Peroxisome Proliferator-Activated Receptor (PPAR)γ Can Inhibit Chronic Renal Allograft Damage
2010; Elsevier BV; Volume: 176; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2010.090370
ISSN1525-2191
AutoresÉva Kiss, Zoran V. Popović, Jens Bedke, Judith E. Adams, Mahnaz Bonrouhi, Andrea Bábelová, Claudia Schmidt, Frank Edenhofer, Inka Zschiedrich, Sophie Domhan, Amir Abdollahi, Liliana Schäfer, Norbert Gretz, Štefan Porubský, Hermann-Josef Gröne,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoChronic inflammation and fibrosis are the leading causes of chronic allograft failure. The nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ is a transcription factor known to have antidiabetogenic and immune effects, and PPARγ forms obligate heterodimers with the retinoid X receptor (RXR). We have reported that a retinoic acid (RAR)/RXR-agonist can potently influence the course of renal chronic allograft dysfunction. In this study, in a Fischer to Lewis rat renal transplantation model, administration of the PPARγ-agonist, rosiglitazone, independent of dose (3 or 30 mg/kgBW/day), lowered serum creatinine, albuminuria, and chronic allograft damage with a chronic vascular damage score as follows: 35.0 ± 5.8 (controls) vs. 8.1 ± 2.4 (low dose-Rosi; P < 0.05); chronic tubulointerstitial damage score: 13.6 ± 1.8 (controls) vs. 2.6 ± 0.4 (low dose-Rosi; P < 0.01). The deposition of extracellular matrix proteins (collagen, fibronectin, decorin) was strikingly lower. The expression of transforming growth factor-β1 was inhibited, whereas that of bone morphogenic protein-7 (BMP-7) was increased. Intragraft mononuclear cells and activated fibroblast numbers were reduced by 50%. In addition, the migratory and proliferative activity of these cells was significantly inhibited in vitro. PPARγ activation diminished the number of cells expressing the proinflammatory and fibrogenic proteoglycan biglycan. In macrophages its secretion was blocked by rosiglitazone in a predominantly PPARγ-dependent manner. The combination of PPARγ- and RAR/RXR-agonists resulted in additive effects in the inhibition of fibrosis. In summary, PPARγ activation was potently immunosuppressive and antifibrotic in kidney allografts, and these effects were enhanced by a RAR/RXR-agonist. Chronic inflammation and fibrosis are the leading causes of chronic allograft failure. The nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ is a transcription factor known to have antidiabetogenic and immune effects, and PPARγ forms obligate heterodimers with the retinoid X receptor (RXR). We have reported that a retinoic acid (RAR)/RXR-agonist can potently influence the course of renal chronic allograft dysfunction. In this study, in a Fischer to Lewis rat renal transplantation model, administration of the PPARγ-agonist, rosiglitazone, independent of dose (3 or 30 mg/kgBW/day), lowered serum creatinine, albuminuria, and chronic allograft damage with a chronic vascular damage score as follows: 35.0 ± 5.8 (controls) vs. 8.1 ± 2.4 (low dose-Rosi; P < 0.05); chronic tubulointerstitial damage score: 13.6 ± 1.8 (controls) vs. 2.6 ± 0.4 (low dose-Rosi; P < 0.01). The deposition of extracellular matrix proteins (collagen, fibronectin, decorin) was strikingly lower. The expression of transforming growth factor-β1 was inhibited, whereas that of bone morphogenic protein-7 (BMP-7) was increased. Intragraft mononuclear cells and activated fibroblast numbers were reduced by 50%. In addition, the migratory and proliferative activity of these cells was significantly inhibited in vitro. PPARγ activation diminished the number of cells expressing the proinflammatory and fibrogenic proteoglycan