The fungal lactone oxacyclododecindione is a potential new therapeutic substance in the treatment of lupus-associated kidney disease
2014; Elsevier BV; Volume: 86; Issue: 4 Linguagem: Inglês
10.1038/ki.2014.109
ISSN1523-1755
AutoresJenny Henke, Gerhard Erkel, Christoph Brochhausen, Hartmut Kleinert, Andreas Schwarting, Julia Menke, Andrea Pautz,
Tópico(s)Systemic Lupus Erythematosus Research
ResumoRecently oxacyclododecindione (Oxa), a macrocyclic lactone isolated from the imperfect fungus Exserohilum rostratum, has been described as a potent transcription inhibitor of inducible proinflammatory and profibrotic genes in cell culture models. As kidney disease in systemic lupus erythematosus is characterized by aberrant expression of inflammatory mediators and infiltration of immune cells, we investigated the effect of Oxa in MRL-Faslpr mice, a model of systemic lupus erythematosus. These mice develop a spontaneous T-cell and macrophage-dependent autoimmune disease including severe glomerulonephritis that shares features with human lupus. Comparable to the results of in vitro models, we found a reduced expression of the cytokines IFN-γ, IL-6, and TNF-α and the chemokines CCL2, RANTES, and CSF-1 on mRNA and protein level in the kidney of Oxa-treated MRL-Faslpr mice. Accordingly, Oxa treatment reduced the infiltration of immune cells and the frequency of activated proinflammatory T cells in the kidney. Moreover, kidney disease, measured by histopathology, IgG and collagen deposition, and proteinuria, was ameliorated in Oxa-treated MRL-Faslpr mice compared with the control group. Thus, Oxa is a new effective anti-inflammatory compound, which may serve as base structure for the development of new therapeutics for the treatment of chronic inflammatory and/or fibrotic diseases. Recently oxacyclododecindione (Oxa), a macrocyclic lactone isolated from the imperfect fungus Exserohilum rostratum, has been described as a potent transcription inhibitor of inducible proinflammatory and profibrotic genes in cell culture models. As kidney disease in systemic lupus erythematosus is characterized by aberrant expression of inflammatory mediators and infiltration of immune cells, we investigated the effect of Oxa in MRL-Faslpr mice, a model of systemic lupus erythematosus. These mice develop a spontaneous T-cell and macrophage-dependent autoimmune disease including severe glomerulonephritis that shares features with human lupus. Comparable to the results of in vitro models, we found a reduced expression of the cytokines IFN-γ, IL-6, and TNF-α and the chemokines CCL2, RANTES, and CSF-1 on mRNA and protein level in the kidney of Oxa-treated MRL-Faslpr mice. Accordingly, Oxa treatment reduced the infiltration of immune cells and the frequency of activated proinflammatory T cells in the kidney. Moreover, kidney disease, measured by histopathology, IgG and collagen deposition, and proteinuria, was ameliorated in Oxa-treated MRL-Faslpr mice compared with the control group. Thus, Oxa is a new effective anti-inflammatory compound, which may serve as base structure for the development of new therapeutics for the treatment of chronic inflammatory and/or fibrotic diseases. Systemic lupus erythematosus (SLE) is a multisystem disorder characterized by increased production of autoantibodies, leukocyte infiltration, and deposition of immune complexes in various organs. These symptoms can be particularly observed in lupus nephritis, a major clinical manifestation of SLE, which occurs in ∼40% of patients.1.Bomback A.S. Appel G.B. Updates on the treatment of lupus nephritis.J Am Soc Nephrol. 2010; 21: 2028-2035Crossref PubMed Scopus (136) Google Scholar Lupus nephritis is the main cause of morbidity and mortality in SLE patients. Lupus-associated nephritis is driven by proinflammatory cytokines (interferon (IFN)-γ, interleukin (IL)-6, IL-18, tumor necrosis factor (TNF)-α, etc) and chemokines (chemokine (C-C motive) ligand (CCL)-2, RANTES, CXCL10, etc), which are primarily produced by infiltrating monocytes and lymphocytes.2.Tucci M. Ciavarella S. Strippoli S. et al.Oversecretion of cytokines and chemokines in lupus nephritis is regulated by intraparenchymal dendritic cells: a review.Ann N Y Acad Sci. 2009; 1173: 449-457Crossref PubMed Scopus (28) Google Scholar,3.Iwata Y. Furuichi K. Kaneko S. et al.The role of cytokine in the lupus nephritis.J Biomed Biotechnol. 