Nuclear Factor-κB Inhibitors as Potential Novel Anti-Inflammatory Agents for the Treatment of Immune Glomerulonephritis
2002; Elsevier BV; Volume: 161; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64425-2
ISSN1525-2191
AutoresÓscar López-Franco, Yusuke Suzuki, Guillermo Sanjuán, Julia Blanco, Purificación Hernández-Vargas, Yoshikage Yo, Jeffrey B. Kopp, Jesús Egido, Carmen Gómez‐Guerrero,
Tópico(s)Natural product bioactivities and synthesis
ResumoNuclear factor (NF)-κB regulates several genes implicated in the inflammatory response and represents an interesting therapeutic target. We examined the effects of gliotoxin (a fungal metabolite) and parthenolide (a plant extract), which possess anti-inflammatory activities in vitro, on the progression of experimental glomerulonephritis. In the anti-Thy 1.1 rat model, gliotoxin (75 μg/rat/day, 10 days, n = 18 rats) markedly reduced proteinuria, glomerular lesions, and monocyte infiltration. In anti-mesangial cell nephritis in mice, parthenolide (70 μg/mouse/day, 7 days, n = 17 mice) significantly decreased proteinuria, hematuria, and glomerular proliferation. NF-κB activity, localized in glomerular and tubular cells, was attenuated by either gliotoxin or parthenolide, in association with diminished renal expression of monocyte chemoattractant protein-1 and inducible nitric oxide synthase. In cultured mesangial cells and monocytes, gliotoxin and parthenolide inhibited NF-κB activation and expression of inflammatory genes induced by lipopolysaccharide and cytokines, by blocking the phosphorylation/degradation of the IκBα subunit. In summary, gliotoxin and parthenolide prevent proteinuria and renal lesions by inhibiting NF-κB activation and expression of regulated genes. This may represent a novel approach for the treatment of immune and inflammatory renal diseases. Nuclear factor (NF)-κB regulates several genes implicated in the inflammatory response and represents an interesting therapeutic target. We examined the effects of gliotoxin (a fungal metabolite) and parthenolide (a plant extract), which possess anti-inflammatory activities in vitro, on the progression of experimental glomerulonephritis. In the anti-Thy 1.1 rat model, gliotoxin (75 μg/rat/day, 10 days, n = 18 rats) markedly reduced proteinuria, glomerular lesions, and monocyte infiltration. In anti-mesangial cell nephritis in mice, parthenolide (70 μg/mouse/day, 7 days, n = 17 mice) significantly decreased proteinuria, hematuria, and glomerular proliferation. NF-κB activity, localized in glomerular and tubular cells, was attenuated by either gliotoxin or parthenolide, in association with diminished renal expression of monocyte chemoattractant protein-1 and inducible nitric oxide synthase. In cultured mesangial cells and monocytes, gliotoxin and parthenolide inhibited NF-κB activation and expression of inflammatory genes induced by lipopolysaccharide and cytokines, by blocking the phosphorylation/degradation of the IκBα subunit. In summary, gliotoxin and parthenolide prevent proteinuria and renal lesions by inhibiting NF-κB activation and expression of regulated genes. This may represent a novel approach for the treatment of immune and inflammatory renal diseases. Chronic glomerulonephritis is because of the release of many inflammatory mediators that can be produced by resident glomerular cells or infiltrating leukocytes.1Clynes R Dumitru C Ravetch JV Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis.Science. 