RESUMEN
Butyrate is a short-chain fatty acid derived from the metabolism of indigestible carbohydrates by the gut microbiota. Butyrate contributes to gut homeostasis, but it may also control inflammatory responses and host physiology in other tissues. Butyrate inhibits histone deacetylases, thereby affecting gene transcription, and also signals through the metabolite-sensing G protein receptor (GPR)109a. We produced an mAb to mouse GPR109a and found high expression on podocytes in the kidney. Wild-type and Gpr109a-/- mice were induced to develop nephropathy by a single injection of Adriamycin and treated with sodium butyrate or high butyrate-releasing high-amylose maize starch diet. Butyrate improved proteinuria by preserving podocyte at glomerular basement membrane and attenuated glomerulosclerosis and tissue inflammation. This protective phenotype was associated with increased podocyte-related proteins and a normalized pattern of acetylation and methylation at promoter sites of genes essential for podocyte function. We found that GPR109a is expressed by podocytes, and the use of Gpr109a-/- mice showed that the protective effects of butyrate depended on GPR109a expression. A prebiotic diet that releases high amounts of butyrate also proved highly effective for protection against kidney disease. Butyrate and GPR109a play a role in the pathogenesis of kidney disease and provide one of the important molecular connections between diet, the gut microbiota, and kidney disease.-Felizardo, R. J. F., de Almeida, D. C., Pereira, R. L., Watanabe, I. K. M., Doimo, N. T. S., Ribeiro, W. R., Cenedeze, M. A., Hiyane, M. I., Amano, M. T., Braga, T. T., Ferreira, C. M., Parmigiani, R. B., Andrade-Oliveira, V., Volpini, R. A., Vinolo, M. A. R., Mariño, E., Robert, R., Mackay, C. R., Camara, N. O. S. Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.
Asunto(s)
Butiratos/farmacología , Epigénesis Genética , Microbioma Gastrointestinal/fisiología , Enfermedades Renales/prevención & control , Proteinuria/prevención & control , Receptores Acoplados a Proteínas G/genética , Animales , Bacterias/metabolismo , Butiratos/metabolismo , Células Cultivadas , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Sustancias Protectoras/metabolismo , Sustancias Protectoras/farmacología , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.
Asunto(s)
Galactosilceramidas/administración & dosificación , Interferón gamma/genética , Nefritis/tratamiento farmacológico , Insuficiencia Renal/tratamiento farmacológico , Adenina/toxicidad , Animales , Antígenos CD1d/biosíntesis , Antígenos CD1d/genética , Colágeno Tipo I/biosíntesis , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Interleucina-4/biosíntesis , Interleucina-4/genética , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Nefritis/inducido químicamente , Nefritis/genética , Nefritis/patología , Insuficiencia Renal/inducido químicamente , Insuficiencia Renal/genética , Insuficiencia Renal/patología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
BACKGROUND/AIMS: Renal ischaemia-reperfusion injury (IRI) is a systemic inflammatory process in which Th1 responses predominate affecting other organs including the lungs. The present study explored the phagocytic and microbicidal capacity of macrophages in rats with lung inflammation that underwent IRI. METHODS: The alveolar macrophages of rats sensitised to OVA were evaluated for phagocytosis and bacterial killing 24h after antigen challenge in animals with or without prior submission to 60 min of renal ischaemia. RESULTS: Bronchoalveolar lavage had a high level of cellular infiltrate in immunised animals (420%) compared with control animals; IRI significantly reduced this infiltration (52%). Macrophages from animals immunised and challenged with OVA presented a 10x increase in phagocytic capacity compared to the control group, whereas immunised animals subjected to IRI showed a reduction in the phagocytic index of 68%. The killing of Klebsiella pneumoniae by macrophages from immunised animals was higher (56%) compared with the control group but reduced in animals submitted to IRI (45%). Immunised and challenged group showed an increase in gene expression levels of IL-10(450%), HO-1 (259%), INF-γ (460%) and MCP-1 (370%) compared to the immunised group subjected to IRI. CONCLUSIONS: Renal ischaemia and reperfusion injury apparently alters the phagocytic and microbicidal capacity of macrophages, reducing lung inflammation to OVA.