2011; 2011: 594-809Crossref Scopus (37) Google Scholar The current treatment for SLE includes the use of glucocorticoids, antimalarial drugs, and immunosuppressive agents. Recently, belimumab, an antibody interfering with B-cell activation and maturation, was approved as the first biological for SLE.4.Lo M.S. Tsokos G.C. Treatment of systemic lupus erythematosus: new advances in targeted therapy.Ann N Y Acad Sci. 2012; 1247: 138-152Crossref PubMed Scopus (38) Google Scholar Despite the recent advances in SLE therapy, a number of patients fail to respond, respond inadequately to these drugs, or have severe adverse effects. Many pharmaceuticals (e.g., TNF-α antagonists, etc) approved for therapy against other chronic inflammatory diseases seem to be less effective in SLE treatment. Therefore, there is a need to identify new therapeutics and new drug targets to improve the current treatment regimes for SLE. As demonstrated by the action of glucocorticoids, modulation of proinflammatory gene expression is an effective and powerful way to control inflammatory processes in asthma, rheumatoid arthritis, or SLE.5.Mosca M. Tani C. Carli L. et al.Glucocorticoids in systemic lupus erythematosus.Clin Exp Rheumatol. 2011; 29: S126-S129PubMed Google Scholar,6.Barnes P.J. Glucocorticosteroids: current and future directions.Br J Pharmacol. 2011; 163: 29-43Crossref PubMed Scopus (379) Google Scholar We have recently shown that the fungal compounds (S)-curvularin and galiellalactone reduce inflammation in animal models of rheumatoid arthritis and asthma and thereby ameliorate disease symptoms comparable to the action of glucocorticoids.7.Schmidt N. Art J. Forsch I. et al.The anti-inflammatory fungal compound (s)-curvularin reduces proinflammatory gene expression in an in vivo model of rheumatoid arthritis.J Pharmacol Exp Ther. 2012; 343: 106-114Crossref PubMed Scopus (20) Google Scholar,8.Hausding M. Tepe M. Ubel C. et al.Induction of tolerogenic lung CD4+ T cells by local treatment with a pSTAT-3 and pSTAT-5 inhibitor ameliorated experimental allergic asthma.Int Immunol. 2011; 23: 1-15Crossref PubMed Scopus (28) Google Scholar Similar to glucocorticoids, these substances alter the expression of a number of proinflammatory mediators such as cytokines or chemokines. However, the molecular mode of action seems to be independent of the glucocorticoid receptor signaling pathway.9.Schmidt N. Pautz A. Art J. et al.Transcriptional and post-transcriptional regulation of iNOS expression in human chondrocytes.Biochem Pharmacol. 2010; 79: 722-732Crossref PubMed Scopus (51) Google Scholar The recently identified fungal macrocyclic lactone oxacyclododecindione (Oxa) exhibits very potent anti-inflammatory and antifibrotic activities at nanomolar concentrations in cell culture experiments. The molecular mechanisms by which way Oxa interferes with the corresponding signaling pathways are not yet known. Initial analyses of different cellular models hint toward an inhibition of proinflammatory and profibrotic transcription factors.10.Erkel G. Belahmer H. Serwe A. et al.Oxacyclododecindione, a novel inhibitor of IL-4 signaling from Exserohilum rostratum.J Antibiot (Tokyo). 2008; 61: 285-290Crossref PubMed Scopus (20) Google Scholar,11.Rudolph K. Serwe A. Erkel G. Inhibition of TGF-beta signaling by the fungal lactones (S)-curvularin, dehydrocurvularin, oxacyclododecindione and galiellalactone.Cytokine. 