RESUMO
BACKGROUND: Postoperative intra-abdominal adhesion is associated with high morbidity and mortality. Smad7, a protein that occupies a strategic position in fibrogenesis, inhibits the transforming growth factor (TGF) beta/Smad signalling pathway. In this study the therapeutic potential of exogenous Smad7 in preventing fibrogenesis in postoperative intra-abdominal adhesion was investigated. METHODS: Intra-abdominal adhesion was induced in a rodent model by peritoneal abrasion. Smad7 was delivered into the peritoneal cavity by a non-viral ultrasound-microbubble-mediated naked gene transfection system. The effect of Smad7 transgene on adhesion formation was studied by measuring changes in TGF-beta, fibrogenic factors, alpha-SMA and Smad2/3 activation in the anterior abdominal wall. RESULTS: Four weeks after surgical abrasion, all rats developed significant peritoneal adhesion with enhanced TGF-beta expression, increased levels of extracellular matrix components and activated myofibroblasts, accompanied by decreased Smad7 expression and increased Smad2/3 activation. In rats treated with the Smad7 transgene, the incidence and severity of peritoneal adhesion were significantly reduced, with biochemical downregulation of fibrogenic factors and inhibition of Smad2/3 activation. Serial quantitation using magnetic resonance imaging revealed a significant reduction in adhesion areas from day 14 onwards. CONCLUSION: Ultrasound-microbubble-mediated gene transfection provides timely targeted gene delivery for the treatment of postoperative peritoneal adhesions.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/fisiologia , Doenças Peritoneais/prevenção & controle , Proteína Smad7/administração & dosagem , Aderências Teciduais/prevenção & controle , Transgenes/fisiologia , Parede Abdominal , Animais , Matriz Extracelular/patologia , Imuno-Histoquímica , Masculino , Microbolhas , Doenças Peritoneais/metabolismo , Doenças Peritoneais/patologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad7/genética , Aderências Teciduais/metabolismo , Aderências Teciduais/patologia , Fator de Crescimento Transformador beta/biossíntese , Regulação para CimaRESUMO
Aldehyde dehydrogenase (ALDH) activity is used to define normal hematopoietic stem cell (HSC), but its link to leukemic stem cells (LSC) in acute myeloid leukemia (AML) is currently unknown. We hypothesize that ALDH activity in AML might be correlated with the presence of LSC. Fifty-eight bone marrow (BM) samples were collected from AML (n=43), acute lymphoblastic leukemia (ALL) (n=8) and normal cases (n=7). In 14 AML cases, a high SSC(lo)ALDH(br) cell population was identified (ALDH(+)AML) (median: 14.89%, range: 5.65-48.01%), with the majority of the SSC(lo)ALDH(br) cells coexpressing CD34(+). In another 29 cases, there was undetectable (n=23) or rare (< or =5%) (n=6) SSC(lo)ALDH(br) population (ALDH(-)AML). Among other clinicopathologic variables, ALDH(+)AML was significantly associated with adverse cytogenetic abnormalities. CD34(+) BM cells from ALDH(+)AML engrafted significantly better in NOD/SCID mice (ALDH(+)AML: injected bone 21.11+/-9.07%; uninjected bone 1.52+/-0.75% vs ALDH(-)AML: injected bone 1.77+/-1.66% (P=0.05); uninjected bone 0.23+/-0.23% (P=0.03)) with the engrafting cells showing molecular and cytogenetic aberrations identical to the original clones. Normal BM contained a small SSC(lo)ALDH(br) population (median: 2.92%, range: 0.92-5.79%), but none of the ALL cases showed this fraction. In conclusion, SSC(lo)ALDH(br) cells in ALDH(+)AML might denote primitive LSC and confer an inferior prognosis in patients.
