive anti-oxidant. Here, we show that treatment with H2 effectively protects the lungs of mice from severe damage caused by oral administration of gefitinib after intraperitoneal injection of naphthalene, the toxicity of which is related to oxidative stress. Drinking H2-rich water ad libitum mitigated naphthalene/gefitinib-induced weight loss and significantly improved survival, which was associated with a decrease in lung inflammation and inflammatory cytokines in the bronchoalveolar lavage fluid. Naphthalene decreased glutathione in the lung, increased malondialdehyde in the plasma, and increased 4-hydroxy-2-nonenal production in airway cells, all of which were mitigated by H2-rich water, indicating that the H2-rich water reverses cellular damage to the bronchial wall caused by oxidative stress. Finally, treatment with H2 did not interfere with the anti-tumor effects of gefitinib on a lung cancer cell line in vitro or on tumor-bearing mice in vivo. These results indicate that H2-rich water has the potential to improve quality of life during gefitinib therapy by mitigating lung injury without impairing anti-tumor activity.

Optimal conditions and the advantages of using laser microdissection and liquid chromatography tandem mass spectrometry for diagnosing renal amyloidosis.

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently been utilized to accurately detect the amyloid proteins of renal amyloidosis. The present study investigated the optimal procedures for analyzing samples by LCMS/MS, and the advantage of using this technique to diagnosis renal amyloidosis. METHODS: To detect amyloid proteins, laser microdissected glomeruli from AL (n = 13) or AA (n = 10) renal amyloidosis patients were digested and analyzed by LCMS/MS. To determine the best procedures for analyzing samples by LCMS/MS, we examined the suitability of tissue samples, frozen or formalin-fixed paraffin-embedded (FFPE), the number of dissected glomeruli required for analysis (2, 10, or 50 glomeruli), and the amount of trypsin with or without dithiothreitol (DTT). We additionally compared the detection of amyloid proteins between immunostaining and LCMS/MS. RESULTS: Examining 10 dissected glomeruli from FFPE sections digested with trypsin 3 µL (0.1 mg/mL) without DDT made it possible to detect amyloid protein in all 10 AA and in 10 out of 12 AL amyloidosis cases. All AA amyloidosis cases were diagnosed using immunohistochemistry for amyloid A. With immunostaining, however, there were several inconclusive immunoglobulin and/or their light chain staining noted in the AA or AL amyloidosis cases. Even so, LCMS/MS was able to accurately detect amyloid protein in renal amyloidosis. CONCLUSION: The use of 10 laser microdissected glomeruli (170,000-220,000 µm2) with amyloid deposition from FFPE sections digested with trypsin 3 µL (0.1 mg/mL) allowed the accurate detection of amyloid protein in AA and AL amyloidosis.

Uterine leiomyosarcoma with osteoclast-like giant cells associated with high expression of receptor activator of nuclear factor κB ligand.

The occurrence of osteoclast-like giant cells (OLGCs) in uterine leiomyosarcomas (LMSs) is a rare phenomenon. The nature of OLGCs and the significance of their accumulation in these tumors are poorly understood. Recent studies revealed that the formation of osteoclasts requires a specific cytokine, receptor activator of nuclear factor κB ligand (RANKL), in bone. In this study, we investigated the expression of RANKL in 2 cases of uterine LMS with OLGCs by means of immunohistochemistry and compared the extent of RANKL expression with that in conventional uterine LMSs and leiomyomas by using real-time reverse-transcription quantitative polymerase chain reaction. Our cases of uterine LMS with OLGCs showed markedly high expression of RANKL messenger RNA with clear RANKL immunoreactivity compared with messenger RNA expression and immunoreactivity of conventional uterine LMSs and leiomyomas. These findings suggest that the tumors producing RANKL may account for accumulation of OLGCs in tumor tissue because of RANKL-related osteoclastogenesis.

Glomerular basement membrane injuries in IgA nephropathy evaluated by double immunostaining for α5(IV) and α2(IV) chains of type IV collagen and low-vacuum scanning electron microscopy.