2013; 61: 285-296Crossref PubMed Scopus (31) Google Scholar According to the in vitro data, Oxa seems to be a promising new substance for the treatment of chronic inflammatory diseases such as SLE, but until now no data exist on whether Oxa ameliorates chronic inflammation in vivo. To test the novel therapeutic in an in vivo model of SLE we chose the MRL-Faslpr mouse strain. These mice spontaneously develop a lupus-like syndrome very similar to human disease. As in humans it is an inflammatory multiorgan disease with severe glomerulonephritis, circulating autoantibodies, splenomegaly, lymphadenopathy, skin lesions, and arthritis. Comparable to human lupus, one major cause of mortality in MRL-Faslpr mice is renal failure due to progressive inflammation.12.Theofilopoulos A.N. Dixon F.J. Etiopathogenesis of murine SLE.Immunol Rev. 1981; 55: 179-216Crossref PubMed Scopus (406) Google Scholar, 13.Kelley V.E. Roths J.B. Interaction of mutant lpr gene with background strain influences renal disease.Clin Immunol Immunopathol. 1985; 37: 220-229Crossref PubMed Scopus (153) Google Scholar, 14.Kelley V.R. Wuthrich R.P. Cytokines in the pathogenesis of systemic lupus erythematosus.Semin Nephrol. 1999; 19: 57-66PubMed Google Scholar Dysregulated cytokine production contributes to immune dysfunction, mediates tissue inflammation, and causes organ damage. Inflammatory cytokines such as type I and type II IFNs, IL-6, and TNF-α, as well as immunomodulatory cytokines such as IL-10 and transforming growth factor-β, have been identified as important factors in SLE. The expression of these cytokines correlates with the disease activity and severity of glomerulonephritis.15.Chun H.Y. Chung J.W. Kim H.A. et al.Cytokine IL-6 and IL-10 as biomarkers in systemic lupus erythematosus.J Clin Immunol. 2007; 27: 461-466Crossref PubMed Scopus (295) Google Scholar, 16.Baechler E.C. Batliwalla F.M. Karypis G. et al.Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.Proc Natl Acad Sci USA. 2003; 100: 2610-2615Crossref PubMed Scopus (1730) Google Scholar, 17.Aringer M. Smolen J.S. Cytokine expression in lupus kidneys.Lupus. 2005; 14: 13-18Crossref PubMed Scopus (101) Google Scholar, 18.Uhm W.S. Na K. 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Tyden H. et al.Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus.Arthritis Res Ther. 2011; 13: R60Crossref PubMed Scopus (46) Google Scholar which stimulates monocytes23.Vogl T. Tenbrock K. Ludwig S. et al.Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock.Nat Med. 2007; 13: 1042-1049Crossref PubMed Scopus (1024) Google Scholar and cytokine production,24.Sunahori K. Yamamura M. Yamana J. et al.The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis.Arthritis Res Ther. 2006; 8: R69Crossref PubMed Scopus (230) Google Scholar have been observed in SLE patients. Furthermore, during glomerulonephritis, enhanced expression of osteopontin (SPP1) was detected.25.Masutani K. Akahoshi M. Tsuruya K. et al.Predominance of Th1 immune response in diffuse proliferative lupus nephritis.Arthritis Rheum. 2001; 44: 2097-2106Crossref PubMed Scopus (209) Google Scholar There is evidence that SSP1 promotes inflammatory immune responses by activation of T cells and macrophage migration.26.O'Regan A.W. Nau G.J. Chupp G.L. et al.Osteopontin (Eta-1) in cell-mediated immunity: teaching an old dog new tricks.Immunol Today. 2000; 21: 475-478Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar Our goal was to test the hypothesis that treatment with the anti-inflammatory compound Oxa ameliorates MRL-Faslpr lupus nephritis. We detected that Oxa treatment decreased the expression of important proinflammatory cytokines and chemokines involved in the onset and progression of glomerulonephritis in lupus disease. Immunohistochemical analyses revealed a reduced infiltration of immune cells into the kidney. These anti-inflammatory and immunomodulatory effects resulted in an improvement in the clinical indicators of lupus nephritis. We determined less proteinuria and double-stranded DNA (dsDNA) antibodies, a decrease in renal IgG deposition, and less renal fibrosis in Oxa-treated MRL-Faslpr mice. In in vitro studies in the human hepatocarcinoma cell line HepG2 and the breast cancer cell line MDA-MB-231, nanomolar concentrations of Oxa inhibited the expression of signal transducer and activator of transcription-2, -3, -6- and transforming growth factor-β-dependent genes.11.Rudolph K. Serwe A. Erkel G. Inhibition of TGF-beta signaling by the fungal lactones (S)-curvularin, dehydrocurvularin, oxacyclododecindione and galiellalactone.Cytokine. 