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• W2134037340 endingPage "1975" @default.
• W2134037340 startingPage "1965" @default.
• W2134037340 abstract "Shigatoxin (Stx) is the offending agent of post-diarrheal hemolytic uremic syndrome, characterized by glomerular ischemic changes preceding microvascular thrombosis. Because podocytes are highly sensitive to Stx cytotoxicity and represent a source of vasoactive molecules, we studied whether Stx-2 modulated the production of endothelin-1 (ET-1), taken as candidate mediator of podocyte dysfunction. Stx-2 enhanced ET-1 mRNA and protein expression via activation of nuclear factor κB (NF-κB) and activator protein-1 (Ap-1) to the extent that transfection with the dominant-negative mutant of IκB-kinase 2 or with Ap-1 decoy oligodeoxynucleotides reduced ET-1 mRNA levels. We propose a role for p38 and p42/44 mitogen-activated protein kinases (MAPKs) in mediating NF-κB-dependent gene transcription induced by Stx-2, based on data that Stx-2 phosphorylated p38 and p42/44 MAPKs and that MAPK inhibitors reduced transcription of NF-κB promoter/luciferase reporter gene construct induced by Stx-2. Stx-2 caused F-actin redistribution and intercellular gaps via production of ET-1 acting on ETA receptor, because cytoskeleton changes were prevented by ETA receptor blockade. Exogenous ET-1 induced cytoskeleton rearrangement and intercellular gaps via phosphatidylinositol-3 kinase and Rho-kinase pathway and increased protein permeability across the podocyte monolayer. These data suggest that the podocyte is a target of Stx, a novel stimulus for the synthesis of ET-1, which may control cytoskeleton remodeling and glomerular permeability in an autocrine fashion. Shigatoxin (Stx) is the offending agent of post-diarrheal hemolytic uremic syndrome, characterized by glomerular ischemic changes preceding microvascular thrombosis. Because podocytes are highly sensitive to Stx cytotoxicity and represent a source of vasoactive molecules, we studied whether Stx-2 modulated the production of endothelin-1 (ET-1), taken as candidate mediator of podocyte dysfunction. Stx-2 enhanced ET-1 mRNA and protein expression via activation of nuclear factor κB (NF-κB) and activator protein-1 (Ap-1) to the extent that transfection with the dominant-negative mutant of IκB-kinase 2 or with Ap-1 decoy oligodeoxynucleotides reduced ET-1 mRNA levels. We propose a role for p38 and p42/44 mitogen-activated protein kinases (MAPKs) in mediating NF-κB-dependent gene transcription induced by Stx-2, based on data that Stx-2 phosphorylated p38 and p42/44 MAPKs and that MAPK inhibitors reduced transcription of NF-κB promoter/luciferase reporter gene construct induced by Stx-2. Stx-2 caused F-actin redistribution and intercellular gaps via production of ET-1 acting on ETA receptor, because cytoskeleton changes were prevented by ETA receptor blockade. Exogenous ET-1 induced cytoskeleton rearrangement and intercellular gaps via phosphatidylinositol-3 kinase and Rho-kinase pathway and increased protein permeability across the podocyte monolayer. These data suggest that the podocyte is a target of Stx, a novel stimulus for the synthesis of ET-1, which may control cytoskeleton remodeling and glomerular permeability in an autocrine fashion. Shigatoxin (Stx)-producing Escherichia coli has been strongly indicated as the offending agent of typical postdiarrheal hemolytic uremic syndrome (D+HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure that mainly affects infants and small children.1Ruggenenti P Noris M Remuzzi G Thrombotic microangiopathy, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura.Kidney Int. 2001; 60: 831-846Crossref PubMed Scopus (398) Google Scholar, 2Andreoli SP The pathophysiology of the hemolytic uremic syndrome.Curr Opin Nephrol Hypertens. 