RESUMO
Immune homeostasis maintenance throughout pregnancy is critical for normal fetal development. Trophoblast cells differentiate into an invasive phenotype and contribute to the transformation of maternal arteries and the functional shaping of decidual leukocyte populations. Insufficient trophoblast invasion, inadequate vascular remodeling, and a loss of immunologic homeostasis are associated with pregnancy complications, such as preeclampsia and intrauterine growth restriction. Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide synthetized in trophoblasts at the maternal-placental interface. It regulates the function of trophoblast cells and their interaction with decidual leukocytes. By means of a murine model of pregnancy in normal maternal background with VIP-deficient trophoblast cells, here we demonstrate that trophoblast VIP is critical for trophoblast function: VIP gene haploinsufficiency results in lower matrix metalloproteinase 9 expression, and reduced migration and invasion capacities. A reduced number of regulatory T cells at the implantation sites along with a lower expression of proangiogenic and antiinflammatory markers were also observed. Findings detected in the implantation sites at early stages were followed by an abnormal placental structure and lower fetal weight. This effect was overcome by VIP treatment of the early pregnant mice. Our results support the relevance of trophoblast-synthesized VIP as a critical factor in vivo for trophoblast-cell function and immune homeostasis maintenance in mouse pregnancy.-Hauk, V., Vota, D., Gallino, L., Calo, G., Paparini, D., Merech, F., Ochoa, F., Zotta, E., Ramhorst, R., Waschek, J., Leirós, C. P. Trophoblast VIP deficiency entails immune homeostasis loss and adverse pregnancy outcome in mice.
Assuntos
Homeostase/imunologia , Resultado da Gravidez , Trofoblastos/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Movimento Celular , Feminino , Desenvolvimento Fetal , Masculino , Troca Materno-Fetal , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placenta/metabolismo , Gravidez , Linfócitos T Reguladores/imunologia , Trofoblastos/citologia , Peptídeo Intestinal Vasoativo/genéticaRESUMO
We investigated the effect of ovariectomy(oVx) on renal and systemic hemodynamic, electrolyte excretion and total and dephosphorylated Na(+),K(+)-ATPase α1 subunit (t-d-NKA) in normotensive Wistar rats under a normal sodium (NS, 0.24%) or high sodium (HS, 1%) intake versus intact female (IF). On NS intake, t-d-NKA was higher in oVx rats and overexpressed in the thick ascending limbs (P < .01 vs. IF) and renal plasma flow was increased. On HS intake, oVx rats maintained a greater dephosphorylated NKA, excreted less sodium, and developed arterial hypertension (134 ± 4 vs. IF 112 ± 5 mm Hg, P < .05). Sodium load caused salt-sensitive hypertension in oVx Wistar rats.
Assuntos
Hipertensão/enzimologia , Hipertensão/etiologia , Rim/enzimologia , Ovariectomia/efeitos adversos , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Pressão Sanguínea , Feminino , Hipertensão/fisiopatologia , Alça do Néfron/enzimologia , Natriurese , Ovário/fisiologia , Ratos , Ratos Wistar , Fluxo Plasmático Renal , Sódio na Dieta/administração & dosagemRESUMO
OBJECTIVES: Cardiopulmonary bypass (CPB) use is an essential strategy for many cardiovascular surgeries. However, its use and duration have been associated with a higher rate of postoperative complications, such as low cardiac output syndrome due to myocardial oedema and dysfunction. Though Aquaporin water channels have been implicated in myocardial water balance, their specific role in this clinical scenario has not been established. METHODS: In a consecutive study of 17 patients with severe aortic stenosis undergoing aortic valve replacement surgery, 2 myocardial biopsies of the left ventricle were taken: 1 before and 1 after CPB use. Sociodemographic, clinical and laboratory data were collected. Western blot and immunohistochemistry studies were performed. RESULTS: After CPB use, there was a mean increase of â¼62% in Aquaporin 1 protein levels (P = 0.001) and a mean reduction of â¼38% in Aquaporin 4 protein levels (P = 0.030). In immunohistochemistry assays, Aquaporin 1 was found lining small blood vessels, while Aquaporin 4 formed a circular label in cardiomyocytes. There were no changes in the localization of either protein following CPB use. During the observed on-pump time interval, there was a 1.7%/min mean increase in Aquaporin 1 (P = 0.021) and a 2.5%/min mean decrease in Aquaporin 4 (P = 0.018). Myocardial interstitial oedema increased by 42% (95% confidence interval 31-54%) after CPB use. Patients who developed low cardiac output syndrome were in the upper half of the median percentage change of Aquaporin expression. CONCLUSION: Time-dependent changes in cardiac Aquaporin expression may be associated with myocardial oedema and dysfunction related to CPB use.