Asunto(s)
Lesión Renal Aguda/inmunología , Macrófagos Alveolares/fisiología , Fagocitosis/fisiología , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Animales , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Klebsiella pneumoniae/fisiología , Macrófagos Alveolares/citología , Macrófagos Alveolares/inmunología , Masculino , Óxido Nítrico/metabolismo , Ovalbúmina/inmunología , Ratas , Ratas Wistar , Daño por Reperfusión/inmunología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patologíaRESUMEN
There is a consensus in the scientific literature that supports the importance of the kallikrein kinin and renin angiotensin systems in renal physiology, but few studies have investigated their importance after renal transplantation. The aim of this study was to investigate the clinical effects of the insertion/deletion polymorphism in the angiotensin I-converting enzyme (ACE) gene and the +9/-9 polymorphism in the kinin B2 receptor (B2R) gene in kidney-transplanted patients (n=215 ACE, n=203 B2R) compared with 443 healthy individuals. Demographic results showed that there is a higher frequency of the D allele (high plasma ACE activity) and +9 allele (lower B2R expression) in transplant patients compared with control individuals. We also observed a higher frequency of these alleles in patients who had an elevated level of plasma creatinine. At day 7 post-transplantation, we found a higher prevalence of individuals with the DD genotype with elevated plasma creatinine level. Furthermore, individuals with the DD genotype had a higher chronic allograft dysfunction and graft loss compared with the II patient genotype, which showed no loss of graft. Taken together, our data suggest that the DD genotype is an indicator of an unfavorable prognosis following renal transplantation and could be related to kinin modulation.
Asunto(s)
Trasplante de Riñón , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Polimorfismo Genético , Receptor de Bradiquinina B2/genética , Receptor de Bradiquinina B2/metabolismo , Adulto , Femenino , Eliminación de Gen , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Mutagénesis Insercional , PronósticoRESUMEN
The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors.
Asunto(s)
Riñón/lesiones , Pulmón/inmunología , Daño por Reperfusión/inmunología , Balance Th1 - Th2 , Animales , Recuento de Células Sanguíneas , Líquido del Lavado Bronquioalveolar/inmunología , Creatinina/sangre , Ciclooxigenasa 2/metabolismo , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Hipersensibilidad/patología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interleucinas/inmunología , Interleucinas/metabolismo , Riñón/inmunología , Riñón/patología , Pulmón/metabolismo , Pulmón/patología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Moco/inmunología , Neutrófilos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , FosforilaciónRESUMEN
OBJECTIVE AND DESIGN: This work explored the role of inhibition of cyclooxygenases (COXs) in modulating the inflammatory response triggered by acute kidney injury. MATERIAL: C57Bl/6 mice were used. TREATMENT: Animals were treated or not with indomethacin (IMT) prior to injury (days -1 and 0). METHODS: Animals were subjected to 45 min of renal pedicle occlusion and sacrificed at 24 h after reperfusion. Serum creatinine and blood urea nitrogen, reactive oxygen species (ROS), kidney myeloperoxidase (MPO) activity, and prostaglandin E2 (PGE(2)) levels were analyzed. Tumor necrosis factor (TNF)-alpha, t-bet, interleukin (IL)-10, IL-1beta, heme oxygenase (HO)-1, and prostaglandin E synthase (PGES) messenger RNA (mRNA) were studied. Cytokines were quantified in serum. RESULTS: IMT-treated animals presented better renal function with less acute tubular necrosis and reduced ROS and MPO production. Moreover, the treatment was associated with lower expression of TNF-alpha, PGE(2), PGES, and t-bet and upregulation of HO-1 and IL-10. This profile was mirrored in serum, where inhibition of COXs significantly decreased interferon (IFN)-gamma, TNF-alpha, and IL-12 p70 and upregulated IL-10. CONCLUSIONS: COXs seem to play an important role in renal ischemia and reperfusion injury, involving the secretion of pro-inflammatory cytokines, activation of neutrophils, and ROS production. Inhibition of COX pathway is intrinsically involved with cytoprotection.