Assuntos
Aldeído Desidrogenase/análise , Leucemia Mieloide/patologia , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Doença Aguda , Adulto , Idoso , Animais , Antígenos CD34/análise , Exame de Medula Óssea , Estudos de Casos e Controles , Feminino , Humanos , Leucemia Mieloide/diagnóstico , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , PrognósticoRESUMO
AIMS: To investigate the association of megsin A23167G polymorphism with susceptibility and progression of IgA nephropathy in Chinese population. METHODS: 435 IgA nephropathy patients and their family members were recruited for a family-based association study. Genotypes of megsin A23167G were determined by direct sequencing. The results were analyzed by transmission disequilibrium test (TDT) and haplotype relative risk (HRR). Clinical data and histological scores of renal lesions were compared between patients with different genotypes. According to disease stability, IgA nephropathy patients were divided into progressive group and stable group. The distribution of genotype frequencies were compared between the 2 groups. RESULTS: TDT revealed that megsin 23167G alleles were transmitted more frequently from heterozygous parents to patients than expected (classical TDT: chi2 = 5.435, p = 0.020, extended TDT: chi2 = 5.017, p = 0.025). HRR analyses showed significant differences between transmitted and nontransmitted allele frequencies (chi2 = 7.006, p = 0.008, HRR = 1.762). The scores of glomerular index and glomerular sclerosis index were higher in GG genotype patients than those in other genotypes (F = 4.570, p = 0.033, F = 4.324, p = 0.038, respectively). The distribution frequency of GG genotype in the progressive group was higher than that of the stable group (chi2 = 4.370, p = 0.037). No statistical difference was found in tubulo-interstitial index, vascular index and clinical characteristics between the 2 groups. CONCLUSION: The polymorphism of megsin A23167G is associated with susceptibility and progression of IgA nephropathy in Chinese population. GG genotype is associated with severe histological lesions and progression of the disease.
Assuntos
DNA/genética , Predisposição Genética para Doença , Glomerulonefrite por IGA/genética , Polimorfismo Genético , Serpinas/genética , Adulto , Alelos , China/epidemiologia , Progressão da Doença , Feminino , Frequência do Gene , Glomerulonefrite por IGA/epidemiologia , Humanos , Masculino , Reação em Cadeia da PolimeraseRESUMO
Studies on the role of Wnt/ß-catenin signaling in different forms of kidney disease have yielded discrepant results. Here, we report the biphasic change of renal ß-catenin expression in mice with overload proteinuria in which ß-catenin was upregulated at the early stage (4 weeks after disease induction) but abrogated at the late phase (8 weeks). Acute albuminuria was observed at 1 week after bovine serum albumin injection, followed by partial remission at 4 weeks that coincided with overexpression of renal tubular ß-catenin. Interestingly, a rebound in albuminuria at 8 weeks was accompanied by downregulated tubular ß-catenin expression and heightened tubular apoptosis. In addition, there was an inverse relationship between Dickkopf-3 (Dkk-3) and renal tubular ß-catenin expression at these time points. In vitro, a similar trend in ß-catenin expression was observed in human kidney-2 (HK-2) cells with acute (upregulation) and prolonged (downregulation) exposure to albumin. Induction of a proapoptotic phenotype by albumin was significantly enhanced by silencing ß-catenin in HK-2 cells. Finally, Dkk-3 expression and secretion was increased after prolonged exposure to albumin, leading to the suppression of intracellular ß-catenin signaling pathway. The effect of Dkk-3 on ß-catenin signaling was confirmed by incubation with exogenous Dkk-3 in HK-2 cells. Taken together, these data suggest that downregulation of tubular ß-catenin signaling induced by Dkk-3 has a detrimental role in chronic proteinuria, partially through the increase in apoptosis.