2013; 61: 285-296Crossref PubMed Scopus (31) Google Scholar,27.Elzner S. Schmidt D. Schollmeyer D. et al.Inhibitors of inducible NO synthase expression: total synthesis of (S)-curvularin and its ring homologues.ChemMedChem. 2008; 3: 924-939Crossref PubMed Scopus (28) Google Scholar As these signaling pathways are important for the onset and progression of lupus nephritis, we tested whether Oxa ameliorates glomerulonephritis in MRL-Faslpr mice, a well-accepted lupus model. In our experiments, 2-month-old female MRL-Faslpr mice with mild disease symptoms such as low-grade proteinurea (Figure 5)28.Tesch G.H. Maifert S. Schwarting A. et al.Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas(lpr) mice.J Exp Med. 1999; 190: 1813-1824Crossref PubMed Scopus (275) Google Scholar,29.Menke J. Rabacal W.A. Byrne K.T. et al.Circulating CSF-1 promotes monocyte and macrophage phenotypes that enhance lupus nephritis.J Am Soc Nephrol. 2009; 20: 2581-2592Crossref PubMed Scopus (78) Google Scholar were injected intraperitoneally with 1mg/kg Oxa or phosphate-buffered saline (PBS)/10% EtOH as solvent control every other day for 5 weeks. To rank the anti-inflammatory potency of Oxa, 5mg/kg of the glucocorticoid dexamethasone was used as a positive control. First, we analyzed the expression of the proinflammatory mediators IFN-γ, IL-6, and TNF-α, known to be elevated in the sera of SLE patients with active disease and glomerulonephritis. The mRNA expression of these inflammation markers was significantly reduced by Oxa treatment in the kidney of MRL-Faslpr mice. The effects of Oxa were comparable to those of dexamethasone (Figure 1a). For TNF-α, we confirmed the data on protein level by western blot analyses. Although the effects of Oxa and dexamethasone on TNF-α protein expression in total kidney lysates were less pronounced, we still detected a significant reduction (Figure 1b). Furthermore, the mRNA expression of S100A8 and SPP1, two additional marker molecules for inflammation and glomerulonephritis, was reduced by Oxa treatment. In case of SPP1 the effect was comparable to that of the dexamethasone control group, but virtually no effect of dexamethasone on S100A8 expression was observed (Figure 1c). In enzyme-linked immunosorbent assay (ELISA) experiments we also detected reduced S100A8 protein expression in the total kidney lysates of Oxa-treated animals, confirming our data described above (Figure 1d). To analyze the effects of Oxa on the synthesis of a broader range of cytokines and chemokines implicated in the pathogenesis of SLE, we performed a proteome profile analysis with a mouse cytokine/chemokine array (R&D Systems). This western blot-based technique allows the simultaneous detection of about 40 different proinflammatory mediators. For these experiments we isolated protein from total kidney lysates from Oxa- or PBS/EtOH-treated mice. The quantitative analyses revealed a moderate reduction in CCL4 and CXCL9 expression by Oxa. The effects were more pronounced for CCL2, RANTES, and CXCL12. Here, application of Oxa decreased the expression to half of that of the solvent control group (Figure 2a). Similar results were obtained for different cytokines. Consistent with the results described above (Figure 1), we detected a reduction in IFN-γ and TNF-α protein expression in Oxa-treated animals (Figure 2b). The expression of further lupus-relevant cytokines IL-16, IL-17, IL-23, and IL-27 was decreased compared with the control group. Moreover, upon Oxa treatment we detected reduced expression of CSF-1, a factor required for macrophage survival, differentiation, and proliferation and therefore involved in the pathogenesis of lupus nephritis.30.Lenda D.M. Stanley E.R. Kelley V.R. Negative role of colony-stimulating factor-1 in macrophage, T cell, and B cell mediated autoimmune disease in MRL-Fas(lpr) mice.J Immunol. 