1998; 279: 1052-1054Crossref PubMed Scopus (534) Google Scholar The mechanisms for gene expression of these inflammatory proteins have been extensively studied, with special attention to transcription factors, such as nuclear factor (NF)-κB. NF-κB can be activated in several cell types by numerous physiological and pathological stimuli, such as cytokines, mitogens, virus, mechanical and oxidative stress, and chemical agents.2Chen F Castranova V Shi X Demers LM New insights into the role of nuclear factor-κB, a ubiquitous transcription factor in the initiation of diseases.Clin Chem. 1999; 45: 7-17Crossref PubMed Scopus (657) Google Scholar, 3Lee JI Burckart GJ Nuclear factor-κB: important transcription factor and therapeutic target.J Clin Pharmacol. 1998; 38: 981-993Crossref PubMed Scopus (322) Google Scholar Studies in experimental and human nephritis have provided evidence supporting the involvement of NF-κB in the pathogenesis of glomerulonephritis.4Guijarro C Egido J Transcription factor-κB (NF-κB) and renal disease.Kidney Int. 2001; 59: 415-424Crossref PubMed Scopus (450) Google Scholar, 5Sakurai H Shigemori N Hisada Y Ishizuka T Kawashima K Sugita T Suppression of NF-κB and AP-1 activation by glucocorticoids in experimental glomerulonephritis in rats.Biochim Biophys Acta. 1997; 1362: 252-262Crossref PubMed Scopus (65) Google Scholar, 6Gómez-Guerrero C Duque N Casado MT Pastor C Blanco J Mampaso F Vivanco F Egido J Administration of IgG Fc fragments prevents glomerular injury in experimental immune complex nephritis.J Immunol. 2000; 164: 2092-2101PubMed Google Scholar In addition, expression of NF-κB-dependent genes, such as chemokines, adhesion molecules, inducible nitric oxide synthase (iNOS), and cyclooxygenase has been demonstrated in cultured glomerular cells, including mesangial cells (MCs).7Rovin BH Dikerson JA Tan LC Hebert CA Activation of nuclear factor-κB correlates with MCP-1 expression by human mesangial cells.Kidney Int. 1995; 48: 1263-1271Crossref PubMed Scopus (182) Google Scholar, 8Duque N Gómez-Guerrero C Egido J Interaction of IgA with Fcα receptors of human mesangial cells activates transcription factor nuclear factor-κB and induces expression and synthesis of monocyte chemoattractant protein-1, IL-8 and IFN-inducible protein 10.J Immunol. 1997; 159: 3474-3482PubMed Google Scholar, 9Saura M Zaragoza C Diaz-Cazorla M Hernandez-Perera O Eng E Lowenstein CJ Perez-Sala D Lamas S Involvement of transcriptional mechanisms in the inhibition of NOS2 expression by dexamethasone in rat mesangial cells.Kidney Int. 1998; 53: 38-49Crossref PubMed Scopus (31) Google Scholar The family of NF-κB proteins shares a conserved central region, the Rel domain, involved in the dimerization and interaction with the inhibitory subunit IκB, nuclear translocation, and DNA binding. 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Because many forms of primary glomerulonephritis do not consistently respond to current therapy, in this study we attempted novel therapeutic approaches for these diseases, focusing on NF-κB as a possible target. Our purpose is to modify in vivo the expression of proinflammatory genes by using novel NF-κB inhibitors. First, we evaluated the effects of gliotoxin and parthenolide on NF-κB activation and expression of regulated genes (MCP-1 and iNOS) in cultured MCs and monocytes stimulated with inflammatory stimuli. Second, we analyzed whether these inhibitors modulate (or prevent) the initiation and progression of glomerular injury in two experimental models of mesangial proliferative glomerulonephritis. The administration of these agents could represent a novel therapeutic approach to progressive glomerulonephritis. Gliotoxin, parthenolide, and lipopolysaccharide (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO). The cytokines [human tumor necrosis factor-α (TNF-α), human interferon-γ (IFN-γ) and human interleukin-1β (IL-1β)] were purchased from Immunogenex Corp. (Los Angeles, CA). The specific