Assuntos
Apoptose , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Nefropatias/metabolismo , Túbulos Renais/metabolismo , Proteinúria/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular , Quimiocinas , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Nefropatias/genética , Nefropatias/patologia , Túbulos Renais/patologia , Camundongos , Proteinúria/genética , Proteinúria/patologia , beta Catenina/genéticaRESUMO
Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and statins have renoprotective effects. We studied the cellular mechanisms for this effect in adriamycin-treated mice receiving captopril, losartan, simvastatin, or their combinations. The mice developed albuminuria, renal insufficiency, and parenchymal inflammation/fibrosis accompanied by overexpression of intrarenal converting enzyme and angiotensin II. Only captopril consistently improved these abnormalities and reduced the cortical expression of several proinflammatory and profibrotic factors including transforming growth factor-beta (TGF-beta). These effects were independent of blood pressure, accompanied by increased urine N-acetylseryl-aspartyl-lysyl-proline (Ac-SDKP) levels, and the restoration of renal angiotensin-converting enzyme and angiotensin II to baseline levels. Losartan or simvastatin alone or together had no effect, and their addition to captopril did not enhance protection. In vitro, angiotensin II stimulated TGF-beta in renal tubular cells via mitogen-activated protein kinase (MAPK) signaling. Captopril or Ac-SDKP suppressed angiotensin II-induced MAPK activation and TGF-beta secretion. Angiotensin-converting enzyme inhibition confers renoprotection in adriamycin nephropathy by reducing intrarenal angiotensin II and augmenting Ac-SDKP expression that together attenuate MAPK signaling and its downstream proinflammatory and fibrogenic properties. The addition of receptor blocker and/or statin failed to potentiate such effects.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Rim/efeitos dos fármacos , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Colágeno Tipo I/metabolismo , Doxorrubicina/efeitos adversos , Quimioterapia Combinada , Células Epiteliais/efeitos dos fármacos , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Rim/imunologia , Rim/metabolismo , Rim/patologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Peptidil Dipeptidase A/metabolismo , Fenótipo , Fator de Crescimento Placentário , Proteínas da Gravidez/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P<2.2e-16) were found. Marked overexpression of BRE was detected in majority of the tumors, whereas most non-tumoral regions expressed the same low level of the protein as in normal livers. To investigate whether BRE overexpression could promote cell survival in vivo, liver-specific transgenic BRE mice were generated and found to be significantly resistant to Fas-mediated lethal hepatic apoptosis. The transgenic model also revealed post-transcriptional regulation of Bre level in the liver, which was not observed in HCC and non-HCC cell lines. Indeed, all cell lines analysed express high levels of BRE. In conclusion, BRE is antiapoptotic in vivo, and may promote tumorigenesis when overexpressed.
Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Proteínas Reguladoras de Apoptose/fisiologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/imunologiaRESUMO
We hypothesize an interaction between T cells/monocytes and the tubules in the development of tubulointerstitial injury in chronic proteinuric nephropathy. We established in vitro co-culture systems of proximal tubular epithelial cells (PTEC) and T cells/monocytes to study the contribution of soluble factors and cell-to-cell contact in the development of tubulointerstitial injury. The release of monocyte chemoattractant protein-1 (MCP1 or CCL2), Regulated upon Activation, normal T cell Expressed and Secreted (RANTES or CCL5), soluble intracellular adhesion molecules-1 (sICAM-1), or interleukin-6 (IL-6) was increased in PTEC following apical exposure to human serum albumin (HSA). The release of CCL2, CCL5, or sICAM-1 from PTEC was enhanced by contact of monocytes/T cells on the basolateral surface. Tumor necrosis factor-alpha (TNF-alpha) and IL-1beta are important soluble factors as suggested by the blocking effect of antibodies (Abs) against TNF-alpha or IL-1beta but not against other cytokines. The percentage of CD4+ T cells expressing both chemokine receptors, CCR2 and CCR5, was increased after culturing with supernatant from the apical or basolateral surface of PTEC following apical exposure to HSA. However, only CCR2 was upregulated in CD8+ T cells, whereas CCR5 expression was increased in monocytes. The chemotaxis of CD4+ or CD8+ T cells to supernatant from PTEC upon apical exposure to HSA was reduced with neutralizing Abs against CCL5 and/or CCL2, whereas the chemotaxis of monocytes was only reduced by anti-CCL5 but not by anti-CCL2. In summary, chemokines released by HSA-activated PTEC are amplified by monocytes/T cells. Mediators released by HSA-activated PTEC can differentially modulate the expression of chemokine receptors in monocytes/T cells and hence, alter their chemotaxis towards activated PTEC. These interactions are pivotal in the development of tubulointerstitial injury.