2004; 173: 4744-4754Crossref PubMed Scopus (85) Google Scholar In summary, in the kidney of MRL-Faslpr mice Oxa reduced the expression of a number of proinflammatory genes involved in the pathogenesis of lupus and lupus glomerulonephritis. Renal injury in MRL-Faslpr mice is complex and consists of glomerular, interstitial, and perivascular infiltration of monocytes and lymphocytes, and concomitant immune complex deposition. To determine whether Oxa ameliorates kidney injury, we compared the renal histopathology of Oxa-treated MRL-Faslpr mice with that of solvent control–treated MRL-Faslpr mice. These analyses demonstrated that the severity of interstitial and glomerular pathology was decreased by Oxa treatment. The same tendency was observed for perivascular areas (Figure 3a). In accordance, we detected reduced kidney infiltration of CD4+ T cells, CD68+ monocytes/macrophages, and CD20+ B cells (Figure 3b). Thus, we can conclude that Oxa treatment prevents the characteristic massive accumulation of leukocytes in the kidney of MRL-Faslpr mice. Moreover, in accordance with the proteome profile results (Figure 2), we detected a lower frequency of CD4+IFN-γ-, CD4+IL-6-, and CD4+IL-17 cells in the kidney of Oxa-treated MRL-Faslpr mice, demonstrating the anti-inflammatory effect of Oxa in this in vivo model (Figure 4a and b). Furthermore, tubular epithelial cells showed a lower IL-6 expression. Unexpectedly, flow cytometry and immunostaining results revealed an Oxa-mediated increase in the number of CD4+FoxP3 cells in the kidney of MRL-Faslpr mice (Figure 4a and b). This may indicate an immunomodulatory capacity of Oxa. We measured different clinical parameters to determine whether the Oxa-induced alterations in proinflammatory gene expression and renal infiltration of immune cells improve renal function. First, we determined the progression of proteinuria semiquantitatively with urine test strips twice a week during the treatment period. These results showed a delayed increase in proteinuria in Oxa-treated MRL-Faslpr mice compared with the control group (Figure 5a). In addition, we analyzed albuminuria in ELISA experiments in urinary samples normalized to urine-creatinine from days 0, 22, 29, and 35 after first Oxa administration. Here, we detected over time decreased urine albumin/creatinine ratios in the Oxa group (Figure 5b). Blood urea nitrogen and creatinine showed comparable levels in Oxa-treated versus PBS-treated mice (Supplementary Figure S1 online). Download .ppt (.17 MB) Help with ppt files Supplementary Figure Another important clinical indicator for the severity of lupus nephritis is renal IgG deposition. Consistent with our findings, we detected by fluorescence staining of serial kidney sections significantly fewer IgG deposits in the glomeruli of Oxa-treated MRL-Faslpr mice compared with the control group (Figure 6a). In addition, the evaluation of IgG subclasses in serum (Figure 6b) and circulating dsDNA antibodies (Figure 6c) showed a reduction of these parameters in Oxa-treated mice. Moreover, a slight decrease in the amount of C3 deposition in the glomeruli indicates a reduced activation of the complement system upon Oxa treatment (Figure 6d). In accordance, we detected also an increase in systemic complement C3 levels (Figure 6e) in Oxa-treated mice. Thus, these results imply that Oxa-induced changes ameliorate kidney disease and thereby disease progression in MRL-Faslpr mice. In the advanced state of glomerulonephritis, fibrotic processes contribute to renal failure. As cell culture experiments reveal potent antifibrotic properties of Oxa, we decided to examine whether this also works in vivo. Therefore, we stained kidney sections of both experimental groups with the Elastic van Gieson staining method. Because of the time point of analysis (13-week-old mice), we detected only mild kidney fibrosis. Nevertheless, in the Oxa-treated group we saw significantly less renal collagen deposition (red colored) compared with the untreated control group (Figure 7). Again, this may imply that Oxa improves renal function in the lupus model of MRL-Faslpr mice and thereby prevents disease progression. In summary, our data demonstrate that Oxa reduced the expression of a number of proinflammatory genes in the inflamed kidney of