1999; 8: 459-464Crossref PubMed Scopus (48) Google Scholar, 3Karmali MA Infection by Shiga toxin-producing Escherichia coli: an overview.Mol Biotechnol. 2004; 26: 117-122Crossref PubMed Scopus (198) Google Scholar The kidney is the privileged organ of Stx toxicity because it expresses high levels of the specific receptor glycosphingolipid globotriaosyl ceramide (Gb3).4Lingwood CA Role of verotoxin receptors in pathogenesis.Trends Microbiol. 1996; 4: 147-153Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 5Lingwood CA Verotoxin-binding in human renal sections.Nephron. 1994; 66: 21-28Crossref PubMed Scopus (180) Google Scholar The characteristic lesion consists of swelling and detachment of glomerular endothelial cells that have been extensively recognized as the main target of Stx.6Habib R Pathology of the hemolytic uremic syndrome.in: Kaplan BS Trompeter RS Moake JL Hemolytic Uremic Syndrome and Thrombotic Thrombocytopenic Purpura. M. Dekker, Inc., New York1992: 315-353Google Scholar Retraction and collapse of the capillary tuft in the glomerulus are prominent and typically occur in association with fusion of foot processes and swelling of podocytes.6Habib R Pathology of the hemolytic uremic syndrome.in: Kaplan BS Trompeter RS Moake JL Hemolytic Uremic Syndrome and Thrombotic Thrombocytopenic Purpura. M. Dekker, Inc., New York1992: 315-353Google Scholar, 7Striker GE Striker LJ D'Agati V Renal lesions in hypertension.in: Livolsi VA The Renal Biopsy Major Problems in Pathology. W.B. Saunders Company, Philadelphia1997: 258-268Google Scholar Although ischemic lesion in the glomerular microcirculation can significantly contribute to renal dysfunction, the precise role of podocyte injury in the toxic response to Stx and the underlying cellular and molecular mechanisms have not been established yet.Recent studies have indicated that glomerular visceral epithelial cells (podocytes) are sensitive to the toxic effects of Stx-1 and −2 isoforms because they express Gb3 and bind Stx, as documented either in cultured cells8Hughes AK Stricklett PK Schmid D Kohan DE Cytotoxic effect of Shiga toxin-1 on human glomerular epithelial cells.Kidney Int. 2000; 57: 2350-2359Crossref PubMed Scopus (66) Google Scholar or in human renal biopsies.9Ergonul Z Clayton F Fogo AB Kohan DE Shigatoxin-1 binding and receptor expression in human kidneys do not change with age.Pediatr Nephrol. 2003; 18: 246-253PubMed Google Scholar In vitro, Stx-1 activates podocytes to release inflammatory cytokines like interleukin (IL)-1 and tumor necrosis factor (TNF), which remarkably increased the cellular content of Gb3 receptor, thereby enhancing renal toxin responsiveness.8Hughes AK Stricklett PK Schmid D Kohan DE Cytotoxic effect of Shiga toxin-1 on human glomerular epithelial cells.Kidney Int. 2000; 57: 2350-2359Crossref PubMed Scopus (66) Google Scholar, 10Hughes AK Stricklett PK Kohan DE Shiga toxin-1 regulation of cytokine production by human glomerular epithelial cells.Nephron. 2001; 88: 14-23Crossref PubMed Scopus (37) Google Scholar In an experimental model of HUS in baboons, swelling of podocytes with osmophilic inclusions was found in association with the typical glomerular endothelial lesions after intravenous infusion of Stx-1.11Taylor Jr, FB Tesh VL DeBault L Li A Chang AC Kosanke SD Pysher TJ Siegler RL Characterization of the baboon responses to Shiga-like toxin: descriptive study of a new primate model of toxic responses to Stx-1.Am J Pathol. 