RESUMEN
Mercuric chloride (HgCl2), which induces kidney toxicity, constitutes a potential threat to human health. In addition to direct toxic effects, kidney inflammatory events take place during the HgCl2-induced nephropathy. There is no information currently available about the role of angiotensin II (Ang II) in this inflammatory process. Accordingly, the aim of this study was to determine the expression of Ang II and Ang II-associated inflammatory molecules, i.e. intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and mono-cyte/macrophage infiltration (ED-1), in HgCl2-induced nephropathy. Three groups of Sprague Dawley rats that were to receive HgCl2 (2.5 mg HgCl2/kg BW, by gavage) were utilized: one had received Losartan at 30 mg/kg BW; one had received Enalapril at 30 mg/kg BW; and one had received distilled water, in each case daily for 3 days prior to the HgCl2 exposure. For these studies, an extra set of controls treated with saline solution in place of HgCl2 and water in place of the test drugs was employed. Renal biopsies were obtained 96 h after HgCl2 injection and the expressions of Ang II, ICAM-1, iNOS, and ED-1 were analyzed by indirect immunoflourescence while tubular damage was assessed via histopathology. An increased expression of Ang II, ICAM-1, iNOS, and ED-1 as well as increases in tubular necrosis were observed in all HgCl2-animals. Treatments with Losartan or Enalapril diminished the induced expressions as well as the extent of tubular damage. The data here suggest that Ang II is involved in the pro-inflammatory events during HgCl2-induced nephropathy, and that this is probably mediated, in part, by Ang II receptors Type 1 (AT-1).
Asunto(s)
Angiotensina II/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Cloruro de Mercurio/toxicidad , Nefritis/inducido químicamente , Nefritis/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/administración & dosificación , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Animales , Movimiento Celular/efectos de los fármacos , Enalapril/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Losartán/administración & dosificación , Macrófagos/efectos de los fármacos , Masculino , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Renal inflammation and oxidative stress are constantly present in experimental hypertension. Since the spontaneously hypertensive rat (SHR) has reduced levels of hepatocyte growth factor (HGF), which suppresses the activation of the proinflammatory nuclear transcription factor kappa B (NF-κB), we speculated that HGF deficiency could play a key role in the pathogenesis of hypertension in the SHR. To test this hypothesis we increased HGF in the SHR by HGF gene delivery. We found that kidneys of 15-week-old SHR had an important reduction in HGF mRNA and protein expression. Adult SHRs were randomly assigned to receive weekly hydrodynamic injection (1mg/kg) of a naked plasmid containing human HGF (hHGF) gene associated with a cytomegalovirus promoter (pCMV-HGF) or empty vector (pcDNA3.1) during 6weeks. WKY rats treated with pcDNA3.1 and pCMV-HGF served as controls. The kidneys in the hypertensive SHR untreated and treated with the empty vector had increased NF-κB activation, elevated mRNA and protein expression of RANTES, MCP-1 and IL-6 and increased oxidative stress. Activity of Na(+)-ATPase was increased while activity of Na(+), K(+)-ATPase was normal. hHGF gene therapy normalized renal NF-κB activity, proinflammatory cytokines, antioxidant status (GSH, SOD and CAT), Na(+)-ATPase activity, reduced renal injury and ameliorated hypertension. Our results suggest that reduction in HGF production plays a major role in the pathogenesis of hypertension in the SHR and increasing HGF is a potential therapeutic target in the treatment of hypertension.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Factor de Crecimiento de Hepatocito/biosíntesis , Hipertensión/genética , Mediadores de Inflamación/metabolismo , Animales , Antioxidantes/metabolismo , Peso Corporal , Quimiocina CCL2/metabolismo , Quimiocina CCL5/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Hipertensión/enzimología , Hipertensión/metabolismo , Inflamación/genética , Inflamación/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Masculino , FN-kappa B/metabolismo , Estrés Oxidativo/genética , ARN Mensajero/genética , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , TransgenesRESUMEN
Hyperglycemia during diabetes is one of the causes of encephalopathy. However, diabetes causes chronic inflammatory complications and among them is peripheral neuropathy. Since, diabetes is one of the major risk factors for cerebrovascular disease, inflammatory process could take place in central nervous system (CNS). To test that hypothesis, experiments to determine inflammatory events in CNS during streptozotocin-induced diabetes were performed. Diabetes was induced by intravenous injection of streptozotocin (STZ). Brain angiotensin II (Ang II), monocyte/macrophage (ED-1 positive cells), CD8, the intercellular adhesion molecule-1 (ICAM-1), the lymphocyte function-associated antigen-1 (LFA-1) and superoxide anion were determined by hystochemical and immunohistochemical methods. Nitric oxide (NO), malondialdehyde (MDA) and catalase activity were measured in brain homogenates by enzymatic and biochemical methods. This research showed increased expressions of Ang II, ICAM-1, LFA-1 and CD8 positive cells in diverse zones of cerebrum and cerebellum of diabetic rats (week 8). Treatment of diabetic animals with losartan or enalapril reduced the expression of those molecules. Values of lipid peroxidation, nitrite content and superoxide anion expression remained similar to control rats. Only decreased activity of catalase was observed in diabetic animals, but losartan or enalapril failed to modify catalase activity. This study suggests the presence of Ang II-mediated brain inflammatory events in diabetes probably mediated by AT1 receptors.