MRL-Faslpr mice and led to a decrease in the invasion of immune cells. Beside fibrotic processes, different clinical parameters of lupus nephritis were also improved upon Oxa treatment. Thus, Oxa administration might be an intriguing new approach for the treatment of lupus glomerulonephritis. Current SLE treatment approaches comprise nonsteroidal anti-inflammatory drugs, antimalarial agents, immunosuppressive drugs (e.g., cyclophosphamide, azathioprine, and mycophenolate mofetil), and glucocorticoids. Glucocorticoids still represent the mainstay of treatment for controlling disease flares in SLE patients. They have potent anti-inflammatory and immunosuppressive effects, which are mediated by their ability to induce or repress the expression of different target genes. However, the long-term usage of glucocorticoids is limited because of a number of severe side effects such as osteoporosis, insulin resistance, or skin thinning.6.Barnes P.J. Glucocorticosteroids: current and future directions.Br J Pharmacol. 2011; 163: 29-43Crossref PubMed Scopus (379) Google Scholar Moreover, there are a significant number of patients who fail to respond to steroid therapy. Therefore, it is necessary to develop new therapeutic approaches for chronic inflammatory diseases. We have recently shown that fungal compounds such as (S)-curvularin improved chronic inflammation in a murine model of rheumatoid arthritis. As far as we know, these effects rely on transcription-based inhibition of proinflammatory gene expression.7.Schmidt N. Art J. Forsch I. et al.The anti-inflammatory fungal compound (s)-curvularin reduces proinflammatory gene expression in an in vivo model of rheumatoid arthritis.J Pharmacol Exp Ther. 2012; 343: 106-114Crossref PubMed Scopus (20) Google Scholar,31.Yao Y. Hausding M. Erkel G. et al.Sporogen, S14-95, and S-curvularin, three inhibitors of human inducible nitric-oxide synthase expression isolated from fungi.Mol Pharmacol. 2003; 63: 383-391Crossref PubMed Scopus (47) Google Scholar Therefore, modulation of proinflammatory gene expression seems to be a promising strategy for the treatment of chronic inflammatory diseases. This is supported by the development of the Janus kinase inhibitor CP-690.550 (tofacitinib), which represses proinflammatory gene expression by inhibition of the Janus kinase/signal transducer and activator of transcription pathway. Tofacitinib successfully passed phase III clinical trials for the treatment of rheumatoid arthritis32.Flanagan M.E. Blumenkopf T.A. Brissette W.H. et al.Discovery of CP-690,550: a potent and selective Janus kinase (JAK) inhibitor for the treatment of autoimmune diseases and organ transplant rejection.J Med Chem. 2010; 53: 8468-8484Crossref PubMed Scopus (273) Google Scholar and is currently in clinical trials for the treatment of SLE. In the present study, we evaluated for the first time the anti-inflammatory effects of Oxa in the SLE model, the MRL-Faslpr mouse in vivo. We presented evidence that Oxa improves clinical, immunological, and histological parameters of lupus nephritis in MRL-Faslpr mice. The Oxa-mediated reduction in proteinuria, dsDNA antibodies, and renal IgG deposition clearly hints toward a protection of the renal integrity and therefore of function in MRL-Faslpr mice. It is true that some renal function parameters (blood urea nitrogen/creatinine) are not affected by Oxa treatment. However, these parameters start to increase between 3 and 4 months of age, whereas proteinuria is detectable much earlier. Hence, our findings of increasing proteinuria during the treatment period, while renal function parameters are still unaffected, are consistent and give no evidence of the side effects of Oxa treatment. The amelioration of renal disease in MRL-Faslpr mice following Oxa treatment compared with control mice seems to be accompanied by a decreased humoral response, as demonstrated by reduced serum titers of IgG subclasses and decreased incidence and severity of glomerular IgG deposits. These data suggest that antibody-mediated mechanisms may be responsible, at least in part, for the glomerulonephritis in MRL-Faslpr mice, as the role of IgGs in mediating kidney and systemic disease is discussed controversially.33.Seredkina N. Van Der Vlag J. Berden J. et al.Lupus nephritis: enigmas, conflicting models and an emerging concept.Mol Med. 2013; 19: 161-169Crossref PubMed Scopus (44) Google Scholar Furthermore, Oxa treatment diminished renal collagen deposition demonstrating its antifibrotic effectivity also in vivo.11.Rudolph K. Serwe A. Erkel G. Inhibition of TGF-beta signaling by the fungal lactones (S)-curvularin, dehydrocurvularin, oxacyclododecindione and galiellalactone.Cytokine. 