BACKGROUND: The glomerulus contains well-developed capillaries, which are at risk of injury due to high hydrostatic pressure, hyperfiltration, hypertension and inflammation. However, the pathological alterations of the injured glomerular basement membrane (GBM), the main component of the glomerular filtration barrier, are still uncertain in cases of glomerulonephritis. METHODS: We examined the alterations of the GBM in 50 renal biopsy cases with IgA nephropathy (31.8 ± 17.6 years old) using double immunostaining for the α2(IV) and α5(IV) chains of type IV collagen, and examining the ultrastructural alterations by transmission electron microscopy (TEM) and low-vacuum scanning electron microscopy (LV-SEM). RESULTS: The GBM of IgA nephropathy cases showed various morphological and qualitative alterations. In the TEM findings, thinning, gaps, rupture, thickening with a lamellar and reticular structure and double contours were detected in the GBM. Double immunostaining for α5(IV) and α2(IV) showed thickening of the GBM with reduced α5(IV) and increased α2(IV), or mosaic images of α5(IV) and α2(IV), and holes, fractures, spiny projections and rupture of α5(IV) in the GBM. In addition, LV-SEM showed an etched image and multiple holes in a widening and wavy GBM. These findings might be associated with the development of a brittle GBM in IgA nephropathy. CONCLUSION: Glomerular basement membrane alterations were frequently noted in IgA nephropathy, and were easily evaluated by double immunostaining for α2(IV) and α5(IV) of type IV collagen and LV-SEM. The application of these analyses to human renal biopsy specimens may enhance our understanding of the alterations of the GBM that occur in human glomerular diseases.

Role of survivin in acute lung injury: epithelial cells of mice and humans.

Survivin, an inhibitor of apoptosis, regulates cell division and is a potential target for anticancer drugs because many cancers express high survivin levels. However, whether survivin would be toxic to human lung cells and tissues has not been determined. This report clarified the involvement of survivin in acute lung injury. We used immunohistochemical analysis, immunoelectron microscopy, and real-time reverse transcription-quantitative polymerase chain reaction to study survivin expression and localization in injured mouse and human lungs. We also used cultured human lung epithelial cells (BEAS-2B and A549) to study survivin cytoprotection. Nuclei and cytoplasm of epithelial cells in day 3 and day 7 models of bleomycin-injured lung showed survivin-positive results, which is consistent with upregulated survivin mRNA expression. These nuclei also evidenced double positive findings for proliferating cell nuclear antigen and survivin. Day 7 models had similar Smac/DIABLO-positive and survivin-positive cell distributions. The cytoplasm and nuclei of epithelial cells in lesions with diffuse alveolar damage manifested strong survivin-positive findings. Bleomycin stimulation in both epithelial cell lines upregulated expression of survivin and apoptosis-related molecules. Suppression of survivin expression with small interfering RNA rendered human lung epithelial cells susceptible to bleomycin-induced damage, with markedly upregulated activation of caspase-3, caspase-7, poly (ADP-ribose) polymerase, and lactate dehydrogenase activity and an increased number of dead cells compared with mock small interfering RNA-treated cells. Overexpression of survivin via transfection resulted in these epithelial cells being resistant to bleomycin-induced cell damage, with reduced activation of apoptosis-related molecules and lactate dehydrogenase activity and fewer dead cells compared with results for mock-transfected cells. Survivin, acting at the epithelial cell level that depends partly on apoptosis inhibition, is therefore a key mediator of cytoprotection in acute lung injury. Understanding the precise role of survivin in normal lung cells is required for the development of therapeutic survivin.

The difference of neovascularization in early intra-alveolar fibrosis between nonspecific interstitial pneumonia and usual interstitial pneumonia.

Of the idiopathic interstitial pneumonias (IIPs), usual interstitial pneumonia (UIP) and diffuse alveolar damage (DAD) usually have poor prognoses. The prognoses of cryptogenic organizing pneumonia (COP) and nonspecific interstitial pneumonia (NSIP) are usually more favorable. Although several reports have described neovascularization in COP and UIP, this aspect of UIP has not been compared with NSIP. In this study, we evaluated neovascularization in intra-alveolar fibrotic lesion of cases of fibrosing NSIP (f-NSIP) (n = 26) and UIP (n = 25). In the f-NSIP group, a considerable degree of neovascularization was observed compared to the UIP group and bud type intra-alveolar fibrosis showed a greater degree of neovascularization compared to the mural-incorporation and obliterative types of intra-alveolar fibrosis. Real-time reverse transcription polymerase chain reaction revealed a significantly greater expression of VEGF-A mRNA in f-NSIP than in UIP. The expression of matrix metalloproteinase-2 (MMP-2) mRNA also showed significantly higher in f-NSIP than UIP. The greater VEGF-A and MMP-2 expression may play a role in the pathogenesis of neovascularization in early intra-alveolar fibrotic lesions in f-NSIP.

Involvement of matrix metalloproteinase-2 in the development of renal interstitial fibrosis in mouse obstructive nephropathy.