Asunto(s)
Angiotensina II/metabolismo , Encéfalo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Neuropatías Diabéticas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Animales , Glucemia/metabolismo , Antígenos CD8/metabolismo , Catalasa/metabolismo , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Macrófagos/metabolismo , Masculino , Monocitos/metabolismo , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismoRESUMEN
Depression has been associated to inflammatory and oxidative events. Previous report has shown renal oxidative stress in patients with depression. In order to analyze if depressive status is related to renal oxidative and inflammatory events, Sprague Dawley rats were submitted to forced swimming test (FST) and the renal oxidative metabolism, monocyte-macrophage infiltration and Angiotensin II (Ang II) expression were determined. Rats were submitted to FST daily (30 min) for 15 days. Motor activity was analyzed before FST. Kidney sections were homogenized to measure nitric oxide (NO), malondialdehyde (MDA), reduced glutathione (GSH) and catalase activity by enzymatic and biochemical methods. Renal frozen sections were studied for superoxide anion (O2-), monocyte/macrophage infiltration and Ang II expression by histochemical and immunofluorescence methods. In addition, three groups of FST rats were treated with losartan, sertraline or water for 18 days with further renal O2-analysis. In the FST group, struggle time, motor activity, food intake and body weight gain were found decreased. Increased number of glomerular, interstitial and tubular O2-positive cells was observed in FST rats. High renal content of nitrite/nitrate (NO), MDA and decreased amount of GSH were found in FST rats. Values of renal ED-1 or Ang II positive cells in FST rats remained similar to controls; however, AT1 receptor blocking (losartan) and sertraline reduced both depressive-like behavior and renal O2-expression. These data suggests that depression-like behavior in rats is involved in kidney oxidative stress probably mediated by AT1 receptors.
Asunto(s)
Depresión/patología , Depresión/fisiopatología , Riñón/metabolismo , Estrés Oxidativo/fisiología , Angiotensina II/metabolismo , Animales , Modelos Animales de Enfermedad , Glutatión/metabolismo , Riñón/patología , Macrófagos/metabolismo , Masculino , Malondialdehído/metabolismo , Monocitos/metabolismo , Actividad Motora/fisiología , Óxido Nítrico/metabolismo , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismo , Natación/psicología , Factores de TiempoRESUMEN
INTRODUCTION: Nephrotic syndrome induced by adriamycin (ADR) is an experimental model of glomerulosclerosis in humans. The AT(1) receptor for angiotensin II (Ang II) is involved in the renal expression of the nuclear factor-kappa B (NF-ΚB) during this nephrosis. NF-ΚB is a transcription factor for proinflammatory effects of Ang II; however, there is no information about the role of this receptor in the renal proinflammatory events in ADR nephrosis. MATERIALS AND METHODS: To determine the role of Ang II in ADR nephrosis, Sprague-Dawley rats were treated with ADR (6 mg/kg iv). One ADR group received oral losartan treatment (15 mg/kg gavage) 3 days before ADR injection and then daily for 4 weeks, and the other group water. Animals were sacrificed at week 4 and renal macrophage infiltration, ICAM-1, superoxide anion (O(2(-))) and Ang II expressions were analysed by indirect immunofluorescence and histochemical techniques. RESULTS: ADR rats showed increased expression of ICAM-1, Ang II, O(2(-)) and macrophage infiltration, events that were diminished by losartan treatment. Ang II expression remained unaltered after antagonist treatment. Proteinuria was reduced after 3 weeks of treatment. CONCLUSIONS: These data suggest that Ang II plays a role in the inflammatory events during ADR-induced nephrosis, probably mediated by AT(1) receptors.