2013; 61: 285-296Crossref PubMed Scopus (31) Google Scholar The improvement in different clinical parameters in combination with the observed reduction of critical mediators of glomerulonephritis strongly indicates the potency of Oxa as a potential new drug for the treatment of lupus nephritis. The beneficial effects detected in our analyses are most probably linked to the reduced expression of TNF-α, IL-6, and IFN-γ in the kidney of Oxa-treated MRL-Faslpr mice. It is well known that these cytokines are important mediators of inflammation and contribute to the development of lupus nephritis shown by investigating the impact of single cytokine deletions in MRL-Faslpr mice on disease progression.3.Iwata Y. Furuichi K. Kaneko S. et al.The role of cytokine in the lupus nephritis.J Biomed Biotechnol. 2011; 2011: 594-809Crossref Scopus (37) Google Scholar Moreover, upon Oxa treatment less infiltration of lymphocytes and macrophages into the kidney was observed. This may be due to an Oxa-mediated reduction in protein expression of different chemokines (e.g., CCL2, RANTES) as detected in our proteome profile analyses. Knockout of CCL2 expression has been demonstrated to reduce T-cell and macrophage recruitment into the kidney of MRL-Faslpr mice and thereby ameliorate disease progression.28.Tesch G.H. Maifert S. Schwarting A. et al.Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas(lpr) mice.J Exp Med. 1999; 190: 1813-1824Crossref PubMed Scopus (275) Google Scholar In addition, increased CCL2 levels correlate with the activity and severity of lupus nephritis in humans.34.Alzawawy A. Zohary M. Ablordiny M. et al.Estimation of monocyte-chemoattractantprotein-1 (Mcp-1) level in patients with lupus nephritis.Int J Rheum Dis. 2009; 12: 311-318Crossref PubMed Scopus (26) Google Scholar RANTES is a chemokine that attracts primarily T cells or eosinophils to inflammatory sites. A recent publication demonstrated an increased serum concentration of RANTES in SLE patients, indicating a function of this chemokine in the pathogenesis of SLE.35.Lu M.M. Wang J. Pan H.F. et al.Increased serum RANTES in patients with systemic lupus erythematosus.Rheumatol Int. 2012; 32: 1231-1233Crossref PubMed Scopus (27) Google Scholar Therefore, the transcriptional repression of both chemokines by Oxa could contribute to disease amelioration in our in vivo model. Macrophages seem to be key mediators of lupus nephritis and are found in great numbers in inflamed kidneys.36.Schiffer L. Bethunaickan R. Ramanujam M. et al.Activated renal macrophages are markers of disease onset and disease remission in lupus nephritis.J Immunol. 2008; 180: 1938-1947Crossref PubMed Scopus (178) Google Scholar CSF-1, which is required for macrophage differentiation, survival, and proliferation, is known to be elevated in lupus nephritis in MRL-Faslpr mice. In addition, increased serum levels of CSF-1 correlate with human lupus disease activity.29.Menke J. Rabacal W.A. Byrne K.T. et al.Circulating CSF-1 promotes monocyte and macrophage phenotypes that enhance lupus nephritis.J Am Soc Nephrol. 2009; 20: 2581-2592Crossref PubMed Scopus (78) Google Scholar In the kidney, protein expression of CSF-1 was reduced by Oxa. Again this might be a beneficial effect of Oxa to reduce inflammatory processes in lupus nephritis. The most pronounced effect of Oxa on cytokine expression was on IL-16. This cytokine, which induces chemotaxis of CD4+ T cells and monocytes, has been shown to b
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