Objective: Aquaporin-1 (AQP1) is expressed in the heart and it has been reported to transport nit... more Objective: Aquaporin-1 (AQP1) is expressed in the heart and it has been reported to transport nitric oxide (NO), an important regulator of cardiac function. Our aim was to study AQP1 abundance and localization, NO synthase (NOS) activity and AQP1 nitrosylation in response to osmotic stress induced by water restriction during postnatal growth. Design and method: Male Sprague-Dawley rats aged 25 and 50 days (n = 10) were divided in: R: water restriction 3 days; C: water ad libitum 3 days. NOS activity (14C-Arginine), AQP1 protein levels (Western Blot) and localization (immunohistochemistry) and AQP1 nitrosylation (colocalization of immunofluorescence signals of AQP1 and nitrosylated cysteine by confocal microscopy) were determined in cardiac tissue. We also evaluated the effects of NO donor sodium nitroprusside (SNP) on osmotic water permeability of cardiac membrane vesicles expressing AQP1 by stopped-flow spectrometry. Results: Water restriction induced a dehydration state in both age groups. Cardiac AQP1 was localized in the endocardium and endothelium in both age groups in control animals. Water restriction did not change AQP1 abundance or localization in the 25-day-old R group; however, in the 50-day-old group, AQP1 protein levels were increased and immunohistochemistry showed its localization on cardiomyocyte plasma membrane after water restriction. Cardiac NOS activity was increased in the youngest R group but it did not change in the 50-day-old R group. AQP1 nitrosylation was increased in R25 group, whereas there are no significant differences in colocalization of fluorescent signals between C or R animals aged 50 days. On the other hand, cardiac membrane vesicles expressing AQP1 presented a high water permeability coefficient (Pf: 326+/-17 &mgr;m/s, n = 6) indicated water transport by aquaporins and pretreatment with SNP decreased water permeability (Pf: 102+/-4 &mgr;m/s, n = 5). Conclusions: Cardiac NO system and AQP1 abundance and localization during osmotic stress in vivo depend on postnatal age. Increased activity of cardiac NO system in the youngest group may induce AQP1 nitrosylation, decreasing osmotic water permeability of cardiac membranes and having a negative impact on cardiac water balance. In the 50-day-old group, changes in AQP1 abundance and localization may contribute to maintaining cardiac water homeostasis during hypovolemic state.
The syncytiotrophoblast (SCT), a multinucleated epithelium forming the outer layer of chorionic v... more The syncytiotrophoblast (SCT), a multinucleated epithelium forming the outer layer of chorionic villi, acts in human placenta as a transporting barrier regulating the transference of nutrients, solutes and water between maternal and fetal blood. Electrolyte homeostasis and extracellular fluid volume are maintained primarily by regulated Na+ transport. The present study was conducted to analyze the presence of the epithelial Na channel (ENaC) in placental tissue from normal and pre-eclamptic women and in BeWo cell, a model of a human SCT. Changes in the expression of these proteins during sodium transport across the placenta may be related to the pathogeny of pre-eclampsia. The role that ENaC and Na+ transport deregulation play on human placental tissues still remains unknown although in aldosterone-responsive epithelial cells (kidney, colon), abnormalities upregulating its activity lead to increased Na+ uptake and hypertension (i.e. Liddle's syndrome) whereas a diminished channel activity can result in the pseudohypoaldosteronisn syndrome with salt loss and hypotension. Our results show that ENaC is expressed in the apical membrane of normal syncytiotrophoblast. The amplified fragment of alpha-ENaC was cloned and sequenced having a 100% identity with the sequence of (alpha-ENaC obtained from GenBank (SCNN1A, accession number Z92981). We found that the transcription of the alpha-ENaC mRNA was not detectable in preeclamptic placentas and the protein was not observed with immunohistochemistry staining, probably indicating a low protein expression level. In BeWo cells ENac was found and its expression is regulated by aldosterone, vasopressin, progesterone and estradiol. With patch clamp techniques we studied the currents trough ENaO channels in Bewo cells. We observed currents that were blocked by 10 microM amiloride in cells incubated in 100 nM aldosterone for 12 hs. The amplitude of this current was 20-fold the basal current, a reversal potential of 3 mV and a conductance of 127 +/- 26 pS/pF with pulses between -60 and -140 mV. These characteristics are similar to those reported in ENaC channels in several tissues. Although their roles in placenta are still poorly understood, the differences in the expression of ENaC in pre-eclamptic placentas may have consequences for ion transport and these data could lead to future studies concerning the mechanism involved in the pathophysiology of pre-eclampsia.