Asunto(s)
Angiotensina II/metabolismo , Mediadores de Inflamación/metabolismo , Nefrosis/metabolismo , Nefrosis/patología , Animales , Colesterol/sangre , Modelos Animales de Enfermedad , Doxorrubicina , Endotelina-1/metabolismo , Técnica del Anticuerpo Fluorescente , Inflamación/complicaciones , Inflamación/patología , Riñón/efectos de los fármacos , Riñón/patología , Losartán/farmacología , Masculino , Nefrosis/sangre , Nefrosis/inducido químicamente , Proteinuria/sangre , Proteinuria/complicaciones , Proteinuria/patología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Triglicéridos/sangre , Aumento de Peso/efectos de los fármacosRESUMEN
OBJECTIVES: Previous reports have shown that the depressive status in humans and experimental animals is associated with decreased immune response. Since monocyte chemotaxis and expression of CD11a are pivotal mechanisms in immune response, impairment of these events could explain the diminished immune response in depression. METHODS: To test this, rats were submitted to the forced swimming test (FST) for 3 and 15 days. Animals were sacrificed at days 4 (3 days' FST), 16 (15 days' FST) and 30 (15 days' FST and 15 days of recovery time). At these times, a blood sample was obtained for serum and leukocyte isolation. Mononuclear leukocytes were obtained by Histopaque gradient. Chemotaxis responsiveness was determined in Boyden chambers using zymosan-activated rat serum. Cellular CD11a expression and serum CD11a were determined by immunofluorescence and ELISA, respectively. RESULTS: Decreased chemotaxis was observed in FST animals at days 4 and 16 with total recovery at day 30. Diminished expression of cellular CD11a was observed at day 16 and remained decreased at day 30. There were no significant differences in serum CD11a content. CONCLUSION: Decreased chemotactic response and expression of CD11a found in this experimental model of depression could be important mechanisms to induce impairment immune response in experimental and clinical depression.
Asunto(s)
Antígeno CD11a/biosíntesis , Quimiotaxis de Leucocito/inmunología , Tolerancia Inmunológica , Monocitos/inmunología , Animales , Antígeno CD11a/sangre , Antígeno CD11a/genética , Células Cultivadas , Quimiotaxis de Leucocito/genética , Trastorno Depresivo/inmunología , Trastorno Depresivo/metabolismo , Trastorno Depresivo/psicología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/inmunología , Tolerancia Inmunológica/genética , Inmunidad Innata/genética , Masculino , Monocitos/metabolismo , Ratas , Ratas Sprague-Dawley , Estrés Psicológico/inmunología , Estrés Psicológico/metabolismo , Estrés Psicológico/psicología , Natación/psicología , Factores de TiempoRESUMEN
Autonomic and peripheral neuropathies are well-described complications in diabetes. Diabetes mellitus is also associated to central nervous system damage. This little-known complication is characterized by impairment of brain functions and electrophysiological changes associated with neurochemical and structural abnormalities. The purpose of this study was to investigate brain structural and ultrastructural changes in rats with streptozotocin-induced diabetes. Cerebral cortex, hypothalamus, and cerebellum were obtained from controls and 8 weeks diabetic rats. Light and electron microscope studies showed degenerative changes of neurons and glia, perivascular and mitochondrial swelling, disarrangement of myelin sheath, increased area of myelinated axons, presynaptic vesicle dispersion in swollen axonal boutoms, fragmentation of neurofilaments, and oligodendrocyte abnormalities. In addition, depressive mood was observed in diabetic animals. The brain morphological alterations observed in diabetic animals could be related to brain pathologic process leading to abnormal function, cellular death, and depressive behavioral.