1999; 154: 1285-1299Abstract Full Text Full Text PDF PubMed Scopus (123) Google ScholarPodocytes represent a crucial component of the glomerular filter barrier. They are highly specialized cells endowed with foot processes that, through a contractile structure composed of actin and associated proteins connected to the glomerular basement membrane, stabilize glomerular architecture by counteracting the distension of the basement membrane.12Barisoni L Kopp JB Update in podocyte biology: putting one's best foot forward.Curr Opin Nephrol Hypertens. 2003; 12: 251-258Crossref PubMed Scopus (31) Google Scholar, 13Pavenstädt H Roles of the podocyte in glomerular function.Am J Physiol Renal Physiol. 2000; 278: F173-F179PubMed Google Scholar The contractile apparatus of the foot processes responds to vasoactive hormones, thus controlling the glomerular capillary surface area and the ultrafiltration coefficient. Podocytes are an important source of the vasoconstrictor peptide endothelin-1 (ET-1),14Cybulsky AV Stewart DJ Cybulsky MI Glomerular epithelial cells produce endothelin-1.J Am Soc Nephrol. 1993; 3: 1398-1404PubMed Google Scholar, 15Kasinath BS Fried TA Davalath S Marsden PA Glomerular epithelial cells synthesize endothelin peptides.Am J Pathol. 1992; 141: 279-283PubMed Google Scholar, 16Morigi M Buelli S Angioletti S Zanchi C Longaretti L Zoja C Galbusera M Gastoldi S Mundel P Remuzzi G Benigni A In response to protein load podocytes reorganize cytoskeleton and modulate ET-1 gene: implication for permselective dysfunction of chronic nephropathies.Am J Pathol. 2005; 166: 1309-1320Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar which is recognized to play a key role in the control of glomerular hemodynamics. They constitutively express pre-proET-1 mRNA and synthesize the mature peptide, the generation of which is markedly up-regulated by transforming growth factor-β, membrane attack complex, and thrombin.14Cybulsky AV Stewart DJ Cybulsky MI Glomerular epithelial cells produce endothelin-1.J Am Soc Nephrol. 1993; 3: 1398-1404PubMed Google Scholar Studies have shown that rat podocytes are targets of ET-1 because they express ETA and ETB receptors.17Yamamoto T Hirohama T Uemura H Endothelin B receptor-like immunoreactivity in podocytes of the rat kidney.Arch Histol Cytol. 2002; 65: 245-250Crossref PubMed Scopus (20) Google Scholar, 18Ortmann J Amann K Brandes RP Kretzler M Munter K Parekh N Traupe T Lange M Lattmann T Barton M Role of podocytes for reversal of glomerulosclerosis and proteinuria in the aging kidney after endothelin inhibition.Hypertension. 2004; 44: 974-981Crossref PubMed Scopus (129) Google ScholarIn an attempt to identify possible mechanisms evoked by Stx that could contribute to podocyte dysfunction, we tested in cultured murine podocytes whether Stx-2 induced the expression and synthesis of ET-1, instrumental for cytoskeletal remodeling and cellular retraction. Intracellular signals involved in ET-1 gene regulation were also investigated.Materials and MethodsCell Culture and IncubationImmortalized mouse podocytes (obtained from Dr. Peter Mundel, Department of Medicine, Mount Sinai School of Medicine, New York, NY) were grown as previously described.19Mundel P Reiser J Zuniga Mejia Borja A Pavenstadt H Davidson GR Kriz W Zeller R Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (759) Google Scholar Briefly, cells were cultured under growth-permissive conditions on rat tail collagen type I-coated plastic dishes (BD Bioscience, Bedford, MA) at 33°C in RPMI 1640 (Invitrogen, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Invitrogen), 10 U/ml mouse recombinant interferon-γ (Sigma Chemical Co., St. Louis, MO), and 100 U/ml penicillin plus 0.1 mg/ml streptomycin (Sigma). To induce differentiation, podocytes were maintained in nonpermissive conditions at 37°C without interferon-γ for 14 days and used for the experiments. In this culture condition, cells stopped proliferating and were