biglycan. In macrophages its secretion was blocked by rosiglitazone in a predominantly PPARγ-dependent manner. The combination of PPARγ- and RAR/RXR-agonists resulted in additive effects in the inhibition of fibrosis. In summary, PPARγ activation was potently immunosuppressive and antifibrotic in kidney allografts, and these effects were enhanced by a RAR/RXR-agonist. Present immunosuppressive strategies are unable to prevent graft loss by atrophy and fibrosis that occur during chronic renal allograft dysfunction; the cellular and molecular mechanisms involved are complex.1Liu Y Renal fibrosis: new insights into the pathogenesis and therapeutics.Kidney Int. 2006; 69: 213-217Crossref PubMed Scopus (882) Google Scholar A persistent activity of T cells and monocytes/macrophages seems to be relevant for the subsequent activation and proliferation of (myo)fibroblasts and endothelial cells.2Yates PJ Nicholson ML The aetiology and pathogenesis of chronic allograft nephropathy.Transpl Immunol. 2006; 16: 148-157Crossref PubMed Scopus (78) Google Scholar, 3Joosten SA Sijpkens YW van Kooten C Paul LC Chronic renal allograft rejection: pathophysiologic considerations.Kidney Int. 2005; 68: 1-13Crossref PubMed Scopus (165) Google Scholar Proinflammatory and fibrogenic mediators such as transforming growth factor (TGF)-β, platelet-derived growth factor, endothelin and angiotensin II are synthesized and secreted by both tissue-infiltrating inflammatory mononuclear cells and tubular epithelia.1Liu Y Renal fibrosis: new insights into the pathogenesis and therapeutics.Kidney Int. 2006; 69: 213-217Crossref PubMed Scopus (882) Google Scholar, 3Joosten SA Sijpkens YW van Kooten C Paul LC Chronic renal allograft rejection: pathophysiologic considerations.Kidney Int. 2005; 68: 1-13Crossref PubMed Scopus (165) Google Scholar In addition, authors have recently reported that the small leucine-rich proteoglycans (SLRPs), biglycan (BGN), and decorin are not only structural components of the extracellular matrix; they may be involved in the synthesis of cytokines/chemokines and growth factors including basic fibroblast growth factor and TGF-β.4Kresse H Schönherr E Proteoglycans of the extracellular matrix and growth control.J Cell Physiol. 2001; 189: 266-274Crossref PubMed Scopus (339) Google Scholar, 5Kinsella MG Bressler SL Wight TN The regulated synthesis of versican, decorin, and biglycan: extracellular matrix proteoglycans that influence cellular phenotype.Crit Rev Eukaryot Gene Expr. 2004; 14: 203-234Crossref PubMed Scopus (139) Google Scholar We have reported that BGN is an endogenous ligand of the innate immunity receptors toll-like receptor (TLR) 2 and 4 and an activator of the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, thereby increasing the synthesis and secretion of chemokines and collagen in monocytes/macrophages and fibroblasts.6Schaefer L Babelova A Kiss E Hausser HJ Baliova M Krzyzankova M Marsche G Young MF Mihalik D Götte M Malle E Schaefer RM Gröne HJ The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4 and 2 in macrophages.J Clin Invest. 2005; 115: 2223-2233Crossref PubMed Scopus (637) Google Scholar, 7Babelova A Moreth K Tsalastra-Greul W Zeng-Brouwers J Eickelberg O Young MF Bruckner P Pfeilschifter J Schaefer RM Gröne HJ Schaefer L Biglycan, a danger signal that activates the NLRP3 inflammasome via toll-like and P2X receptors.J Biol Chem. 2009; 284: 24035-24048Crossref PubMed Scopus (336) Google Scholar Adenovirus-mediated gene transfer of BGN has been shown to induce a fibroblastic response in the lung, indicating a role of BGN in fibrogenesis.8Sime PJ Sarnstrand B Xing Z Graham F Fisher L Gauldie J Adenovirus-mediated gene transfer of the proteoglycan biglycan induces fibroblastic responses in the lung.Chest. 