Asunto(s)
Cerebelo/ultraestructura , Corteza Cerebral/ultraestructura , Diabetes Mellitus Experimental/patología , Hipotálamo/ultraestructura , Animales , Apoptosis , Axones/patología , Depresión , Etiquetado Corte-Fin in Situ , Masculino , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Dilatación Mitocondrial , Vaina de Mielina/ultraestructura , Oligodendroglía/ultraestructura , Ratas , Ratas Sprague-Dawley , Sinapsis/ultraestructuraRESUMEN
Depression is frequently observed among patients with diabetes and depressive status has been associated to activation of inflammatory processes, suggesting a role of depression in the inflammatory events observed in diabetes. To test that proposal, it was studied the effect of depression induced by forced swimming test (FST) on the evolution of early diabetic nephropathy. Diabetes was induced by streptozotocin injection. Rats were submitted to FST for 15 days. Struggle time was determined during FST and motor activity previously to FST. Nitric oxide, malondialdehyde, reduced glutathione and catalase activity were measured in kidney homogenates by enzymatic and biochemical methods. Superoxide anion, monocyte/macrophage (ED-1 positive cells) and RAGE were determined by histochemical and immunohistochemical methods. Diabetic rats had decreased struggle time and locomotor activity at day 1 of FST. Both control and diabetic rats had those parameters decreased at day 15. Renal oxidative stress, RAGE expression and ED-1 cells were observed increased in diabetic animals. Those parameters were not significantly altered by FST. The depressive status does not alter oxidative and immune parameters during the early renal changes of diabetic nephropathy.
Asunto(s)
Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/psicología , Nefropatías Diabéticas/inmunología , Nefropatías Diabéticas/psicología , Riñón/inmunología , Animales , Citocinas/sangre , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/patología , Técnica del Anticuerpo Fluorescente , Glutatión/metabolismo , Peroxidación de Lípido/inmunología , Macrófagos/inmunología , Masculino , Malondialdehído/metabolismo , Monocitos/inmunología , Motivación , Actividad Motora/fisiología , Estrés Oxidativo/inmunología , Psiconeuroinmunología , Ratas , Ratas Sprague-Dawley , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/metabolismo , Superóxidos/metabolismoRESUMEN
Previous reports have shown that angiotensin II and oxidative stress may be important features in acute poststreptococcal glomerulonephritis (APSGN) and that streptococcal erythrogenic toxin type B (ETB) and its precursor (ETBP) may have an important role in the pathogenesis of APSGN. The aim of this study was to determine the effect of ETBP on the production of angiotensin II and oxidative stress in rat mesangial cells and human mononuclear leukocytes. Mesangial cells and leukocytes were isolated from digested glomeruli and by histopaque gradient, respectively, while ETBP was isolated from nephritogenic streptococcus cultures using a cation exchange column. Angiotensin II was determined by an enzyme-linked immunosorbent assay and by cytometrics. Superoxide anion, reduced glutathione, nitrites, lipid peroxidation and catalase activity were determined by cytochemical, biochemical and enzymatic assays. Inducible nitric oxide synthase expression was determined by cytometrics. An increased production of angiotensin II was observed in ETBP-treated mesangial cell and leukocyte cultures. The ETBP induced an elevated production of superoxide anions and nitrites in mesangial cells and superoxide anions in leukocytes, while this streptococcal protein decreased the expression of inducible nitric oxide synthase in leukocytes. The ETBP was capable of inducing an increased production of angiotensin II and increased oxidative stress, both of which may be important mediators of inflammatory events in the renal tissue and during APSGN.