identified as differentiated podocytes by their arborized morphology and the presence of high levels of synaptopodin, determined using indirect immunofluorescence microscopy. Cells were routinely maintained for 24 hours in serum-free medium before all of the experiments.To investigate the effect of Stx-2 on the expression of the ET-1 gene, differentiated podocytes were exposed for 3, 6, and 24 hours to medium alone or to Stx-2, 50 pmol/L and 1 nmol/L (Toxin Technology Inc., Sarasota, FL). Preliminary experiments showed that these concentrations did not affect podocyte viability until 48 hours of incubation, as evaluated by viable cell count using trypan blue dye exclusion (Sigma). ET-1 mRNA transcript levels were measured by Northern blot analysis and real-time PCR. To exclude any possible effect of lipopolysaccharide (LPS) contamination of Stx-2 preparation on ET-1 mRNA expression, additional experiments were performed in podocytes incubated with Stx-2 in the presence of the LPS inhibitor polymyxin B, 10 μg/ml (Sigma). This concentration was chosen on the basis of previous experiments20Forestal CA Benach JL Carbonara C Italo JK Lisinski TJ Furie MB Francisella tularensis selectively induces proinflammatory changes in endothelial cells.J Immunol. 2003; 171: 2563-2570PubMed Google Scholar showing that 20 μg of polymyxin B was capable of neutralizing the effect of 1 ng of purified E. coli LPS. Because the Limulus test assay (Cambrex, Walkersville, MD) revealed the presence of 117 pg LPS/μg Stx-2 protein preparation, the concentration of polymyxin B used here far exceeded that needed to inhibit the detected LPS traces. The time course of ET-1 protein synthesis was assessed by radioimmunoassay (RIA) in supernatants of podocytes exposed to both concentrations of Stx-2.To study intracellular signaling pathways that regulate ET-1 gene transcription in Stx-2-loaded podocytes, we first assessed the potential role of nuclear factor κB (NF-κB) and activator protein-1 (Ap-1) by determining the activity of both transcription factors in nuclear extracts from podocytes exposed for 30 minutes to Stx-2 (50 pmol/L and 1 nmol/L) and by evaluating the effect of NF-κB and Ap-1 inhibition on ET-1 gene expression. Podocytes were transfected for 3 hours with a dominant-negative mutant of the IκB kinase 2 (IKK2),21Oitzinger W Hofer-Warbinek R Schmid JA Koshelnick Y Binder BR de Martin R Adenovirus-mediated expression of a mutant IkappaB kinase 2 inhibits the response of endothelial cells to inflammatory stimuli.Blood. 2001; 97: 1611-1617Crossref PubMed Scopus (72) Google Scholar a kinase that acts as an upstream activator of NF-κB, and then exposed to Stx-2 (50 pmol/L) for 24 hours. In other experiments, podocytes were transfected for 2 hours with double-stranded oligodeoxynucleotide (ODN)22Viedt C Dechend R Fei J Hansch GM Kreuzer J Orth SR MCP-1 induces inflammatory activation of human tubular epithelial cells: involvement of the transcription factors, nuclear factor-kappaB and activating protein-1.J Am Soc Nephrol. 2002; 13: 1534-1547Crossref PubMed Scopus (170) Google Scholar that scavenge Ap-1 activity by competitive reaction or with mutated control ODNs and then exposed to the toxin for 6 hours. Then, we studied whether Stx-2 induced activation/phosphorylation of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, known activators of NF-κB and Ap-1, in podocytes treated with 50 pmol/L Stx-2 for 15, 30, 60, and 180 minutes. To elucidate whether MAPKs were involved in NF-κB regulation, podocytes were transfected with NF-κB luciferase reporter gene and incubated with Stx-2 (50 pmol/L, 6 hours) in the presence or absence of the p38 inhibitor SB-202190 (20 μmol/L)23Donadelli R Zanchi C Morigi M Buelli S Batani C Tomasoni S Corna D Rottoli D Benigni A Abbate M Remuzzi G Zoja C Protein overload induces fractalkine upregulation in proximal tubular cells through nuclear factor kappaB- and p38 mitogen-activated protein kinase-dependent pathways.J Am Soc Nephrol. 