Asunto(s)
Lesión Renal Aguda/metabolismo , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Indometacina/farmacología , Inflamación/metabolismo , Animales , Citocinas/sangre , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interleucina-10/metabolismo , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/metabolismoRESUMEN
Ischemia and reperfusion injury (IRI) contributes to the development of chronic interstitial fibrosis/tubular atrophy in renal allograft patients. Cyclooxygenase (COX) 1 and 2 actively participate in acute ischemic injury by activating endothelial cells and inducing oxidative stress. Furthermore, blockade of COX 1 and 2 has been associated with organ improvement after ischemic damage. The aim of this study was to evaluate the role of COX 1 and 2 in the development of fibrosis by performing a COX 1 and 2 blockade immediately before IRI. We subjected C57Bl/6 male mice to 60 min of unilateral renal pedicle occlusion. Prior to surgery mice were either treated with indomethacin (IMT) at days -1 and 0 or were untreated. Blood and kidney samples were collected 6 wks after IRI. Kidney samples were analyzed by real-time reverse transcription-polymerase chain reaction for expression of transforming growth factor beta (TGF-beta), monocyte chemoattractant protein 1 (MCP-1), osteopontin (OPN), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta, IL-10, heme oxygenase 1 (HO-1), vimentin, connective-tissue growth factor (CTGF), collagen I, and bone morphogenic protein 7 (BMP-7). To assess tissue fibrosis we performed morphometric analyses and Sirius red staining. We also performed immunohistochemical analysis of anti-actin smooth muscle. Renal function did not significantly differ between groups. Animals pretreated with IMT showed significantly less interstitial fibrosis than nontreated animals. Gene transcript analyses showed decreased expression of TGF-beta, MCP-1, TNF-alpha, IL-1-beta, vimentin, collagen I, CTGF, and IL-10 mRNA (all P < 0.05). Moreover, HO-1 mRNA was increased in animals pretreated with IMT (P < 0.05). Conversely, IMT treatment decreased osteopontin expression and enhanced BMP-7 expression, although these levels did not reach statistical significance when compared with control expression levels. The blockade of COX 1 and 2 resulted in less tissue fibrosis, which was associated with a decrease in proinflammatory cytokines and enhancement of the protective cellular response.
Asunto(s)
Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Riñón/irrigación sanguínea , Riñón/patología , Daño por Reperfusión/metabolismo , Animales , Proteína Morfogenética Ósea 7/genética , Quimiocina CCL2/genética , Colágeno Tipo I/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Fibrosis , Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Inmunohistoquímica , Indometacina/farmacología , Interleucina-10/genética , Interleucina-1beta/genética , Riñón/metabolismo , Pruebas de Función Renal , Masculino , Ratones , Osteopontina/genética , Daño por Reperfusión/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/genética , Factor de Necrosis Tumoral alfa/genética , Vimentina/genéticaRESUMEN
BACKGROUND: Tim-3 was recently described as a Th1-specific molecule, participating in the regulation of immune responses and in the induction of allograft tolerance. Here, we studied Tim-3 mRNA expression together with molecular markers of T-cell activation and cytotoxicity, in rejected human kidney grafts. METHODS: Twenty human kidney grafts that had undergone nephrectomy due to an irreversible acute rejection episode were studied. We quantified intragraft expression of Tim-3, granzyme B, perforin, IFN-gamma and Fas-ligand mRNA by real-time RT-PCR, with probes and primers TaqMan. Protocol biopsies were studied as controls. Statistical analyses were performed to compare groups, and to investigate the potential association with gene transcripts measures and rejection. RESULTS: All molecules studied were up-regulated in the rejection group compared with controls (p<0.001). Acute rejection type III (Banff 97) profiles were associated with higher values, where granzyme B and perforin presented the highest (5672.51+/-9002.16 and 1866.59+/-2426.38, respectively) and Tim-3 had the lowest ones (166.62+/-174.94). Tim-3 had also a lower expression in those patients that did not respond to anti-rejection therapy. There was a positive correlation between Tim-3 and IFN-gamma mRNA expression levels (r(2)=0.73; p<0.001). CONCLUSIONS: Our results corroborate the concept that acute rejection is an active process, where inflammatory as well as regulatory factors have their roles. Severe episodes of acute rejection were associated with higher expression of cytotoxic molecules and lower expression of potential regulatory molecule.