1997; 111: 137SPubMed Google ScholarLigand activated transcription factors of the nuclear receptor superfamily might regulate these complex inflammatory/fibrotic networks.9Hong C Tontonoz P Coordination of inflammation and metabolism by PPAR and LXR nuclear receptors.Curr Opin Genet Dev. 2008; 18: 461-467Crossref PubMed Scopus (178) Google Scholar Thiazolidinediones, such as rosiglitazone, are high-affinity ligands for peroxisome proliferator-activated receptor (PPAR)γ, a member of the nuclear receptor family. They are used as insulin-sensitizing drugs in type 2 diabetes. In addition, it has been demonstrated that PPARγ-agonists suppress the synthesis and release of immunomodulatory cytokines/chemokines (eg, interleukin-1β, tumor necrosis factor-α, and interferon-γ) from various cell types and modulate cell cycle, differentiation, and apoptosis.10Lehrke M Lazar MA The many faces of PPARgamma.Cell. 2005; 123: 993-999Abstract Full Text Full Text PDF PubMed Scopus (1135) Google Scholar, 11Straus DS Glass CK Anti-inflammatory actions of PPAR ligands: new insights on cellular and molecular mechanisms.Trends Immunol. 2007; 28: 551-558Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar After ligand-induced activation, PPARγ heterodimerizes with the retinoid X receptor (RXR). PPARγ/RXR heterodimers bind to specific DNA sequences in promoter regions of target genes, regulating genes of lipid and glucose homeostasis.10Lehrke M Lazar MA The many faces of PPARgamma.Cell. 2005; 123: 993-999Abstract Full Text Full Text PDF PubMed Scopus (1135) Google Scholar Anti-inflammatory effects of PPARγ probably result by transrepression, impeding the activity of transcription factors such as activator protein-1 (AP-1), signal transducer and activator of transcription (STAT), and nuclear factor ®B.11Straus DS Glass CK Anti-inflammatory actions of PPAR ligands: new insights on cellular and molecular mechanisms.Trends Immunol. 2007; 28: 551-558Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 12Pascual G Glass CK Nuclear receptors versus inflammation: mechanisms of transrepression.Trends Endocrinol Metab. 2006; 17: 321-327Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar PPARγ-independent anti-inflammatory effects of thiazolidinediones have been also reported.13Chawla A Barak Y Nagy L Liao D Tontonoz P Evans RM PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation.Nat Med. 2001; 7: 48-52Crossref PubMed Scopus (957) Google Scholar, 14Rossi A Kapahi P Natoli G Takahashi T Chen Y Karin M Santoro MG Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase.Nature. 2000; 403: 103-108Crossref PubMed Scopus (1200) Google ScholarWe have demonstrated that retinoids, which act through retinoic acid receptors (RAR) and RXRs, potently block inflammatory and fibrosing processes in renal allograft rejection.15Adams J Kiss E Arroyo AB Bonrouhi M Sun Q Li Z Gretz N Schnitger A Zouboulis CC Wiesel M Wagner J Nelson PJ Gröne HJ 13-cis retinoic acid inhibits development and induces regression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 16Kiss E Adams J Grone HJ Wagner J Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.Transplantation. 