2003; 14: 2436-2446Crossref PubMed Scopus (114) Google Scholar or the p42/44 inhibitor PD-98059 (10 μmol/L).24Hannken T Schroeder R Zahner G Stahl RA Wolf G Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1): role in angiotensin II-mediated hypertrophy of proximal tubular cells.J Am Soc Nephrol. 2000; 11: 1387-1397Crossref PubMed Google ScholarThe effect of Stx-2 on F-actin cytoskeletal rearrangement and gap formation was assessed in differentiated podocytes exposed for 15 hours to Stx-2 (50 pmol/L). To investigate the role of ET-1 in Stx-2-induced cytoskeleton rearrangement, we first studied whether murine podocytes expressed ETA and ETB receptor mRNA and protein by real-time PCR and immunofluorescence studies. The effect of Stx-2 on ETA and ETB receptor expression was also evaluated. Then, podocytes were treated with the ETA receptor antagonist LU-302146 (1 μmol/L; Knoll AG, Ludwighafen, Germany),25Brehm BR Klaussner M Wolf SC Chronic elevated endothelin-1 concentrations regulate mitogen-activated protein kinases ERK 1 and ERK 2 in vascular smooth muscle cells.Clin Sci (Lond). 2002; 103: 137S-140SPubMed Google Scholar added 1 hour before and during 15 hours of Stx-2 incubation, and F-actin changes and gap formation were assessed.Finally, the effect of exogenous ET-126Simonson MS Dunn MJ Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates beta-adrenergic-mediated cyclic adenosine monophosphate accumulation.J Clin Invest. 1990; 85: 790-797Crossref PubMed Scopus (142) Google Scholar on cytoskeletal rearrangement and permeability to protein was evaluated. The involvement of phosphatidylinositol-3 kinase (PI3K) and Rho kinase in ET-1-induced F-actin redistribution was studied by incubating cells with ET-1 for 6 hours in the presence of the specific inhibitors wortmannin (100 nmol/L; Sigma)27Vinci MC Visentin B Cusinato F Nardelli GB Trevisi L Luciani S Effect of vascular endothelial growth factor and epidermal growth factor on iatrogenic apoptosis in human endothelial cells.Biochem Pharmacol. 2004; 67: 277-284Crossref PubMed Scopus (29) Google Scholar and Y27632 (1 μmol/L; Sigma),28Fukuda T Takekoshi K Nanmoku T Ishii K Isobe K Kawakami Y Inhibition of the RhoA/Rho kinase system attenuates catecholamine biosynthesis in PC 12 rat pheochromocytoma cells.Biochim Biophys Acta. 2005; 1726: 28-33Crossref PubMed Scopus (13) Google Scholar respectively, added 30 minutes before and during ET-1 stimulation.Flow Cytometry AnalysisThe surface expression of Stx receptor Gb3 (CD77) was evaluated by flow cytometry analysis (FACSort; Becton, Dickinson and Company, San Jose, CA) in differentiated podocytes treated with medium alone, α-galactosidase (7.5 U/ml), or IL-1 β (100 U/ml, 24 hours). Unstimulated human umbilical vein endothelial cells were used as positive control. At the end of incubations, cells in suspension were exposed for 45 minutes at 4°C to fluorescein isothiocyanate (FITC)-labeled mouse anti-human CD77 monoclonal antibody (25 μg/ml; BD Biosciences Pharmingen, Milan, Italy). Unstimulated cells incubated with FITC-conjugated mouse IgM (BD Biosciences Pharmingen) were used as negative control. Then cells were washed and fixed with 2% paraformaldehyde plus 4% sucrose in phosphate-buffered saline (PBS; 10 minutes, 4°C) and assayed within 1 hour.Northern Blot AnalysisTotal RNA was isolated from podocytes by the guanidium isothiocyanate/cesium chloride procedure. Fifteen micrograms of total RNA was then fractionated on 1.2% agarose gel and blotted onto synthetic