Asunto(s)
Angiotensina II/biosíntesis , Proteínas Bacterianas/farmacología , Exotoxinas/farmacología , Mesangio Glomerular/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Animales , Catalasa/metabolismo , Separación Celular , Células Cultivadas , Mesangio Glomerular/metabolismo , Mesangio Glomerular/patología , Glutatión/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Peroxidación de Lípido/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Melatonin seems to be an important stimulatory factor of the immune system. This indolamine is capable of inducing activation of leukocytes. Tissue leukocyte infiltration is a key feature of inflammatory and immune responses; however, there is no information about the effect of melatonin on leukocyte chemotaxis. Therefore, the aim of this study was to examine the in vitro and in vivo effects of melatonin on leukocyte chemotaxis, on modulation of leukocyte chemotaxis to other chemoattractants and on the in vivo induction of leukocyte chemokines. Neutrophils and mononuclear leukocytes (PBMC) were isolated by a discontinuous gradient on Hystopaque. Chemotaxis was performed in blind well Boyden's chambers. In vivo chemotaxis was determined after intraperitoneal injection of melatonin into rats. Leukocyte chemotactic response and leukocyte chemokine expression were determined in human volunteers treated with 20 mg daily of melatonin. Increased neutrophils and PBMC chemotaxis in response to 1.2 nm melatonin was observed in vitro. Peritoneal leukocytes were found increased after melatonin injection. Humans treated with melatonin showed an increased neutrophil chemotactic response to a physiological chemoattractant and increased expression of intracellular chemokines; however, decreased chemotactic response and no chemokine expression were observed in PBMC. These data suggest that melatonin could have a relevant role during the tissue leukocyte infiltration in inflammatory and immune responses.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Leucocitos/citología , Leucocitos/efectos de los fármacos , Melatonina/farmacología , Adulto , Animales , Humanos , Masculino , Persona de Mediana Edad , Ratas , Ratas Sprague-DawleyRESUMEN
Previous reports have shown the presence of streptococcal erythrogenic exotoxin type B (ETB), leukocyte infiltration, interleukin-8 (IL-8), transforming growth factor-beta (TGF-beta) and glomerular proliferation in renal biopsies from patients with acute post-streptococcal glomerulonephritis (APSGN). In addition, increased levels of plasma interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha), and urinary IL-6, have also been reported in this disease. To determine the effect of streptococcal proteins on leukocyte proliferation and leukocyte production of IL-6, TNFalpha, IL-8 and TGF-beta1, we cultured human mononuclear leukocytes with ETB or ETB precursor (ETBP). After 24 h, 48 h and 96 h, culture supernatants were assessed for cytokines by enzyme-linked immunosorbent assay (ELISA), and for leukocyte proliferation by a monoclonal antibody anti-proliferating cellular nuclear antigen (PCNA). A significant increase in all cytokines was found in ETB- or ETBP-treated cultures when compared with controls. A polyclonal anti-ETB antibody diminished the cytokine stimulatory effect of ETB. An increased number of PCNA-positive cells was observed in ETB or ETBP treated cultures at 48 h and 96 h. Cytokine production and proliferation were not correlated. The stimulatory effect of streptococcal exotoxin B on leukocyte cytokine production may be relevant in renal tissue during the course of APSGN.
Asunto(s)
Proteínas Bacterianas/farmacología , Exotoxinas/farmacología , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Factor de Crecimiento Transformador beta1/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Células Cultivadas , HumanosRESUMEN
Acute poststreptococcal glomerulonephritis (APSGN) is a consequence of the immune response to streptococcal antigens with further in situ antigen-antibody interaction and deposition of circulating immune complexes, resulting in the activation of complement and the inflammatory process. These events are related to a previous antibody response. However, early renal events, when circulating streptococcal antigens bind to the kidney during streptococcal infection, remain unknown. Cationic streptococcal erythrogenic toxin type B (ETB) and its precursor (ETBP) are largely produced by nephritogenic streptococci and have high affinity for anionic glomerular structures. Renal deposition of ETB/ETBP makes conceivable a possible interaction between these streptococcal proteins with intrinsic glomerular cells or infiltrating leukocytes. Since ETB/ETBP are chemotactic for leukocytes and capable of inducing proliferation, cytokine and chemokine production, expression of adhesion molecules and apoptosis in renal cells and leukocytes, the early presence of these proteins could be a relevant event before and during antigen-antibody interaction takes place in renal tissues.