2003; 76: 480-489Crossref PubMed Scopus (36) Google Scholar We have also found that rosiglitazone could ameliorate rejection phenomena in acute models of heart and renal transplantation (manuscript in preparation). On the basis of these results, we postulated that PPARγ-agonists could prevent chronic dysfunction of kidney allografts by inhibiting proinflammatory and fibrogenic mediators.The effects of rosiglitazone in a chronic Fisher344 donor→Lewis recipient renal transplantation model have been investigated. Rosiglitazone led to a significant preservation of renal function and morphology. Mononuclear cell infiltration and secretion of cytokines/chemokines as well as proliferation were reduced in kidney allografts. In vitro, rosiglitazone inhibited the proliferation of monocytes/macrophages, endothelial cells, and fibroblasts as well as their migration in co-culture experiments. In addition, the PPARγ ligand, rosiglitazone, suppressed the expression of the SLRPs decorin and BGN in the treated allografts and inhibited the secretion of BGN by interleukin-6 stimulated macrophages in a mainly PPARγ-dependent fashion. The effectiveness of rosiglitazone in reducing chronic allograft damage was significantly increased by addition of an RXR agonist.Materials and MethodsAnimalsMale inbred Lewis (LEW, RT11) and Fisher (F344, RT11v1) rats were purchased from Charles River GmbH (Sulzfeld, Germany). Fisher or Lewis rats were used as recipients of Fisher kidney grafts. Donors and recipients weighed about 200 to 220 g at the time of renal transplantation.Conditional PPARγ-Deficient MiceHomozygous floxed PPARγ (B6.129-Ppargtm2Rev/J; PPARγfl/fl) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred in our animal facility. PPARγfl/fl mice were crossed with a transgenic mouse containing the Cre recombinase gene under the control of the murine M lysozyme promoter (LysCre) to achieve a selective deficiency of PPARγ in macrophages (PPARγfl/fl/LysCre; PPARγ−/−). C57Bl/6 wild-type mice were used as controls, as macrophages of PPARγfl/fl, and LysCre mice had shown similar characteristics of cytokine secretion.17Malur A Mccoy AJ Arce S Barna BP Kavuru MS Malur AG Thomassen MJ Deletion of PPAR gamma in alveolar macrophages is associated with a Th-1 pulmonary inflammatory response.J Immunol. 2009; 182: 5816-5822Crossref PubMed Scopus (94) Google Scholar Animal experiments were performed according to German laws on animal protection.Kidney TransplantationTransplantation was performed under ether drop anesthesia.15Adams J Kiss E Arroyo AB Bonrouhi M Sun Q Li Z Gretz N Schnitger A Zouboulis CC Wiesel M Wagner J Nelson PJ Gröne HJ 13-cis retinoic acid inhibits development and induces regression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 16Kiss E Adams J Grone HJ Wagner J Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.Transplantation. 2003; 76: 480-489Crossref PubMed Scopus (36) Google Scholar The left kidney of the donor F344 rat was isolated, perfused with ice-cold isotonic sodium chloride solution, excised, and transplanted orthotopically into a weight-matched F344 (isograft) or LEW recipient (allograft). In the recipient the left renal vein and artery were mobilized and clamped, the ureter was cut, and the left kidney was excised. End-to-end anastomosis of renal vessels and of ureter, without ureteral stenting, were performed with 10–0 nonabsorbable nylon sutures. Total ischemic time of the donor kidney varied between 30 and 45 minutes. The right kidney was left in place to enhance rejection and damage to the transplant by avoidance of potential endogenous immunosuppressive effects of renal insufficiency and by a reduction of the work load of the transplanted kidney;15Adams J Kiss E Arroyo AB Bonrouhi M Sun Q Li Z Gretz N Schnitger A Zouboulis CC Wiesel M Wagner J Nelson PJ Gröne HJ 13-cis retinoic acid inhibits development and induces regression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 18Bedke J Kiss E Schaefer L Behnes CL Bonrouhi M Gretz N Horuk R Diedrichs-Moehring M Wildner G Nelson PJ Gröne HJ Beneficial effects of CCR1 blockade on the progression of chronic renal allograft damage.Am J Transplant. 