membranes (Zeta-probe; Bio-Rad, Richmond, CA). ET-1 mRNA was detected by using a 319-bp fragment of rat ET-1 cDNA. The probe was labeled with [α-32P]dCTP by the random-primed method. Hybridization was performed overnight at 60°C in 0.25 mol/L Na2HPO4, pH 7.2, and 7% sodium dodecyl sulfate (SDS). Filters were washed twice for 30 minutes with 20 mmol/L Na2HPO4, pH 7.2, and 5% SDS and twice for 10 minutes with 20 mmol/L Na2HPO4, pH 7.2, and 1% SDS at 60°C. Membranes were subsequently probed with β-actin cDNA, taken as internal standard of equal loading of the samples on the membrane. Expression of ET-1 mRNA was corrected for β-actin expression and quantitated densitometrically.Quantitative Real-Time PCRTotal RNA was extracted from podocytes by the guanidium isothiocyanate/cesium chloride procedure. Contaminating genomic DNA was removed by RNase-free DNase (Promega, Ingelheim, Germany) for 1 hour at 37°C. The purified RNA (1 μg) was reverse transcribed using random examers (50 ng) and 200 U of SuperScript II RT (Life Technologies, San Giuliano Milanese, Italy) for 1 hour at 42°C. No enzyme was added for reverse transcriptase-negative controls.Real-time PCR was performed with ABI PRISM 5700 Sequence Detection System (PE Biosystems, Warrington, UK) using heat-activated TaqDNA polymerase (Amplitaq Gold; PE Biosystems).16Morigi M Buelli S Angioletti S Zanchi C Longaretti L Zoja C Galbusera M Gastoldi S Mundel P Remuzzi G Benigni A In response to protein load podocytes reorganize cytoskeleton and modulate ET-1 gene: implication for permselective dysfunction of chronic nephropathies.Am J Pathol. 2005; 166: 1309-1320Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar The SYBR Green I PCR Reagents kit was used according to the manufacturer's protocol. After an initial hold of 2 minutes at 50°C and 10 minutes at 95°C, the samples were cycled 40 times at 95°C for 15 seconds and 60°C for 60 seconds. Fluorescence detection, defined as threshold cycle (Ct), was chosen in the exponential phase of the PCR and used for relative quantification of the target gene. The comparative Ct method normalizes the number of target gene copies to the housekeeping gene as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ΔCt). Gene expression was then evaluated by the quantification of cDNA corresponding with the target gene relative to a calibrator sample serving as a physiological reference (eg, untreated cells, ΔΔCt). On the basis of exponential amplification of target gene as well as calibrator, the amount of amplified molecules at the Ct is given by 2−ΔΔCt.The following specific primers (300 nmol/L) were used. Mouse ET-1: sense 5′-AACTACGAAGGTTGGAGGCCA-3′, antisense 5′-CACGAAAAGATGCCTTGATGC-3′; mouse ETA receptor: sense 5′-CTTGCGGATCGCCCTTAGT-3′, antisense 5′-TTTGCCACTTCTCGACGCT-3′; mouse ETB receptor: sense 5′-CCTACAAGTTGCTCGCAGAGG-3′, antisense 5′-GCTTACACATCTCAGCTCCAAATG-3′; and GAPDH: sense 5′-TCATCCCTGCATCCACTGGT-3′, antisense 5′-CTGGGATGACCTTGCCCAC-3′. All primers were obtained from Sigma Genosys (Cambridgeshire, UK). True identity of the amplification products was ensured by primer specificity for mouse sequences, the presence of a single dissociation curve at a constant T melting, and the lack of genomic DNA contamination or primer dimers in the reverse transcriptase-negative control sample.RadioimmunoassayET-1 production was assayed in podocyte supernatants by RIA. Either standard compounds or unknown samples (100 μl) were mixed with 100 μl of antiserum (Peninsula Laboratories Inc., Belmont, CA) diluted in phosphate buffer, pH 7.2 (RIA buffer) at a final dilution of 1:72,000. The reaction mixture was incubated for 24 hours at 4°C, then 15,000 cpm of 125I-labeled ET-1 in 100 μl was added, and the incubation