We have previously described that the GH-induced JAK2/STAT5 signalling pathway is desensitized in... more We have previously described that the GH-induced JAK2/STAT5 signalling pathway is desensitized in the liver of transgenic mice overexpressing GH. Therefore, in the present study we have evaluated other GH signalling pathways that could be activated in GH-transgenic mice liver. Animals ...
Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical prese... more Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence.
Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by he... more Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by hemolytic anemia, thrombocytopenia and renal failure. The inflammatory response mediated by polymorphonuclear neutrophils (PMNs) and monocytes is essential to HUS onset. Still, the role of anti-inflammatory cytokines is less clear. The deficiency of IL-10, an anti-inflammatory cytokine, leads to severe pathology in bacterial infections but also to beneficial effects in models of sterile injury. The aim of this work was to analyze the role of IL-10 during HUS. Control and IL-10 lacking mice (IL-10−/−) were intravenously injected with Stx type 2 (Stx2) and survival rate was evaluated. PMN and circulating and renal pro- and anti-inflammatory factors were analyzed by FACS and enzyme-linked immunosorbent assay (ELISA) respectively. IL-10−/− mice showed a higher survival associated with lower renal damage reflected by reduced plasma urea and creatinine levels than control mice. Circulating PMN i...
Background. Despite enzyme replacement therapy, Fabry nephropathy still progresses. Podocyturia i... more Background. Despite enzyme replacement therapy, Fabry nephropathy still progresses. Podocyturia is an irreversible event that antedates proteinuria and leads to chronic renal failure. We evaluated a potential mechanism of podocyte detachment via the expression of the urokinase-type Plasminogen Activator Receptor (uPAR) in urinary podocytes of Fabry patients.Methods. This is a cross-sectional study that included controls (n=20) and Fabry patients (n=44) either untreated (n=23) or treated with agalsidase-β(n=21).Variables. Variables are estimated glomerular filtration rate (eGFR), urinary protein : creatinine ratio, and urinary uPAR+ podocyte : creatinine ratio. uPAR mRNA expression in response to lyso-Gb3, a bioactive glycolipid accumulated in Fabry disease, was studied in cultured human podocytes.Results. Controls and Fabry patients had similar age, gender, and renal function. Urinary uPAR+ podocytes were higher in patients than in controls. Untreated patients were significantly you...
Objective: Aquaporin-1 (AQP1) is expressed in the heart and it has been reported to transport nit... more Objective: Aquaporin-1 (AQP1) is expressed in the heart and it has been reported to transport nitric oxide (NO), an important regulator of cardiac function. Our aim was to study AQP1 abundance and localization, NO synthase (NOS) activity and AQP1 nitrosylation in response to osmotic stress induced by water restriction during postnatal growth. Design and method: Male Sprague-Dawley rats aged 25 and 50 days (n = 10) were divided in: R: water restriction 3 days; C: water ad libitum 3 days. NOS activity (14C-Arginine), AQP1 protein levels (Western Blot) and localization (immunohistochemistry) and AQP1 nitrosylation (colocalization of immunofluorescence signals of AQP1 and nitrosylated cysteine by confocal microscopy) were determined in cardiac tissue. We also evaluated the effects of NO donor sodium nitroprusside (SNP) on osmotic water permeability of cardiac membrane vesicles expressing AQP1 by stopped-flow spectrometry. Results: Water restriction induced a dehydration state in both age groups. Cardiac AQP1 was localized in the endocardium and endothelium in both age groups in control animals. Water restriction did not change AQP1 abundance or localization in the 25-day-old R group; however, in the 50-day-old group, AQP1 protein levels were increased and immunohistochemistry showed its localization on cardiomyocyte plasma membrane after water restriction. Cardiac NOS activity was increased in the youngest R group but it did not change in the 50-day-old R group. AQP1 nitrosylation was increased in R25 group, whereas there are no significant differences in colocalization of fluorescent signals between C or R animals aged 50 days. On the other hand, cardiac membrane vesicles expressing AQP1 presented a high water permeability coefficient (Pf: 326+/-17 &mgr;m/s, n = 6) indicated water transport by aquaporins and pretreatment with SNP decreased water permeability (Pf: 102+/-4 &mgr;m/s, n = 5). Conclusions: Cardiac NO system and AQP1 abundance and localization during osmotic stress in vivo depend on postnatal age. Increased activity of cardiac NO system in the youngest group may induce AQP1 nitrosylation, decreasing osmotic water permeability of cardiac membranes and having a negative impact on cardiac water balance. In the 50-day-old group, changes in AQP1 abundance and localization may contribute to maintaining cardiac water homeostasis during hypovolemic state.