2007; 7: 527-537Crossref PubMed Scopus (48) Google Scholar to obtain functional parameters of the graft at the end of the experiments, the right kidney was removed 48 hours before sacrifice. All transplant kidneys with hydronephrosis, which was evaluated both macroscopically and by light microscopy, were excluded from the experimental groups.Experimental ProtocolAnimals were randomly allocated to five experimental groups: (1) control group, 56 days (n = 20), F344 to LEW allografts fed with standard rat chow; (2) Rosiglitazone (Rosi) groups: (a) high dose (HD)-Rosi, 56 days (n = 9), and (b) low dose (LD)-Rosi, 56 days (n = 8), in which animals were treated with HD (30 mg/kgBW/day) or LD (3 mg/kgBW/day) to study the effects of rosiglitazone on renal allograft rejection.To evaluate the effects of a combination of two ligands to RXR-heterodimers on chronic allograft dysfunction, two other groups were studied: (3) Isotretinoin (Iso), 56 days (n = 8), and (4) Rosi + Iso, 56 days (n = 8), in which animals were treated with isotretinoin (0.2 mg/kgBW/day) or a combination of rosiglitazone (3 mg/kgBW/day) and isotretinoin (0.2 mg/kgBW/day). The dose of isotretinoin used here corresponded to a 10-fold lower dose as that shown to be immunosuppressive in our previous experiments.16Kiss E Adams J Grone HJ Wagner J Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.Transplantation. 2003; 76: 480-489Crossref PubMed Scopus (36) Google ScholarRosiglitazone (Avandia; GlaxoSmithKline, Münich, Germany) and isotretinoin (Roaccutan; Ratiopharm, Ulm, Germany) were administered orally for 8 weeks starting at the day of renal transplantation. None of the recipients were treated with any other immunosuppressant.In addition, F344 to F344 isograft transplantation was performed and evaluated after 56 days (n = 5).Graft Functional Parameters and Systolic Arterial Blood PressureFor measurements of serum creatinine and albuminuria, rats were kept in metabolic cages 24 hours before the end of the experiment. Serum creatinine (enzymatic determination), urea nitrogen, cholesterol, tiglycerides, Glutamix oxalacetic transaminase (GOT), Glutamic pyruvic transaminase (GPT), AP, Lactate dehydrogenase (LDH), and urine (albuminuria) samples were analyzed by using a Hitachi 9-17-E autoanalyzer (Hitachi, Frankfurt/M, Germany).19Keppler A Gretz N Schmidt R Kloetzer HM Groene HJ Lelongt B Meyer M Sadick M Pill J Plasma creatinine determination in mice and rats: an enzymatic method compares favorably with a high-performance liquid chromatography assay.Kidney Int. 2007; 71: 74-78Crossref PubMed Scopus (101) Google Scholar Systolic blood pressure was determined before sacrifice by tail cuff plethysmography under light ether anesthesia.20Grone HJ Helmchen U Impairment and recovery of the clipped kidney in two kidney, one clip hypertensive rats during and after antihypertensive therapy.Lab Invest. 1986; 54: 645-655PubMed Google ScholarHistological AnalysisRenal allografts were removed in deep anesthesia, quickly blotted free of blood, weighed, and processed as required for histology, immunohistology, and molecular analysis. For histology, the kidneys were cut into 1-mm coronal slices and either immersion fixed in 4% formaldehyde in PBS (99 mmol/L NaH2PO4, 108 mmol/L, NaH2PO4, and 248 mmol/L NaCl) at ph 7.35 for 24 hours at 4°C or fixed in methacarn (60% methanol, 30% chloroform, and 10% acetic acid) for 8 hours and then embedded in paraffin. In addition, tissue slices were snap frozen in liquid nitrogen and stored at −80°C.Light microscopy was performed on 3-μm sections stained by PAS. In brief, kidneys were