was prolonged for 24 hours at 4°C. Separation of free ET-1 from antibody-bound 125I-labeled ET-1 was achieved by the addition of a second antibody (500 μl of immunoprecipitating mixture consisting of a sheep anti-rabbit IgG and polyethylene glycol) for 30 minutes at room temperature. Finally, 500 μl of RIA buffer was added to stop the reaction, and the immunoprecipitates were centrifuged at 5000 × g for 30 minutes. Supernatants were discarded, and pellet radioactivity was detected by gamma counter (Beckman, Irvine, CA). Results were expressed as picograms per 106 cells. The minimum detectable concentration was 0.4 pg/tube. Nonspecific binding did not exceed 2% of total radioactivity.The cross-reactivity of the antibody with other endothelins is as follows: endothelin-2, 46.9%; endothelin-3, 17%; and big endothelin-1, 9.4%. Intra-assay and interassay variability averaged 10 and 12%, respectively, over a range between 0.4 and 100 pg/tube.Preparation of Nuclear Extracts and Electrophoretic Mobility Shift AssayNuclear extracts were prepared from podocytes with the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce/Celbio, Pero, Italy) according to the manufacturer's instructions. To minimize proteolysis, all buffers contained protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany). The protein concentration was determined by the Bradford assay using the Bio-Rad protein assay reagent.Electrophoretic mobility shift assays were performed as previously described23Donadelli R Zanchi C Morigi M Buelli S Batani C Tomasoni S Corna D Rottoli D Benigni A Abbate M Remuzzi G Zoja C Protein overload induces fractalkine upregulation in proximal tubular cells through nuclear factor kappaB- and p38 mitogen-activated protein kinase-dependent pathways.J Am Soc Nephrol. 2003; 14: 2436-2446Crossref PubMed Scopus (114) Google Scholar with the κB DNA sequence of the immunoglobulin gene (5′-CCGGTCAGAGGGGACTTTCCGAGACT-3′) and consensus binding site for Ap-1 (5′-CGCTTGATGACTCAGCCGGAA-3′). Nuclear extracts (3 μg) were incubated with 50,000 cpm of 32P-labeled oligonucleotide in a binding reaction mixture [10 mmol/L Tris-HCl, pH 7.5, 80 mmol/L NaCl, 1 mmol/L ethylenediamine tetraacetic acid, 1 mmol/L dithiothreitol, 5% glycerol, and 1.5 μg of poly(dI-dC)] for 30 minutes on ice. In competition studies, a 100-fold molar excess of unlabeled oligonucleotide was added to the binding reaction mixture before the addition of the labeled κB or Ap-1 probes. For densitometric analysis, the volume density for each band was determined in arbitrary units. The sum of the volume density of bands for a single sample was used as an indirect measure of NF-κB or Ap-1 activation and expressed as a fold increase of the mean densitometry of respective control (represented as 1).Adenoviral Vector-Mediated Gene Transfer in PodocytesReplication-deficient adenovirus encoding for kinase-defective dominant-negative form of human IKK2 (AdV-dnIKK2) was a kind gift of Dr. R. de Martin (University of Vienna, Vienna, Austria).21Oitzinger W Hofer-Warbinek R Schmid JA Koshelnick Y Binder BR de Martin R Adenovirus-mediated expression of a mutant IkappaB kinase 2 inhibits the response of endothelial cells to inflammatory stimuli.Blood. 