Asunto(s)
Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Exotoxinas/inmunología , Exotoxinas/metabolismo , Glomerulonefritis/microbiología , Inflamación/fisiopatología , Infecciones Estreptocócicas/complicaciones , Enfermedad Aguda , Apoptosis , Adhesión Celular , Proliferación Celular , Quimiotaxis , Citocinas/metabolismo , Glomerulonefritis/inmunología , Humanos , Riñón/química , Riñón/patología , Infecciones Estreptocócicas/inmunologíaRESUMEN
BACKGROUND/AIMS: Previous reports have shown the presence of streptococcal erythrogenic toxin type B (ETB), IL-8, transforming growth factor-beta (TGF-beta) and glomerular proliferation in renal biopsies from patients with acute poststreptococcal glomerulonephritis (APSGN). In addition, increased levels of plasma IL-6 and tumor necrosis factor-alpha (TNFalpha) and urinary IL-6 have also been reported in this disease. To determine the effect of ETB in mesangial cell cytokine production and proliferation, the concentration of several cytokines (IL-6, IL-1beta, TNFalpha, IL-10, IL-4, RANTES), soluble TNF receptor I (STNFR-I), soluble TNF receptor II (STNFR-II) and proliferation were measured in rat mesangial cells cultures after treatment with ETB or its precursor (ETBP). METHODS: To analyze the levels of cytokines and production of soluble receptors as well as proliferation, rat mesangial cells were cultured with ETB or ETBP (50 microg/ml). After 24, 48 and 96 h of incubation, culture supernatants were assessed for cytokines and receptors by ELISA and for proliferation by incorporation of radioactive thymidine. RESULTS: A significant increase in IL-6 levels was found in mesangial cell cultures treated with either ETBP or ETB when compared with controls. Streptococcal proteins treated mesangial cells also showed elevated levels of proliferation at 96 h. Increased production of IL-6 was not correlated with proliferation. A polyclonal anti-ETB antibody abolished the IL-6 stimulatory effect of ETB on mesangial cells. ETB/ETBP failed to increase the levels of other cytokines and cytokine soluble receptors. CONCLUSION: Streptococcal ETB/ETBP is capable of inducing increased production of IL-6 and proliferation on mesangial cells. These findings could be relevant in a possible early interaction of streptococcal proteins with mesangial cells and during the course of APSGN.
Asunto(s)
Proteínas Bacterianas/farmacología , Exotoxinas/farmacología , Interleucina-6/análisis , Células Mesangiales/química , Células Mesangiales/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/análisis , Células Mesangiales/citología , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Necrosis Tumoral/análisisRESUMEN
Increased apoptosis has been reported in acute puromycin aminonucleoside nephrosis (PAN). The aim of this study was to investigate if increased apoptosis is related to increased expression of apoptosis-associated proteins (AAP) in this model of nephrosis. Sprague-Dawley rats were made nephrotic by intraperitoneal injection of one dose of puromycin aminonucleoside. Renal tissues were obtained at 1, 2 and 7 weeks after injection and apoptosis was investigated by TUNEL and by electron microscopy. Fas, Fas ligand, p53, Bax and Bcl-2 expressions were analyzed by the respective monoclonal and polyclonal antibodies, using indirect immunofluorescence. In the glomerulus of nephrotic animals, increased apoptosis was accompanied with increased expression of p53, Fas and Bax. In the interstitium, high expression of apoptosis, Fas, Fas-L and Bax were observed and in tubules increased apoptosis was accompanied with increased expression of p53, Fas and Fas-L. Bcl-2 was increased in interstitium and tubules during PAN. The incidence of apoptosis during PAN was correlated with the expression of AAP in glomerulus (p53), interstitium (Fas, Fas-L and Bax) and tubules (Fas, Fas-L, p53 and Bcl-2). There was correlation between Fas and Fas-L expression in interstitium and tubules. About 4% of glomerular and 25% of tubular p53 positive cells were apoptotic cells. The data suggest that increased local expression of AAP could contribute to renal apoptosis in the glomerular, interstitial and tubular compartments during this experimental model of nephrosis.