evaluated for evidence of acute and chronic vascular (endothelialitis/vasculitis, fibrointimal thickening), glomerular (glomerulitis, transplant glomerulopathy, glomerulosclerosis), and tubulointerstitial damage (thinning/denudation/necrosis of the tubular epithelia and interstitial edema; tubular atrophy and interstitial fibrosis) on a scale ranging from 0 to 3 as previously described.15Adams J Kiss E Arroyo AB Bonrouhi M Sun Q Li Z Gretz N Schnitger A Zouboulis CC Wiesel M Wagner J Nelson PJ Gröne HJ 13-cis retinoic acid inhibits development and induces regression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 16Kiss E Adams J Grone HJ Wagner J Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.Transplantation. 2003; 76: 480-489Crossref PubMed Scopus (36) Google ScholarTubulointerstitial inflammation was judged as 0 (no mononuclear cells in the interstitium), 0.5 (focal mononuclear cell infiltration in the interstitium), 1 (focal mononuclear infiltration in the interstitium with tubulitis), 2 (diffuse mononuclear cell infiltration of the interstitium), and 3 (diffuse mononuclear cell infiltration of the interstitium with tubulitis). Tubulitis was defined as 1 or more mononuclear cells/tubular cross section. Tubulointerstitial inflammation index was defined as the percentage of fields with respective degree of the injury encountered in 10 fields (objective ×20) of cortex and outer stripe of outer medulla. The tubulointerstitial inflammation score was calculated as the sum of all specific indices, whereby the index of fields with degree 0.5 was multiplied by 0.5, that of degree 1 × 1, that of degree 2 × 2, and that of degree 3 × 3. Vascular and glomerular injuries were scored in an analogous pattern.ImmunohistochemistryImmunohistochemical staining was performed on 30-μm sections of paraffin-embedded tissue except for CD4, which was labeled on frozen sections. Mouse anti-rat monoclonal antibodies against ED1 (Serotec, Oxford, UK) in methacarn-fixed tissue, CD4, CD8 (Serotec), and Ki-67 (clone MIP-5, Dianova, Germany) as well as anti-major histocompatibility complex (MHC)-class-II Ia antibody (OX6; Abcam, Cambridge, UK) in formaldehyde-fixed tissue were used. Formaldehyde-fixed tissues were microwave treated. To detect potential subpopulations of fibroblasts, antibodies to vimentin (from guinea pig; Progen Biotechnik, Heidelberg, Germany), desmin (Dako, Glastrup, Denmark), and α-smooth muscle actin (α-SMA; mouse ascites fluid; Sigma, St. Louis, MO) were used. Biglycan and decorin were stained by using LF-113, a rabbit anti-murine decorin antiserum, and MAY-01, a chicken anti-rat biglycan antiserum, as described previously.21Schaefer L Hausser H Altenburger M Ugorcakova J August C Fisher LW Schaefer RM Kresse H Decorin, biglycan and their endocytosis receptor in rat renal cortex.Kidney Int. 1998; 54: 1529-1541Crossref PubMed Scopus (56) Google ScholarAn alkaline phosphatase anti-alkaline phosphatase detection system was applied (Dako Cytomation A/S). For staining of osteopontin (mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA), fibronectin (rabbit anti-human; Dako), collagen I (rabbit anti-rat; Biogenesis, Poole, UK), and collagen III (Chemicon, Temecula, CA) streptavidin-biotin enhanced horseradish peroxidase immunostaining was performed.Positive glomerular cells were counted in at least 50 glomerular cross sections and given as the mean per glomerular section; interstitial positive cells were counted in 20 high-powered fields (HPFs; ×40) of cortex and outer medulla and recorded as mean per HPF. The intensity of the staining for collagen and fibronectin was evaluated as follows: not detectable (degree 0), faint (degree 