Assuntos
Comunicação Celular/fisiologia , Células Epiteliais/patologia , Túbulos Renais Proximais/patologia , Monócitos/patologia , Proteinúria/patologia , Linfócitos T/patologia , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL5 , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Técnicas de Cocultura , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Monócitos/fisiologia , Proteinúria/fisiopatologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Albumina Sérica/farmacologia , Linfócitos T/fisiologiaRESUMO
Gene transfer into the peritoneal cavity by nonviral methods may provide an effective therapeutic approach for peritoneal diseases. Herein, we investigated the feasibility and the effectiveness of ultrasound-microbubble-mediated delivery of naked plasmid DNA into the peritoneal cavity in rats. Following the intraperitoneal or the intravenous administration of a mixture of plasmid DNA (100 microg) and ultrasound contrast agent microbubbles, an ultrasound transducer was applied on the abdominal wall. The reporter pTRE plasmid encoding Smad7 was used to evaluate transfection efficiency. Smad7 expression was induced by doxycycline in drinking water. We detected less than 10% apoptotic cells and no inflammatory reaction in peritoneal tissues following the ultrasound-microbubble-mediated transfection. More importantly, the insonation significantly improved the transfection efficiency in peritoneal tissues. The transfection efficiency by intraperitoneal delivery route was higher than the intravenous route. The reporter gene, pTRE-Smad7, was readily detected in the parietal peritoneum, mesentery, greater omentum and adipose tissue. The peak of transgene expression occurred 2 days after transfection and the transgene expression diminished in a time-dependent manner thereafter. Overall, the effectiveness and simplicity of the ultrasound-microbubble-mediated system may provide a promising nonviral means for improving gene delivery for treating peritoneal diseases in vivo.
Assuntos
DNA/administração & dosagem , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Transfecção/métodos , Albuminas , Animais , Apoptose , Meios de Contraste , Fluorocarbonos , Expressão Gênica , Injeções Intraperitoneais , Injeções Intravenosas , Microbolhas , Peritônio/patologia , Ratos , Ratos Sprague-Dawley , Proteína Smad7/análise , Proteína Smad7/genética , Fatores de Tempo , Transgenes , UltrassomRESUMO
Marked increase in leptin concentration in spent peritoneal dialysate has been reported following continuous ambulatory peritoneal dialysis treatment. The present study was designed to determine whether functional leptin receptor is expressed by human peritoneal mesothelial cells and if so, the possible implication in dialysis. Expression of leptin receptors in cultured mesothelial cells and omental tissue was examined. The effect of leptin on the production of transforming growth factor-beta (TGF-beta) by mesothelial cells in the presence or absence of high glucose was determined using in vitro culture model of human peritoneal mesothelial cells and adipocytes. The signaling mechanism involved in leptin-induced TGF-beta synthesis by mesothelial cells was studied. Both mRNA and protein of the full-length leptin receptor are constitutively expressed in mesothelial cells. The leptin receptor expression in mesothelial cells was upregulated by glucose but not leptin. In adipocytes, glucose increased the mRNA expression and synthesis of leptin. The Janus kinase-signal transducers and activation (JAK-STAT) signal transduction pathway in mesothelial cells was activated by either exogenous or adipocytes-derived leptin. Exogenous leptin induced the release of TGF-beta by mesothelial cells. The TGF-beta synthesis induced by leptin was amplified by glucose through increased leptin receptor expression. Our novel findings reveal that functional leptin receptor is present on human peritoneal mesothelial cells. The leptin-induced TGF-beta synthesis in mesothelial cells is associated with the expression of leptin receptor and the activation of the JAK-STAT signal transduction pathway.