The syncytiotrophoblast (SCT), a multinucleated epithelium forming the outer layer of chorionic v... more The syncytiotrophoblast (SCT), a multinucleated epithelium forming the outer layer of chorionic villi, acts in human placenta as a transporting barrier regulating the transference of nutrients, solutes and water between maternal and fetal blood. Electrolyte homeostasis and extracellular fluid volume are maintained primarily by regulated Na+ transport. The present study was conducted to analyze the presence of the epithelial Na channel (ENaC) in placental tissue from normal and pre-eclamptic women and in BeWo cell, a model of a human SCT. Changes in the expression of these proteins during sodium transport across the placenta may be related to the pathogeny of pre-eclampsia. The role that ENaC and Na+ transport deregulation play on human placental tissues still remains unknown although in aldosterone-responsive epithelial cells (kidney, colon), abnormalities upregulating its activity lead to increased Na+ uptake and hypertension (i.e. Liddle's syndrome) whereas a diminished channel activity can result in the pseudohypoaldosteronisn syndrome with salt loss and hypotension. Our results show that ENaC is expressed in the apical membrane of normal syncytiotrophoblast. The amplified fragment of alpha-ENaC was cloned and sequenced having a 100% identity with the sequence of (alpha-ENaC obtained from GenBank (SCNN1A, accession number Z92981). We found that the transcription of the alpha-ENaC mRNA was not detectable in preeclamptic placentas and the protein was not observed with immunohistochemistry staining, probably indicating a low protein expression level. In BeWo cells ENac was found and its expression is regulated by aldosterone, vasopressin, progesterone and estradiol. With patch clamp techniques we studied the currents trough ENaO channels in Bewo cells. We observed currents that were blocked by 10 microM amiloride in cells incubated in 100 nM aldosterone for 12 hs. The amplitude of this current was 20-fold the basal current, a reversal potential of 3 mV and a conductance of 127 +/- 26 pS/pF with pulses between -60 and -140 mV. These characteristics are similar to those reported in ENaC channels in several tissues. Although their roles in placenta are still poorly understood, the differences in the expression of ENaC in pre-eclamptic placentas may have consequences for ion transport and these data could lead to future studies concerning the mechanism involved in the pathophysiology of pre-eclampsia.
We have previously described that the GH-induced JAK2/STAT5 signalling pathway is desensitized in... more We have previously described that the GH-induced JAK2/STAT5 signalling pathway is desensitized in the liver of transgenic mice overexpressing GH. Therefore, in the present study we have evaluated other GH signalling pathways that could be activated in GH-transgenic mice liver. Animals ...
Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical prese... more Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence.
Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by he... more Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by hemolytic anemia, thrombocytopenia and renal failure. The inflammatory response mediated by polymorphonuclear neutrophils (PMNs) and monocytes is essential to HUS onset. Still, the role of anti-inflammatory cytokines is less clear. The deficiency of IL-10, an anti-inflammatory cytokine, leads to severe pathology in bacterial infections but also to beneficial effects in models of sterile injury. The aim of this work was to analyze the role of IL-10 during HUS. Control and IL-10 lacking mice (IL-10−/−) were intravenously injected with Stx type 2 (Stx2) and survival rate was evaluated. PMN and circulating and renal pro- and anti-inflammatory factors were analyzed by FACS and enzyme-linked immunosorbent assay (ELISA) respectively. IL-10−/− mice showed a higher survival associated with lower renal damage reflected by reduced plasma urea and creatinine levels than control mice. Circulating PMN i...
Background. Despite enzyme replacement therapy, Fabry nephropathy still progresses. Podocyturia i... more Background. Despite enzyme replacement therapy, Fabry nephropathy still progresses. Podocyturia is an irreversible event that antedates proteinuria and leads to chronic renal failure. We evaluated a potential mechanism of podocyte detachment via the expression of the urokinase-type Plasminogen Activator Receptor (uPAR) in urinary podocytes of Fabry patients.Methods. This is a cross-sectional study that included controls (n=20) and Fabry patients (n=44) either untreated (n=23) or treated with agalsidase-β(n=21).Variables. Variables are estimated glomerular filtration rate (eGFR), urinary protein : creatinine ratio, and urinary uPAR+ podocyte : creatinine ratio. uPAR mRNA expression in response to lyso-Gb3, a bioactive glycolipid accumulated in Fabry disease, was studied in cultured human podocytes.Results. Controls and Fabry patients had similar age, gender, and renal function. Urinary uPAR+ podocytes were higher in patients than in controls. Untreated patients were significantly you...
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