antibodies (Abs) used were as follows: pIκBα, IκBα, p65, and c-Rel (Santa Cruz Biotechnology, Santa Cruz, CA), ED1 (Serotec, Oxford, UK), BrdU (Calbiochem, La Jolla, CA), iNOS and p50 (Chemicon International, Temecula, CA), fluorescein isothiocyanate-labeled IgG (The Binding Site, Birmingham, UK), peroxidase-labeled IgG (Amersham, Buckinghamshire, UK), and alkaline phosphatase-labeled IgG (Roche, Barcelona, Spain). MCs from human and rat kidneys32Gómez-Guerrero C Duque N Egido J Stimulation of FcαR induces tyrosine phosphorylation of phospholipase C-γ1, phosphatidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells.J Immunol. 1996; 156: 4369-4376PubMed Google Scholar were cultured in RPMI 1640 with 25 mmol/L HEPES, pH 7.4, 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mmol/L glutamine (Life Technologies, Paisley, Scotland) and characterized by phase contrast microscopy, positive staining for desmin and vimentin, and negative staining for factor VIII and cytokeratin. The human monocytic cell line THP-1 [American Type Culture Collection (ATCC), Rockville, MD] was cultured in RPMI with 10% FCS. Cells were made quiescent by 24 hours of incubation in medium with 0.5% FCS, then preincubated for 90 minutes with the inhibitors, and washed before stimulation with LPS and cytokines. Cell viability was assessed by trypan blue exclusion. Cell nuclear proteins (10 μg) were incubated in buffer containing 50 μg/ml of poly(dI-dC) (Amersham), and 0.035 pmol of [32P]NF-κB or [32P]AP-1 consensus oligonucleotides, and the reactions were analyzed on a 4% nondenaturing polyacrylamide gel and autoradiographed. Specificity of the binding reaction was confirmed using a 100-fold excess of unlabeled probe. NF-κB subunits were identified by incubation with Abs against p50, p65, and c-Rel.8Duque N Gómez-Guerrero C Egido J Interaction of IgA with Fcα receptors of human mesangial cells activates transcription factor nuclear factor-κB and induces expression and synthesis of monocyte chemoattractant protein-1, IL-8 and IFN-inducible protein 10.J Immunol. 1997; 159: 3474-3482PubMed Google Scholar Cytosolic proteins (20 μg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA). Membranes were blocked with 5% nonfat milk, and then incubated with 10 μg/ml of goat anti-IκBα or 1 μg/ml of monoclonal anti-pIκBα, followed by peroxidase-conjugated secondary Ab and chemiluminescent detection system. Human MCs were pretreated for 90 minutes with serial dilutions of inhibitors, then washed and incubated for an additional 24 hours in medium containing 10% FCS. Cell proliferation was determined in 96-well plates by colorimetric assay of methylene blue incorporation.33Gómez-Guerrero C López-Armada MJ González E Egido J Soluble IgA and IgG aggregates are catabolized by cultured rat mesangial cells and induce production of TNF-α and IL-6, and proliferation.J Immunol. 1994; 153: 5247-5255PubMed Google Scholar Alternatively, cell proliferation was studied after 24 hours of incubation in medium with 10% FCS and inhibitors. Data were expressed as percentage of proliferating cells in relation to control conditions. Total RNA from cells and tissues was obtained by the acid guanidine-thiocyanate-phenol-chloroform method. In cultured cells, MCP-1 mRNA expression was analyzed by Northern blot using cDNA probes for human MCP-1 and 28S ribosomal RNA (JE/pGEM-hJE34 and HHCD07; ATCC). Membranes containing RNA were hybridized, autoradiographed, and densitometered. Relative amounts of MCP-1 were established in relation to 28S. In renal tissues the mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) using the following primers: rat MCP-1 sense, 5′-TTCTGGGCCTGTTGTTCACA-3′; anti-sense, 5′-GGTCACTTCTACAGAAGTCC-3′; mouse MCP-1 sense, 5′-AGCACCAGCCAACTCTCACT-3′; anti-sense, 5′-TCTGGACCCATTCCTTCTTG-3′; rat iNOS sense, 5′-CTTCAACCCCAAGGTTGTCTGCAT-3′; anti-sense, 5′-ATGTCATGAGCAAAGGGGCAGAAC-3′. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. NO released from cells was determined by the accumulation of nitrites and measured by fluorescent reaction with 1 mmol/L of 2,3-diaminonaphthalene.34Misko TP Schilling RJ Salvemini D Moore WM Currie MG A fluorometric assay for the measurement of nitrite in biological samples.Anal Biochem. 1993; 214: 11-16Crossref PubMed Scopus (962) Google Scholar The absorbance at 548 nm was measured and compared with a standard of NaNO2, and data were expressed as nmol NO2−/mg protein. Growth-arrested cells (1 × 105) were co-transfected with piNOS-Luc (a gift from Dr. Santiago Lamas; CIB, Madrid, Spain)9Saura M Zaragoza C Diaz-Cazorla M Hernandez-Perera O Eng E Lowenstein CJ Perez-Sala D Lamas S Involvement of transcriptional mechanisms in the inhibition of NOS2 expression by dexamethasone in rat mesangial cells.Kidney Int. 1998; 53: 38-49Crossref PubMed Scopus (31) Google Scholar and a normalizing reporter vector (pRL-TK with the Renilla luciferase gene; Promega, Madison WI) by using the FuGENE reagent (Roche) and then stimulated for 24 hours. The luciferase activity in cleared lysate was assayed using a luminometer. Luciferase activity was normalized by total protein concentration and variations in transfection efficiency. Monoclonal Ab (IgG1) against rat mesangial antigen Thy 1.1 was isolated from ascitic BALB/c mice injected with hybridome cells OX-7 (European Collection of Cell Cultures, Salisbury, UK) by affinity chromatography on protein G-Sepharose. Anti-murine MC serum was obtained by sheep immunization with protein extract form SV40-transformed mouse cells MES13 (Yo Y, Mobaraki H, Lu H, Braun M, Owens J, King C, Kopp JB, manuscript submitted). Control serum was obtained from sheep before immunization. For all experiments, sera were decomplemented by heating at 56° for 30 minutes. Anti-Thy 1.1 nephritis was induced in male Sprague-Dawley rats (200 g) at day 0 by injection of anti-Thy1.1 IgG (5 mg/kg in 350 μl of phosphate-buffered saline) into a tail vein. Mesangial glomerulonephritis was induced in female C57/BL6 mice (18 to 20 g) at day 0 by injection of sheep anti-murine MC antiserum (200 μl/20 g body weight). Groups of study: I, rats (n = 18) and mice (n = 17) treated daily with gliotoxin (75 μg/200 g body weight, i.p.) and parthenolide (70 μg/20 g body weight, i.p.), respectively; II, rats (n = 20) and mice (n = 10) with spontaneous development of nephritis with injection of vehicle (saline); III and IV, healthy control rats (n = 17) and mice (n = 8) receiving the respective amount of inhibitors and saline. Treatment with gliotoxin or parthenolide was started the day before and followed for 10 or 7 days, respectively. Urine samples were taken at regular intervals starting on day 0. Proteinuria was measured by the sulfosalicylic method (rats) or the Knight's method (mice).35Suzuki Y Shirato I Okumura K Ravetch JV Takai T Tomino Y Ra C Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis.Kidney Int. 1998; 54: 1166-1174Crossref PubMed Scopus (145) Google Scholar Urinary hemoglobin was measured by Aution sticks (Arkray Factory, Shiga, Japan). All studies were performed in accordance to the European Union normative. Animals were sacrificed at days 2, 4, 7, and 10 (rat model) and days 5 and 7 (mouse model). Light microscopic analysis of renal cortex was performed on paraffin sections (5-μm thick) stained with hematoxylin and eosin, and Masson's trichrome. The examination of infiltrating cells (ED1+, rat macrophage) and iNOS production was performed by indirect