Asunto(s)
Apoptosis , Nefrosis/metabolismo , Nefrosis/patología , Animales , Apoptosis/fisiología , Modelos Animales de Enfermedad , Proteína Ligando Fas , Masculino , Glicoproteínas de Membrana/metabolismo , Nefrosis/inducido químicamente , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Puromicina Aminonucleósido , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2 , Receptor fasRESUMEN
Accumulating evidence demonstrates that oxidative stress is one of the underlying mechanisms to induce apoptosis in different biological systems. The aim of this study was to examine the simultaneous presence and correlation between oxidative stress events, apoptosis, apoptosis-associated proteins and monocyte/macrophage infiltration during the course of acute puromycin aminonucleoside nephrosis (PAN). To induce nephrosis, Sprague-Dawley rats were injected intraperitoneally with puromycin aminonucleoside and killed at weeks 1 and 2 of nephrosis. Controls represent animals injected with 0.9% saline solution. Kidney sections were homogenized to measure nitric oxide (NO), malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase activities by appropriate enzymatic and biochemical methods. Renal frozen sections were studied for superoxide anion (O(2) (-)) by a histochemical method, for apoptosis by TUNEL (terminal-deoxynucleotidyl-transferase-mediated dUTP- digoxigenin nick end labelling) and for apoptosis-associated protein expression and monocyte/macrophage infiltration by monoclonal antibodies. Increased renal apoptosis, p53, Bax, Bcl-2 accompanied by increased O(2) (-) and NO generation, lipid peroxidation (MDA) and monocyte/macrophage infiltration were found in nephrotic animals. Renal oxidative stress (O(2) (-), NO and MDA) was correlated with apoptosis, p53 expression, monocyte/macrophage cells and proteinuria. Anti-oxidant molecules (SOD and GSH) remained unchanged apart from a decreased activity of catalase which correlated with glomerular apoptosis. In conclusion, the close correlation between the presence of apoptosis and oxidative events confirms the role of oxidative stress in the apoptosis observed during PAN.
Asunto(s)
Apoptosis , Nefrosis/fisiopatología , Estrés Oxidativo , Animales , Riñón/metabolismo , Macrófagos/patología , Masculino , Monocitos/patología , Nefrosis/inducido químicamente , Nefrosis/patología , Proteinuria/inducido químicamente , Proteinuria/fisiopatología , Puromicina Aminonucleósido , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: The anti-apoptotic properties of melatonin have been demonstrated previously in several in vivo and in vitro studies. Previous reports have shown increased apoptosis during puromycin aminonucleoside nephrosis (PAN). The aim of this study was to determine if melatonin (MEL) can prevent apoptosis and modify oxidative stress, an apoptosis inducer, in this experimental model. METHODS: Rats were injected intraperitoneally with puromycin aminonucleoside. In addition, by the intragastric route they received 1 mg/kg/day of MEL or vehicle 3 days before puromycin injection and throughout the experiment. Animals were sacrificed at weeks 1 and 2 of nephrosis and frozen renal sections were studied for apoptosis by TUNEL, for apoptosis-associated proteins by monoclonal and polyclonal antibodies, and for superoxide anion (O(2)(-)) by a histochemical method. Nitric oxide (NO), malondialdehyde (MDA) and reduced glutathione (GSH), and the activities of superoxide dismutase (SOD) and catalase were measured in homogenized kidney tissue by appropriate biochemical and enzymatic methods. RESULTS: Increases in apoptosis, p53, Fas and Fas-ligand were observed in nephrotic animals. MEL treatment decreased apoptosis at weeks 1 and 2 in the glomerular, interstitial and tubular compartments. This was accompanied by decreased expression of p53 (glomerulus, week 1; tubules, weeks 1 and 2), Fas (glomerulus and interstitium, week 2; tubules, weeks 1 and 2) and Fas-ligand (interstitum and tubules, week 2). Increased expression of Bcl-2-positive cells was observed at week 2 in all renal compartments in MEL-treated animals. High levels of O(2)(-) and NO generation and lipid peroxidation (MDA) were found in nephrotic animals. SOD and GSH remained unchanged, and only decreased catalase activity (week 1) was observed in PAN animals. Tendencies toward decreased values of O(2)(-) and MDA content along with recovery of catalase activity (week 1) were observed in MEL-treated nephrotic animals, but were insignificant in magnitude. MEL, however, did significantly downregulate pro-apoptotic genes and upregulated anti-apoptotic genes. CONCLUSIONS: The data demonstrate that, in PAN, melatonin has anti-apoptotic effects, which might in part be independent of the modulation of the oxidative status.