1), moderate (degree 2), and intense staining (degree 3). A degree-specific staining index was defined as the percentage of the fields with the degree of staining in 20 HPFs (×400) of cortex and outer medulla. The staining score was calculated as the sum of the degree-specific indices by which degree 1 was multiplied by 1, that of degree 2 × 2, and that of degree 3 × 3.Real-Time RT-PCR of Kidney AllograftsTotal RNA was extracted from the kidney allografts by using the method of Chomczynski and Sacchi22Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar (n = 4 to 6 animals per group). RNA quality was characterized by using a RNA6000 Nanochip (Agilent Technologies, Waldbronn, Germany). Ten micrograms of total RNA was digested with DNase I according to standard protocol. Three micrograms of total RNA (DNA free) was used for the first-strand cDNA synthesis by using Superscript II reverse transcriptase and oligo d(T)12-18 as primer (LifeTechnologies, Karlsruhe, Germany). Real-time PCR was performed by LightCycler using LightCyler-FastStart DNA MasterSYBR Green I kit (Roche Diagnostics, Mannheim, Germany) as described.15Adams J Kiss E Arroyo AB Bonrouhi M Sun Q Li Z Gretz N Schnitger A Zouboulis CC Wiesel M Wagner J Nelson PJ Gröne HJ 13-cis retinoic acid inhibits development and induces regression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar The primer sequences for target genes are shown in Table 1. Expression levels of the studied genes are given relative to the expression levels of tubulin.Table 1Sequences of Primers Used for Real-Time RT-PCR Analysis of Kidney AllograftsGeneGene IDSenseAntisenseAmplicon, bpa-Tubulin641585′-TTTGATCTGATGTATGCCAAGC-3′5′-TCCTTCTTCCTCACCCTCAC-3′162CD36291845′-ATTTGTTCTTCCAGCCAACG-3′5′-ATGTCCAGCACACCATACGA-3′114LXR-α588525′-TGATGCTGAATTTGCTCTGC-3′5′-GGCTCACCAGCTTCATTAGC-3′185TGF-β1590865′-GCAACACGTAGAACTCTACC-3′5′-CCCTGTATTCCGTCTCCTTG-3′153PAI-1246175′-TTTGTGTTCCAGTCACACTC-3′5′-ATCTGTCTATCTGCTGCCC-3′153BMP-7852725′-GAAAACAGCAGCAGTGACCA-3′5′-GGTGGCGTTCATGTAGGAGT-3′165 Open table in a new tab Antigen Presentation by Bone Marrow Derived Dendritic CellsBone marrow derived dendritic cells (BMDCs) were cultivated as described in Lutz et al.23Lutz MB Kukutsch N Ogilvie AL Rossner S Koch F Romani N Schuler G An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.J Immunol Methods. 1999; 223: 77-92Crossref PubMed Scopus (2475) Google Scholar Briefly, on day 0, 4 × 106 nucleated bone marrow cells were plated on uncoated Petri dishes (Greiner Bio-One, Frickenhausen, Germany) in Iscove's Modified Dulbecco's Medium with L-Glutamine (PAA, Cölbe, Germany), 10% fetal calf serum (FCS), 1% penicillin/streptomycin, and 50 mmol/L β-mercaptorthanol (all Invitrogen, Karlsruhe, Germany) supplemented with 25 ng/ml granulocyte macrophage colony-stimulating factor (R and D Systems, Wiesbaden, Germany). On day 4 all cells were collected and again 4 × 106 cells were seeded. On day 7 BMDCs were harvested and incubated for an additional 3 hours in pure media or in media supplemented with 100 μmol/L rosiglitazone (Alexis Biochemicals, Grünberg, Germany). Rosiglitazone was dissolved in ethanol. For antigen presentation, 0.5 × 106 BMDCs were first incubated at 37°C in 96-well plates (Greiner Bio-One) in 100-μl media supplemented with endotoxin-free ovalbumin (OVA; Profos, Regensburg, Germany) at 1 mg/ml. After 3 hours, the media was completely removed, and 0.1 × 106 BO17.10 (MHC-II restricted OVA-specific T-cell hybridomas)24Shimonkevitz R Kappler J Marrack P Grey H Antigen recognition by H-2-restricted T cells: cell-free antigen processing.J Exp Med. 1983; 158: 303-316Crossref PubMed Scopus (517) Google Scholar