immunoperoxidase. For determining proliferating cells, mice were intraperitoneally injected with 2 mg of BrdU for 16, 8, and 4 hours before sacrifice, and then immunoperoxidase staining with anti-BrdU was performed on paraffin-embedded samples. Paraffin-embedded tissue sections fixed in 0.5% paraformaldehyde were incubated with 50 pmol of digoxigenin-labeled NF-κB and AP-1 probes in buffer containing 0.25% bovine serum albumin and 1 μg/ml of poly(dI-dC), followed by alkaline phosphatase-conjugated anti-digoxigenin IgG and colorimetric detection. A 200-fold excess of unlabeled probe was used to test the specificity of the technique.36Hernández-Presa MA Gomez-Guerrero C Egido J In situ non-radioactive detection of nuclear factors in paraffin sections by Southwestern histochemistry.Kidney Int. 1999; 55: 209-214Crossref PubMed Scopus (55) Google Scholar Semiquantification of morphological changes in the animal models was made in regard to glomerular lesions (number of glomeruli with hypercellularity, mesangiolysis, or immune deposits/30 glomeruli), tubular lesions (number of tubules with atrophy or degeneration/20 fields at ×40 magnification), and interstitial infiltration (number of focus/20 fields at ×40 magnification). Samples from each animal were examined in a blinded manner, and semiquantitatively graded on a scale from 0 to 3. Quantification of iNOS staining and infiltrating/proliferating cells was made by determining the total number of positive-labeled cells in 20 randomly chosen areas. The results are given as mean ± SD or representative experiments, when indicated. Values were analyzed by ANOVA and Tukey-Kramer tests using Instat (Graphpad Software, San Diego, CA). A P value 75%) at 3 μg/ml, but a toxic effect was noted using concentrations >5 μg/ml (viability around 30%). No toxic effect was observed with parthenolide even at doses up to 6 μg/ml. In proliferation assays (methylene blue incorporation) we observed that preincubation for 90 minutes with serial dilutions of gliotoxin and parthenolide (from 0.75 to 12 μg/ml) did not diminish the number of proliferating MCs (maximal percentages of inhibition: 23 ± 4% and 16 ± 3%, respectively). MC proliferation was only inhibited by using gliotoxin at higher doses and longer incubation periods (24 μg/ml at 24 hours, 66 ± 8% inhibition, n = 4). In these experiments we checked the effects of these agents on the expression of genes transcriptionally regulated by NF-κB, such as MCP-1 and iNOS.7Rovin BH Dikerson JA Tan LC Hebert CA Activation of nuclear factor-κB correlates with MCP-1 expression by human mesangial cells.Kidney Int. 1995; 48: 1263-1271Crossref PubMed Scopus (182) Google Scholar, 8Duque N Gómez-Guerrero C Egido J Interaction of IgA with Fcα receptors of human mesangial cells activates transcription factor nuclear factor-κB and induces expression and synthesis of monocyte chemoattractant protein-1, IL-8 and IFN-inducible protein 10.J Immunol. 1997; 159: 3474-3482PubMed Google Scholar, 9Saura M Zaragoza C Diaz-Cazorla M Hernandez-Perera O Eng E Lowenstein CJ Perez-Sala D Lamas S Involvement of transcriptional mechanisms in the inhibition of NOS2 expression by dexamethasone in rat mesangial cells.Kidney Int. 1998; 53: 38-49Crossref PubMed Scopus (31) Google Scholar In human MCs, preincubation with gliotoxin or parthenolide (1 μg/ml) prevented the MCP-1 mRNA expression induced by LPS/cytokines (72% and 58% inhibition; Figure 2A). In a reported gene assay using human MCs transfected with piNOS-Luc plasmid, both agents inhibited the activation of the iNOS promoter induced by LPS/cytokines (75% inhibition, Figure 2B). According to this data, the mesangial production of nit
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