Asunto(s)
Rechazo de Injerto/inmunología , Trasplante de Riñón/inmunología , Activación de Linfocitos/inmunología , Receptores Virales/biosíntesis , Linfocitos T/inmunología , Adulto , Proteína Ligando Fas/biosíntesis , Femenino , Granzimas/biosíntesis , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Interferón gamma/biosíntesis , Masculino , Glicoproteínas de Membrana/biosíntesis , Proteínas de la Membrana , Persona de Mediana Edad , Perforina , Proteínas Citotóxicas Formadoras de Poros/biosíntesis , ARN Mensajero/análisis , Curva ROC , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HomólogoRESUMEN
Mesenchymal stromal cells (MSCs) are multipotent cells with abilities to exert immunosuppressive response promoting tissue repair. Studies have shown that MSCs can secrete extracellular vesicles (MVs-MSCs) with similar regulatory functions to the parental cells. Furthermore, strong evidence suggesting that MVs-MSCs can modulate several immune cells (i.e., Th1, Th17, and Foxp3+ T cells). However, their precise effect on macrophages (MÏs) remains unexplored. We investigated the immunoregulatory effect of MVs-MSCs on activated M1-MÏs in vitro and in vivo using differentiated bone marrow MÏs and an acute experimental model of thioglycollate-induced peritonitis, respectively. We observed that MVs-MSCs shared surface molecules with MSCs (CD44, CD105, CD90, CD73) and expressed classical microvesicle markers (Annexin V and CD9). The in vitro treatment with MVs-MSCs exerted a regulatory-like phenotype in M1-MÏs, which showed higher CD206 level and reduced CCR7 expression. This was associated with decreased levels of inflammatory molecules (IL-1ß, IL-6, nitric oxide) and increased immunoregulatory markers (IL-10 and Arginase) in M1-MÏs. In addition, we detected that MVs-MSCs promoted the downregulation of inflammatory miRNAs (miR-155 and miR-21), as well as, upregulated its predicted target gene SOCS3 in activated M1-MÏs. In vivo MVs-MSCs treatment reduced the MÏs infiltrate in the peritoneal cavity inducing a M2-like regulatory phenotype in peritoneal MÏs (higher arginase activity and reduced expression of CD86, iNOS, IFN-γ, IL-1ß, TNF-α, IL-1α, and IL-6 molecules). This in vivo immunomodulatory effect of MVs-MSCs on M1-MÏs was partially associated with the upregulation of CX3CR1 in F4/80+/Ly6C+/CCR2+ MÏs subsets. In summary, our findings indicate that MVs-MSCs can modulate an internal program in activated MÏs establishing an alternative regulatory-like phenotype.
RESUMEN
Ischemia-reperfusion injury (IRI) is a common event in organ transplantation, being implicated as a potential contributor for the development of chronic allograft nephropathy. There are new evidences showing a tissue inflammatory response following renal IRI. Cyclooxygenases (COX) 1 and 2 can be detected in tissue submitted to IRI and may have impact on organ function outcome. We evaluated the role of COX inhibition on the renal tissue damage that follows IRI. Mice were submitted to 45 min of renal pedicle ligature and allowed to reperfuse for 24, 48, 72 and 120 h. Blood and kidney samples were collected at reperfusion times. mRNA was extracted from the kidney samples to amplify COX-1, COX-2 and beta-actin genes. Animals were pretreated with indomethacin or rofecoxib before the surgery. Indomethacin treatment induced a better renal function (serum urea) when compared to control animals at 24, 48 and 72 h (219+/-54.5 vs. 338+/-51 mg/dl; 106+/-51 vs. 326+/-86 mg/dl; 94+/-14 vs. 138+/-38 mg/dl, respectively). Surprisingly, rofecoxib use was associated with even better renal improvement following IR. Animals treated with the later drug showed lower urea values at 24 h post reperfusion compared to indomethacin-treated animals (128+/-33 vs. 219+/-54.5 mg/dl, P<0.05). Blockade of COX-1 and -2 resulted in a decrease of tubular necrosis. mRNA COX-2 was up-regulated post IRI and considerable inhibited after indomethacin or rofecoxib treatment. Our data show COX-1/-2 participates in the inflammatory tissue response to IR injury and its inhibition is associated with an improvement in renal function.
Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Isquemia/metabolismo , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Daño por Reperfusión/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Indometacina/farmacología , Isquemia/fisiopatología , Riñón/fisiopatología , Lactonas/farmacología , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/biosíntesis , Daño por Reperfusión/fisiopatología , Sulfonas/farmacologíaRESUMEN
A growing body of evidence demonstrates a correlation between Th2 cytokines and the development of focal and segmental glomerulosclerosis (FSGS). Therefore, we hypothesized that GSL-1, a monoglycosylceramide from Sphingomonas ssp. with pro-Th1 activity on invariant Natural Killer T (iNKT) lymphocytes, could counterbalance the Th2 profile and modulate glomerulosclerosis. Using an adriamycin(ADM)-based model of FSGS, we found that BALB/c mice presented albuminuria and glomerular degeneration in association with a Th2-like pro-fibrogenic profile; these mice also expressed a combination of inflammatory cytokines, such as IL-4, IL-1α, IL-1ß, IL-17, TNF-α, and chemokines, such as RANTES and eotaxin. In addition, we observed a decrease in the mRNA levels of GD3 synthase, the enzyme responsible for GD3 metabolism, a glycolipid associated with podocyte physiology. GSL-1 treatment inhibited ADM-induced renal dysfunction and preserved kidney architecture, a phenomenon associated with the induction of a Th1-like response, increased levels of GD3 synthase transcripts and inhibition of pro-fibrotic transcripts and inflammatory cytokines. TGF-ß analysis revealed increased levels of circulating protein and tissue transcripts in both ADM- and GSL-1-treated mice, suggesting that TGF-ß could be associated with both FSGS pathology and iNKT-mediated immunosuppression; therefore, we analyzed the kidney expression of phosphorylated SMAD2/3 and SMAD7 proteins, molecules associated with the deleterious and protective effects of TGF-ß, respectively. We found high levels of phosphoSMAD2/3 in ADM mice in contrast to the GSL-1 treated group in which SMAD7 expression increased. These data suggest that GSL-1 treatment modulates the downstream signaling of TGF-ß through a renoprotective pathway. Finally, GSL-1 treatment at day 4, a period when proteinuria was already established, was still able to improve renal function, preserve renal structure and inhibit fibrogenic transcripts. In conclusion, our work demonstrates that the iNKT agonist GSL-1 modulates the pathogenesis of ADM-induced glomerulosclerosis and may provide an alternative approach to disease management.
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Ceramidas/farmacología , Regulación de la Expresión Génica/fisiología , Glomeruloesclerosis Focal y Segmentaria/inmunología , Células T Asesinas Naturales/inmunología , Transducción de Señal/fisiología , Sphingomonas/química , Análisis de Varianza , Animales , Western Blotting , Quimiocinas/inmunología , Citocinas/inmunología , Cartilla de ADN/genética , Doxorrubicina/toxicidad , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Células T Asesinas Naturales/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Sialiltransferasas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Células Th2/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
INTRODUCTION: TLR-4 has also been identified as a receptor for endogenous alarmins, which are increased post transplantation. TLR-4 has also been associated with a polymorphism that could impact graft outcome. OBJECTIVE: To assess the expression of TLR-4 in kidney transplant patients carrying or not a polymorphism. METHODS: TLR-4 polymorphism (A299G/T399I) was studied in 200 renal transplant patients. Healthy volunteers were also enrolled as control group. The polymorphism analysis was performed using restriction enzymes technique (RFLP). Functionality of TLR-4 polymorphism was assessed in samples from controls by quantification of TNF-α after LPS stimulus. TLR-4 and -2 expressions were also analyzed by flow cytometry. RESULTS: TLR-4 polymorphism was present in 8.5% of renal transplant patients. This polymorphism was associated with impairment in TNF-α secretion. In general, in renal transplant patients, TLR-4 expression in monocytes and in neutrophils was lower than in health volunteers. TLR-2 and TLR-4 expressions in healthy volunteers with A299G/T399I TLR-4 polymorphism was higher than in wild-type genotype healthy volunteers (p<0.01 and p<0.05, respectively), and also higher than A299G/T399I TLR-4 polymorphism renal transplant patients (p<0.05). TLR-2 expression on neutrophils in wild-type genotype renal transplant patients was higher compared to wild-type genotype healthy volunteers, and was also higher in relation to A299G/T399I kidney transplanted patients (p<0.01). CONCLUSION: Stable renal transplant patients with TLR-4 polymorphism have a lower expression of TLR-4 and TLR-2 receptors in peripheral mononuclear cells, which ultimately indicate a less responsiveness for alarmins.
Asunto(s)
Expresión Génica , Trasplante de Riñón/inmunología , Polimorfismo de Longitud del Fragmento de Restricción , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Adulto , Estudios de Casos y Controles , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Genotipo , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Masculino , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. AIM: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. METHODS: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. RESULTS: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-ß protein production was significantly lower in Hemin-treated animals. CONCLUSION: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.