Renal fibrosis is a common finding in progressive renal diseases. Matrix metalloproteinases (MMPs) are involved in epithelial-to-mesenchymal transition (EMT). We investigated the role of MMP-2 and the effect of inhibition of MMPs on the development of renal fibrosis. Renal fibrosis was induced in MMP-2 wild-type (MMP-2⁺/⁺) mice by unilateral ureteral obstruction (UUO). Renal histopathology, EMT-associated molecules, and activity of MMP-2 and MMP-9 were examined during the development of interstitial fibrosis. UUO-renal fibrosis was also induced in MMP-2 deficient (MMP-2⁻/⁻) and MMP-2⁺/⁺ mice treated with minocycline (inhibitor of MMPs). In MMP-2⁺/⁺ mice, MMP-2 and MMP-9 were expressed in damaged tubules, and their activities increased in a time-dependent manner after UUO. Interstitial fibrosis was noted at day 14, with deposition of types III and I collagens and expression of markers of mesenchymal cells (S100A4, vimentin, α-smooth muscle actin, and heat shock protein-47) in damaged tubular epithelial cells, together with F4/80+ macrophage infiltration. Fibrotic kidneys expressed EMT-associated molecules (ILK, TGF-ß1, Smad, Wnt, ß-catenin, and Snail). In contrast, the kidneys of MMP-2⁻/⁻ mice and minocycline-treated MMP-2⁺/⁺ mice showed amelioration of renal fibrosis with reduced expression of markers of mesenchymal cells in tubular epithelial cells, inhibition of upregulated EMT-associated molecules, and suppression of macrophage infiltration. The results suggested that MMP-2 have a pathogenic role in renal interstitial fibrosis, possibly through the induction of EMT and macrophage infiltration. Inhibition of MMPs may be beneficial therapeutically in renal fibrosis.

Hydrogen therapy attenuates irradiation-induced lung damage by reducing oxidative stress.

Molecular hydrogen (H(2)) is an efficient antioxidant that diffuses rapidly across cell membranes, reduces reactive oxygen species (ROS), such as hydroxyl radicals and peroxynitrite, and suppresses oxidative stress-induced injury in several organs. ROS have been implicated in radiation-induced damage to lungs. Because prompt elimination of irradiation-induced ROS should protect lung tissue from damaging effects of irradiation, we investigated the possibility that H(2) could serve as a radioprotector in the lung. Cells of the human lung epithelial cell line A549 received 10 Gy irradiation with or without H(2) treatment via H(2)-rich PBS or medium. We studied the possible radioprotective effects of H(2) by analyzing ROS and cell damage. Also, C57BL/6J female mice received 15 Gy irradiation to the thorax. Treatment groups inhaled 3% H(2) gas and drank H(2)-enriched water. We evaluated acute and late-irradiation lung damage after H(2) treatment. H(2) reduced the amount of irradiation-induced ROS in A549 cells, as shown by electron spin resonance and fluorescent indicator signals. H(2) also reduced cell damage, measured as levels of oxidative stress and apoptotic markers, and improved cell viability. Within 1 wk after whole thorax irradiation, immunohistochemistry and immunoblotting showed that H(2) treatment reduced oxidative stress and apoptosis, measures of acute damage, in the lungs of mice. At 5 mo after irradiation, chest computed tomography, Ashcroft scores, and type III collagen deposition demonstrated that H(2) treatment reduced lung fibrosis (late damage). This study thus demonstrated that H(2) treatment is valuable for protection against irradiation lung damage with no known toxicity.

Inhibition of matrix metalloproteinases reduces ischemia-reperfusion acute kidney injury.

Matrix metalloproteinases (MMPs) are endopeptidases that degrade extracellular matrix and involved in ischemic organ injuries. The present study was designed to determine the role of MMP-2 in the development of ischemic acute kidney injury (AKI). AKI was induced in MMP-2 wild-type (MMP-2(+/+)) mice by 30, 60, 90, and 120 min renal ischemia and reperfusion. Renal histology, expression and activity of MMP-2 and MMP-9, and renal function were examined during the development of AKI. AKI was also induced in MMP-2-deficient (MMP-2(-/-)) mice and MMP-2(+/+) mice treated with inhibitor of MMPs (minocycline and synthetic peptide MMP inhibitor). In MMP-2(+/+) mice, MMP-2 and MMP-9 activities increased significantly at 2 to 24 h, peaked at 6 h, after reperfusion. Immunohistochemical analysis identified MMP-2 in the interstitium around tubules and peritubular capillaries in the outer medulla. Acute tubular injury (ATI), including apoptosis and necrosis, was evident in the outer medulla at 24 h, along with renal dysfunction. As ischemia period increases, MMP-2 and MMP-9 activities at 6 h and severity of AKI at 24 h increased depending on the duration of ischemia between 30 and 120 min. However, the kidneys of MMP-2(-/-) mice showed minimal ATI; serum creatinine 24 h after reperfusion was significantly low in these mice. Inhibitors of MMPs reduced ATI and improved renal dysfunction at 24 h. We conclude that MMPs, especially MMP-2 have a pathogenic role in ischemia-reperfusion AKI, and that inhibitors of MMPs can protect against ischemic AKI.