were added in pure media or media supplemented either with 10 μmol/L or 100 μmol/L rosiglitazone. In parallel, also BO17.10 cells pretreated with 100 μmol/L rosiglitazone for 3 hours were applied. After overnight incubation, interleukin-2 concentration was measured in the culture media by using interleukin-2 flex set, FACSCalibur (both BD Biosciences, Heildelberg, Germany), and FCAP array software (Soft Flow, Pécs, Hungary).Isolation of Peritoneal Macrophages and Generation of Bone Marrow Derived MacrophagesWild-type and PPARγ−/− (PPARγfl/fl/LysCre) mice were used for the experiments. Peritoneal macrophages were harvested 4 days after injection of thioglycolate and cultured in serum-free RPMI 1640 (Invitrogen) in 6-well plates at a concentration of 1 × 106/well. After 12 hours of incubation at 37C°/5%CO2, the macrophages were thoroughly washed and adherent cells were used for experiments. Purity was controlled by flow cytometry and Giemsa staining.Generation of murine bone marrow derived macrophages was performed according to standard protocols.25Weischenfeldt J Porse B Bone marrow-derived macrophages (BMM): isolation and applications.Cold Spring Harb Protoc. 2008; https://doi.org/10.1101/pdb.prot5080Crossref Scopus (604) Google Scholar, 26Gersuk GM Razai LW Marr KA Methods of in vitro macrophage maturation confer variable inflammatory responses in association with altered expression of cell surface dectin-1.J Immunol Methods. 2008; 329: 157-166Crossref PubMed Scopus (35) Google Scholar In brief, mouse femurs were dissected and each bone was flushed with 10 ml PBS. A bone marrow cell suspension was collected and centrifuged. Pellets were resuspended in RPMI 1640 medium supplemented by 20% Macrophage colony stimulating factor (MCSF)-containing L929 medium. The cells were plated on non-TC (tissue culture) treated 10-cm Petri dishes and incubated at 37°C/5% CO2. Fresh medium was provided at days 3 and 5, and experiments were performed at day 7. After preincubation with 10 or 100 μmol/L rosiglitazone for 1 hour, macrophages were stimulated with human interleukin-6 (10 ng/ml; R and D Systems) for 6 hours.In Vitro Conditional PPARγ Deficiency in Peritoneal MacrophagesConditional deficiency of PPARγ in vitro (PPARγfl/fl/HTNCre) in peritoneal and bone marrow derived macrophages of PPARγfl/fl mice was achieved as described previously.27Peitz M Jäger R Patsch C Jäger A Egert A Schorle H Edenhofer F Enhanced purification of cell-permeant Cre and germline transmission after transduction into mouse embryonic stem cells.Genesis. 2007; 45: 508-517Crossref PubMed Scopus (29) Google Scholar, 28Klotz L Hucke S Thimm D Classen S Gaarz A Schultze J Edenhofer F Kurts C Klockgether T Limmer A Knolle P Burgdorf S Increased antigen cross-presentation but impaired cross-priming after activation of peroxisome proliferator-activated receptor gamma is mediated by up-regulation of B7H1.J Immunol. 2009; 183: 129-136Crossref PubMed Scopus (28) Google Scholar Briefly, thioglycolate-induced peritoneal macrophages from wild-type and PPARγfl/fl mice were treated immediately after isolation with 1 μmol/L of a membrane-permeable His-TAT-NLS-Cre (HTNCre) recombinase (provided by F. Edenhofer, Bonn) in a low serum (1% FCS) medium and washed extensively after 6 hours. The macrophages were then incubated with 10 μmol/L rosiglitazone 1 hour before the interleukin-6 stimulation.Northern Blot and RT-PCR Analysis of MacrophagesTotal RNA was extracted from peritoneal and bone marrow derived macrophages by using TRIzol (Invitrogen). The effectiveness of Cre-mediated recombination was determined by RT-PCR of PPARγ-transcri
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