Asunto(s)
Apoptosis/fisiología , Enfermedades Renales/inducido químicamente , Melatonina/farmacología , Puromicina Aminonucleósido/toxicidad , Animales , Apoptosis/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Enfermedades Renales/patología , Enfermedades Renales/fisiopatología , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Cellular infiltration to renal tissues is an important feature during acute puromycin aminonucleoside nephrosis (PAN) in rats. The mechanisms responsible for this infiltration are poorly understood. To elucidate the participation of adhesion molecules in PAN, nephrosis was induced in rats by intraperitoneal puromycin aminonucleoside injection. Controls represent animals injected with a 0.9% saline solution. ICAM-1 (intercellular adhesion molecule 1), CD18 (beta chain of lymphocyte-function-associated antigen), LCA (leukocyte common antigen), ED1 (monocyte/macrophage marker), and proliferating cell nuclear antigen expressions were evaluated in renal tissues 1, 2, and 7 weeks after injection. Frozen sections from PAN rat kidneys showed increased expressions of ICAM-1 and its ligand, and these findings were associated with increased levels of LCA+ and ED1+ cells in glomerulus and interstitium. The kinetics of leukocyte infiltration was similar to the kinetics of ICAM-1 expression: high values at week 2 which returned to normal values at week 7. Increased glomerular and interstitial proliferative activities (proliferating cell nulear antigen positive cells) were also found at week 2 of nephrosis. There was a correlation between ICAM-1 expression and numbers of LCA+ and ED1+ cells and between numbers of LCA+ cells and proliferating cells in glomerulus and interstitium. Correlations between glomerular and tubular ICAM-1 expression, interstitial leukocyte infiltration, and glomerular, interstitial, and tubular proliferative activities with the proteinuria were also observed during the nephrotic phase. In addition, increased lymphocyte binding to PAN renal tissues was observed, and this binding was diminished by anti-LFA-1beta monoclonal antibody pretreatment of lymphocytes. A similar result was found with anti-ICAM-1 monoclonal antibody pretreatment of renal tissues. Our results suggest that increased expression of ICAM-1 and proliferative activity could be important determinants in the renal hypercellularity found in this experimental model.
Asunto(s)
Antígenos CD18/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Molécula 1 de Adhesión Intercelular/biosíntesis , Nefrosis/inducido químicamente , Nefrosis/metabolismo , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Puromicina Aminonucleósido/farmacología , Enfermedad Aguda , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD18/inmunología , Antígenos CD18/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Glomérulos Renales/fisiopatología , Antígenos Comunes de Leucocito/biosíntesis , Masculino , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Previous reports have demonstrated the presence of streptococcal erythrogenic toxin type B (ETB) as well as proliferation and expression of adhesion molecules along with leukocyte infiltrations in biopsies from patients with acute post-streptococcal glomerulonephritis (APSGN). The purpose of the present study was to correlate infiltrative and proliferative events with interactions between ETB or its precursor (ETBP) and intrinsic mesangial cells. METHODS: Rat mesangial cells were cultured with ETB or ETBP (50 micro g/ml) while measuring production of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) and while examining proliferation and expression of intercellular adhesion molecule-1 (ICAM-1). After 24, 48 and 96 h of incubation, MCP-1 and MIP-2 in culture supernatants were assessed by enzyme-linked immunosorbent assay (ELISA). Cells were assessed for proliferation by incorporation of radioactive thymidine and expression of ICAM-1 was measured by indirect immunofluorescence and by cellular ELISA. RESULTS: Compared with controls, treatment with either ETBP or ETB significantly increased MCP-1 and MIP-2 levels in mesangial cell cultures. Mesangial cells also showed elevated proliferation at 96 h of culture when treated with streptococcal proteins. Although production of MCP-1 and MIP-2 was not correlated with proliferation, treatment with ETBP resulted in a significant correlation between MCP-1 production and proliferation. Immunofluorescence studies revealed an increased expression of ICAM-1 in ETBP/ETB-treated mesangial cells. In addition, cellular ELISA studies showed increased absorbance in cultures treated with ETBP/ETB. Finally, low serum concentrations in the culture medium potentiated the stimulatory effect of ETB on MCP-1 production. CONCLUSIONS: Our findings, by demonstrating a role for cationic streptococcal ETB or ETBP in the induction of chemotactic molecules as well as the proliferation and expression of adhesion molecules, delineate an additional possible pathway for the pathogenesis of APSGN.