Statin attenuates experimental anti-glomerular basement membrane glomerulonephritis together with the augmentation of alternatively activated macrophages.

Macrophages are heterogeneous and include classically activated M1 and alternatively activated M2 macrophages, characterized by pro- and anti-inflammatory functions, respectively. Macrophages that express heme oxygenase-1 also exhibit anti-inflammatory effects. We assessed the anti-inflammatory effects of statin in experimental anti-glomerular basement membrane glomerulonephritis and in vitro, focusing on the macrophage heterogeneity. Rats were induced anti-glomerular basement membrane glomerulonephritis and treated with atorvastatin (20 mg/kg/day) or vehicle (control). Control rats showed infiltration of macrophages in the glomeruli at day 3 and developed crescentic glomerulonephritis by day 7, together with increased mRNA levels of the M1 macrophage-associated cytokines, interferon-gamma, tumor necrosis factor-alpha, and interleukin-12. In contrast, statin reduced the level of proteinuria, reduced infiltration of macrophages in glomeruli with suppression of monocyte chemotactic protein-1 expression, and inhibited the formation of necrotizing and crescentic lesions. The number of glomerular ED3-positive macrophages decreased with down-regulation of M1 macrophage-associated cytokines. Furthermore, statin augmented ED2-positive M2 macrophages with up-regulation of the M2 macrophage-associated chemokines and cytokines, chemokine (C-C motif) Iigand-17 and interleukin-10. Statin also increased the glomerular interleukin-10-expressing heme oxygenase-1-positive macrophages. Statin inhibited macrophage development, and suppressed ED3-positive macrophages, but augmented ED2-positive macrophages in M2-associated cytokine environment in vitro. We conclude that the anti-inflammatory effects of statin in glomerulonephritis are mediated through inhibition of macrophage infiltration as well as augmentation of anti-inflammatory macrophages.

ANG II receptor blockade enhances anti-inflammatory macrophages in anti-glomerular basement membrane glomerulonephritis.

Macrophages are heterogeneous immune cell populations that include classically activated and alternatively activated (M2) macrophages. We examined the anti-inflammatory effect of ANG II type 1 receptor (AT(1)R) blocker (ARB) on glomerular inflammation in a rat model of anti-glomerular basement membrane (GBM) glomerulonephritis (GN). The study focused on infiltrating CD8(+) and CD4(+) cells and macrophages, as well as the heterogeneity of intraglomerular macrophages. Wistar-Kyoto rats were treated with high-dose olmesartan (3 mg.kg(-1).day(-1)), low-dose olmesartan (0.3 mg.kg(-1).day(-1)), or vehicle (control) 7 days before induction of anti-GBM GN. Control rats showed mainly CD8(+) cells and ED1(+) macrophages, with a few CD4(+) cells infiltrating the glomeruli. Necrotizing and crescentic glomerular lesions developed by day 7 with the increase of proteinuria. AT(1)R was expressed on CD8(+) and CD4(+) cells and on ED1(+) macrophages. Low-dose ARB had no anti-inflammatory effects in anti-GBM GN. However, high-dose ARB reduced glomerular infiltration of CD8(+) cells and ED1(+) macrophages and suppressed necrotizing and crescentic lesions by days 5 to 7 (P < 0.05). In addition, high-dose ARB reduced the numbers of ED3(+)-activated macrophages, suppressed glomerular TNF-alpha and IFN-gamma production, and downregulated M1-related chemokine and cytokines (monocyte chemoattractant protein type 1, IL-6, and IL-12). High-dose ARB also enhanced ED2(+) M2 macrophages by day 7 with upregulation of glomerular IL-4 and IL-13 and augmented CCL17, IL-1 receptor antagonist, and IL-10. We concluded that high-dose ARB inhibits glomerular inflammation by increasing the numbers of M2 macrophages and upregulation of anti-inflammatory cytokines and by suppressing M1 macrophage development with downregulation of M1-related proinflammatory cytokines.