Asunto(s)
Fibrosis/metabolismo , Hemo-Oxigenasa 1/biosíntesis , Hemina/farmacología , Túbulos Renales/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Perfilación de la Expresión Génica , Inmunohistoquímica/métodos , Inflamación , Enfermedades Renales/patología , Masculino , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
B lymphocyte infiltration in renal acute allograft rejection has been associated with steroid resistance and poor outcomes. We aimed to measure CD20 mRNA in urine of renal transplant patients with graft dysfunction and correlate with the histological diagnosis and immunohistochemical (IH) staining for CD20. A total of 48 urine samples were analyzed (21 with acute rejection, 10 with chronic allograft nephropathy, 11 with unspecific tubular lesions, 3 with acute pyelonephritis and 3 with polyomavirus nephropathy). Higher urinary CD20 levels associated with a positive IH staining for CD20 (>50 positive cells/HPF) in renal tissue (p=0.04), with a sensitivity of 83.3% and a specificity of 51.6%. Within the acute rejection group, a positive staining for CD20 was not associated with graft loss, steroid resistance or lack of return to basal creatinine after treatment, but was associated with higher serum creatinine at 3 and 6 months, 1 and 2 years after the acute episode (p<0.05). In conclusion, we showed that urinary levels of CD20 detected by RT-PCR had a high sensitivity for CD20+ staining in the corresponding renal tissue, but with a low specificity. Patients with clusters of CD20+ cells >50/HPF had higher serum creatinine after 2 years of follow up.
Asunto(s)
Antígenos CD20/biosíntesis , Linfocitos B/inmunología , Rechazo de Injerto/orina , Trasplante de Riñón/inmunología , ARN Mensajero/orina , Enfermedad Aguda , Adulto , Linfocitos B/metabolismo , Biomarcadores/orina , Enfermedad Crónica , Creatinina/sangre , Creatinina/orina , Femenino , Rechazo de Injerto/patología , Humanos , Trasplante de Riñón/patología , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Trasplante Homólogo/inmunología , Trasplante Homólogo/patologíaRESUMEN
INTRODUCTION: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. METHODS: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. RESULTS: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. CONCLUSION: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN.
Asunto(s)
Rechazo de Injerto/genética , Trasplante de Riñón , Riñón/inmunología , Receptores Toll-Like/genética , Enfermedad Aguda , Adolescente , Adulto , Enfermedad Crónica , Femenino , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Interleucina-6/genética , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Adulto JovenRESUMEN
Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4(+)CTLA4(+)Foxp3(+) cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion, depletion of TCD4(+)CTLA4(+)Foxp3(+) cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1(anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10+/-50.04; PC61: 187.23+/-31.38; DTA-1: 64.53+/-25.65, mg/dL, p<0.05). These data were corroborated by histopathology. We observed an increase of HO-1 expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61(anti-CD25) mAb treatment is deleterious, while DTA-1(anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI.
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Lesión Renal Aguda/inmunología , Daño por Reperfusión/inmunología , Linfocitos T Reguladores/inmunología , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Animales , Nitrógeno de la Urea Sanguínea , Citometría de Flujo , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/genética , Expresión Génica , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/genética , Riñón/inmunología , Riñón/lesiones , Riñón/patología , Depleción Linfocítica/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Antígeno Nuclear de Célula en Proliferación/genética , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previously we have demonstrated that bradykinin B1 receptor deficient mice (B1KO) were protected against renal ischemia and reperfusion injury (IRI). Here, we aimed to analyze the effect of B1 antagonism on renal IRI and to study whether B1R knockout or antagonism could modulate the renal expression of pro and anti-inflammatory molecules. To this end, mice were subjected to 45 minutes ischemia and reperfused at 4, 24, 48 and 120 hours. Wild-type mice were treated intra-peritoneally with antagonists of either B1 (R-954, 200 microg/kg) or B2 receptor (HOE140, 200 microg/kg) 30 minutes prior to ischemia. Blood samples were collected to ascertain serum creatinine level, and kidneys were harvested for gene transcript analyses by real-time PCR. Herein, B1R antagonism (R-954) was able to decrease serum creatinine levels, whereas B2R antagonism had no effect. The protection seen under B1R deletion or antagonism was associated with an increased expression of GATA-3, IL-4 and IL-10 and a decreased T-bet and IL-1beta transcription. Moreover, treatment with R-954 resulted in lower MCP-1, and higher HO-1 expression. Our results demonstrated that bradykinin B1R antagonism is beneficial in renal IRI.