Assuntos
Células Epiteliais/fisiologia , Leptina/fisiologia , Peritônio/citologia , Receptores de Superfície Celular/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Células Cultivadas , Humanos , Receptores para LeptinaRESUMO
We investigated the expression profile of inflammatory cytokines in the lung of lupus-prone MRL/lpr mice and evaluated the therapeutic potential of mycophenolate mofetil (MMF) in reducing pulmonary cytokines in active lupus. Eight-week old female MRL/lpr mice (n = 20) were treated with MMF in vehicle by oral gavage. Disease control MRL/lpr mice (n = 30) or normal control MRL mice (n = 20) received vehicle alone. The mice were sacrificed after eight or 12 weeks of treatment. Gene expression and protein synthesis of IL-1beta, MCP-1 and TGF-beta1 in lung tissues were determined. We found an increase in the gene expression of IL-1beta, MCP-1 and TGF-beta1 in lung tissues of untreated MRL/lpr mice compared with MRL mice at either 16 weeks or 20 weeks of age. MMF treatment significantly prolonged the survival of MRL/lpr mice, down-regulated the gene expression of IL-1beta, MCP-1 and TGF-beta1 in lung tissues at the end of eight or 12 weeks of treatment. Protein synthesis of TGF-beta1 was decreased following eight weeks of MMF treatment. We conclude that MMF treatment can reduce the TGF-beta1 gene expression and protein synthesis in lung tissues of lupus-prone mice. Our findings provide experimental data suggesting a beneficial potential of MMF therapy in pulmonary involvement of lupus.
Assuntos
Imunossupressores/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Ácido Micofenólico/análogos & derivados , Fator de Crescimento Transformador beta/metabolismo , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Feminino , Interleucina-1/genética , Interleucina-1/metabolismo , Lúpus Eritematoso Sistêmico/etiologia , Camundongos , Camundongos Endogâmicos MRL lpr , Ácido Micofenólico/farmacologia , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1RESUMO
OBJECTIVE: To investigate the pathophysiological effect of immunoglobulin G (IgG) from systemic lupus erythematosus (SLE) patients on pleural mesothelial cells and related mechanisms. METHODS: Serum IgG from 28 lupus patients and 13 healthy controls was purified by protein-G affinity chromatography. The concentrations of anti-dsDNA-, anti-histone- and/or anti-nucleohistone-containing IgGs were determined by enzyme-linked immunosorbent assay (ELISA). Lupus patients were divided into an active (n = 12) and an inactive group (n = 16) on the basis of the SLE Disease Activity Index (SLEDAI). The binding of IgG to a human pleural mesothelial cell line (MeT-5A) under different conditions, including pretreatment with DNase and preincubation with exogenous histone, DNA or nucleohistone, was examined using flow cytometry and cellular ELISA. The effect of IgG on MeT-5A cell proliferation was studied using an MTT assay. Gene expression and protein synthesis for interleukin 1beta (IL-1beta), monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor beta1 (TGF-beta1) in MeT-5A cells were determined using reverse transcription-polymerase chain reaction and ELISA. RESULTS: The binding of IgG to MeT-5A cells was higher in the active lupus group than the inactive lupus group (P = 0.047) and controls (P = 0.003). The binding decreased in both lupus groups following pretreatment of MeT-5A cells with DNase. The binding of IgG to MeT-5A cells was greater by 112% in the active lupus group after preincubation with histone (P < 0.001), but not with DNA or nucleohistone. Exposure of MeT-5A cells to IgG from either lupus group induced cell proliferation when compared with IgG from healthy controls (P = 0.04). Gene expression and protein synthesis of MCP-1, TGF-beta1 and IL-1beta in MeT-5A cells were significantly increased after incubation with IgG from patients with active lupus when compared with IgG from the inactive lupus and control groups (P < 0.01). The concentration of anti-dsDNA antibodies correlated with the binding of IgG to MeT-5A cells and the synthesis of cytokines by MeT-5A cells. The serum level of anti-histone antibodies in the active lupus group was higher than that in the inactive group (P = 0.015) and the serum concentration correlated with cell binding and MCP-1 production. CONCLUSIONS: IgG from lupus patients can bind to MeT-5A cells and the binding is modulated by DNA or histone. Binding of anti-dsDNA-containing IgG to MeT-5A cells induces the synthesis of proinflammatory cytokines. Our findings suggest that the binding of anti-dsDNA antibodies, particularly the IgG isotype, to pleural mesothelium plays a direct pathogenetic role in inducing inflammatory injury in the serositis of SLE.