2001; 97: 1611-1617Crossref PubMed Scopus (72) Google Scholar Replication-deficient adenoviral vector having no insert (AdV-0) was from Novartis Pharma (Basel, Switzerland). All viruses used belong to Ad5 serotype.For transfection experiments, podocytes were incubated with adenoviruses at a multiplicity of infection of 50 in RPMI 1640 without serum for 3 hours at 37°C. The adenovirus was washed off, and cells were maintained in serum-free medium for 24 hours. Then cells were exposed to 50 pmol/L Stx-2 for an additional 24 hours and processed for endothelin-1 mRNA expression (real-time PCR analysis). Transfection did not affect cell viability.Ap-1 Decoy ODN TechniqueThe sequences of the phosphorothioate double-stranded ODN against the Ap-1 binding site and the mutated control ODN were as follows: Ap-1 decoy ODN (consensus sequences are underlined), 5′-CGCTTGATGACTCAGCCGGAA-3′ and 3′-GCGAACTACTGAGTCGGCCTT-5′ and mutated control ODN, 5′-CGCTTGATGACTTGGCCGGAA-3′ and 3′-TTCCGGCCAAGTCATCAAGCG-5′.Double-stranded ODNs22Viedt C Dechend R Fei J Hansch GM Kreuzer J Orth SR MCP-1 induces inflammatory activation of human tubular epithelial cells: involvement of the transcription factors, nuclear factor-kappaB and activating protein-1.J Am Soc Nephrol. 2002; 13: 1534-1547Crossref PubMed Scopus (170) Google Scholar were prepared from complementary single-stranded phosphorothioate oligonucleotides by melting at 80°C for 5 minutes followed by a 3- to 4-hour reconstitution period at room temperature. To study the effect of Ap-1 decoy, podocytes were transfected with 200 nmol/L Ap-1 decoy ODN or mutated control ODN in serum-free medium, using Oligofectamine Reagent according to the manufacturer's instructions (Invitrogen, San Giuliano Milanese, Italy). Two hours after transfection, Stx-2 at the final concentration of 50 pmol/L was added to the cells for 6 hours without removing the transfection mixture. Cells were then processed for ET-1 mRNA expression (real-time PCR analysis).Western Blot AnalysesPodocytes were lysed with lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 2 mmol/L ethylenediamine tetraacetic acid, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, and 1 mmol/L β-glycerophosphate) plus phosphatase inhibitors (1 mmol/L Na3VO4 and 50 mmol/L NaF) and protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride and 1 μg/ml leupeptin). Protein concentration was determined by protein assay based on bicinchoninic acid color formation (Pierce, Milan, Italy). Proteins (30 μg) were separated on 10% polyacrylamide gels by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour at room temperature with PBS containing 0.1% Tween 20 and 5% bovine serum albumin (BSA; for phosphorylated protein detection) or 5% nonfat dry milk (for unphosphorylated protein detection) and then incubated overnight at 4°C with the following primary antibodies: mouse monoclonal IgM anti-phospho-p38 (1:300) or mouse monoclonal IgG phospho-p42/44 [Thr202/Tyr204] (1:1000) in PBS plus 1% BSA; mouse monoclonal IgG anti-p38 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse monoclonal IgG anti-p42/44 (1:2000; Cell Signaling Technology Inc., Beverly, MA) in PBS plus 1% nonfat dry milk. After incubation with the secondary antibodies, horseradish peroxidase-conjugated rabbit anti-IgG mouse or goat anti-IgM mouse (Sigma) for 1 hour at room temperature in PBS with 0.1% Tween 20 and 1% BSA or 1% nonfat dry milk, protein bands were detected by Supersignal chemiluminescent substrate (Pierce).Reporter Luciferase Gene AssayPodocytes were transfected with 1 μg of NF-κB luciferase reporter gene (Stratagene; M-Medical, Florence, Italy) by the Superfect transfection reagent following the manufacturer's protocol (Qiagen, Milan, Italy). Three hours after transfection, the reporter gene was washed off, and cells were maintained overnight in fresh medium without serum.23Donadelli R" @default.
• W2134037340 created "2016-06-24" @default.
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• W2134037340 date "2006-12-01" @default.
• W2134037340 modified "2023-10-13" @default.
• W2134037340 title "Shigatoxin-Induced Endothelin-1 Expression in Cultured Podocytes Autocrinally Mediates Actin Remodeling" @default.
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