Assuntos
Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Doenças Pleurais/imunologia , Doença Aguda , Adulto , Anticorpos Antinucleares/sangue , Autoanticorpos/sangue , Estudos de Casos e Controles , Divisão Celular , Doença Crônica , Citocinas/biossíntese , Citocinas/genética , Células Epiteliais/imunologia , Feminino , Citometria de Fluxo , Expressão Gênica , Histonas/imunologia , Humanos , Imunoglobulina G/análise , Imunoglobulina G/farmacologia , Masculino , Pleura/imunologiaRESUMO
Although complement is activated in the peritoneal cavity during chronic peritoneal dialysis (PD), little is known about its role in peritoneal defence and injury related to long-term PD. We examined the impact of glucose and commercial peritoneal dialysis solutions on complement expression in HPMCs obtained by primary culture from omental tissues of consented patients undergoing elective abdominal surgery. Constitutive expression of C3 and C4 mRNA in HPMCs was up-regulated upon exposure to 75 mm glucose in a time-dependent manner. C3 and C4 protein was secreted in both apical and basolateral directions. Glucose doses beyond 100 mm markedly down-regulated C3 and C4 expression, and stimulated LDH release dose-dependently. Such cytotoxic effects were attenuated using equivalent doses of mannitol instead of glucose. Treatment with conventional lactate-buffered dialysis solution gave rise to down-regulation of C3 and C4 expression, and heightened LDH release in HPMCs. These effects correlated with the glucose strength of the solution, persisted despite replacement with a bicarbonate-buffered solution, aggravated by glycated albumin, and were partially abrogated by supplementation with 10% fetal bovine serum in the culture system. Our findings suggest that the artificial conditions imposed by PD lead to alterations in local complement synthesis that have implications for the role of the peritoneal mesothelium in both inflammation and defence.
Assuntos
Complemento C3/biossíntese , Complemento C4/biossíntese , Soluções para Diálise/farmacologia , Diálise Peritoneal , Peritônio/imunologia , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Complemento C3/genética , Complemento C4/genética , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Humanos , Manitol/farmacologia , Peritônio/efeitos dos fármacos , RNA Mensageiro/genéticaRESUMO
Autoclaving peritoneal dialysate fluid (PDF) degrades glucose into glucose degradation products (GDPs) that impair peritoneal mesothelial cell functions. While glycation processes leading to formation of advanced glycation end-products (AGE) were viewed commonly as being mediated by glucose present in the PDF, recent evidence indicates that certain GDPs are even more powerful inducers of AGE formation than glucose per se. In the present study, we examined the expression and modulation of AGE receptors on human peritoneal mesothelial cells (HPMC) cultured with GDPs, conventional PDF or PDF with low GDP content. HPMC cultured with GDPs differentially modulated AGE receptors (including RAGE, AGE-R1, AGE-R2 and AGE-R3) expression in a dose-dependent manner. At subtoxic concentrations, GDPs increased RAGE mRNA expression in HPMC. 2-furaldehyde (FurA), methylglyoxal (M-Glx) and 3,4-dideoxy-glucosone-3-Ene (3,4-DGE) increased the expression of AGE-R1 and RAGE, the receptors that are associated with toxic effects. These three GDPs up-regulated the AGE synthesis by cultured HPMC. In parallel, these GDPs also increased the expression of vascular endothelial growth factor (VEGF) in HPMC. PDF with lower GDP content exerted less cytotoxic effect than traditional heat-sterilized PDF. Both PDF preparations up-regulated the protein expression of RAGE and VEGF. However, the up-regulation of VEGF in HPMC following 24-h culture with conventional PDF was higher than values from HPMC cultured with PDF containing low GDP. We have demonstrated, for the first time, that in addition to RAGE, other AGE receptors including AGE-R1, AGE-R2 and AGE-R3 are expressed on HPMC. Different GDPs exert differential regulation on the expression of these receptors on HPMC. The interactions between GDPs and AGE receptors may bear biological relevance to the intraperitoneal homeostasis and membrane integrity.