Assuntos
Ponte Cardiopulmonar , Próteses Valvulares Cardíacas , Valva Aórtica , Aquaporina 1 , Ponte Cardiopulmonar/efeitos adversos , Humanos , MiocárdioRESUMO
INTRODUCTION: Fabry disease (FD) is a hereditary disorder caused by a deficiency of α-galactosidase A enzyme activity. The transmission of the disorder is linked to the X chromosome. OBJECTIVES: The objectives of the study were: 1. To quantify the presence of podocytes in paediatric patients with FD and compare them with the value of the measured podocyturia in healthy controls. 2. To determine whether a greater podocyturia is related to the onset of pathological albuminuria in patients with FD. 3. To determine the risk factors associated with pathological albuminuria. METHODS: We performed an analytical, observational study of Fabry and control subjects, which were separated into 2groups in accordance with the absence of the disease (control group) or the presence of the disease (Fabry group). RESULTS: We studied 31 patients, 11 with FD and 20 controls, with a mean age of 11.6 years. The difference between the mean time elapsed from the diagnosis of FD to the measurement of podocyturia (40 months) and the onset of pathological albuminuria (34 months) was not significant (p=0.09). Podocytes were identified by staining for the presence of synaptopodin and the mean quantitative differences between both podocyturias were statistically significant (p=0.001). Albuminuria was physiological in 4 of the patients with FD and the relative risk to develop pathological albuminuria according to podocyturia was 1.1 in the control group and 3.9 in the Fabry group, with a coefficient of correlation between podocyturia and albuminuria in the Fabry group of 0.8354. Finally, the 2 risk factors associated with the development of pathological albuminuria were podocyturia (OR: 14) and being aged over 10 years (OR: 18). We found no significant risk with regard to glomerular filtrate renal (GFR) (OR: 0.5) or gender (OR: 1.3). The mean GFR remained within normal values. CONCLUSION: The detection of podocyturia in paediatric patients with FD could be used as an early marker of renal damage, preceding and proportional to the occurrence of pathological albuminuria.
Assuntos
Albuminúria/etiologia , Doença de Fabry/urina , Podócitos , Adolescente , Fatores Etários , Estudos de Casos e Controles , Criança , Pré-Escolar , Doença de Fabry/diagnóstico , Doença de Fabry/patologia , Feminino , Taxa de Filtração Glomerular , Humanos , Masculino , Proteínas dos Microfilamentos/análise , Podócitos/química , Fatores de Risco , Fatores Sexuais , Fatores de TempoRESUMO
BACKGROUND: Urea transport depends on the diffusion through cell membrane and the facilitated urea transport. Two groups of urea transporters (UT-A and UT-B) have been identified in mammals, and both are involved in intrarenal recycling of urea. The aim of our study was to examine the renal urea handling in rats with chronic renal failure (CRF). METHODS: CRF rats were induced by 5/6 nephrectomy followed by a high-protein (HP) diet to increase the progressive loss of renal function for 5 months. Functional studies on water and urea handling were performed. RT-PCR, immunoblotting and immunohistochemistry were used to identify UT-A proteins in remnant kidney. RESULTS: A significant decrease in creatinine clearance consistent with development of CRF was observed. The remnant kidneys were hypertrophied, and total renal mass was increased. Urine production increased markedly, whereas urine osmolality and solute-free water reabsorption decreased significantly. Fractional urea excretion was increased reaching values of 105% -/+ 8%. UT-A protein was localized in pars recta by immunohistochemical studies, and it was identified as UT-A2 in outer medulla from remnant kidneys by RT-PCR and immunoblotting. CONCLUSION: In uremic rats, an urea transporter type UT-A2 was expressed in the pars recta, suggesting a possible relation with the fractional urea excretion increase. This expression may be a consequence of an adaptive mechanism in the handling of urea during development of CRF. Further studies will be necessary to elucidate the contribution of this mechanism to renal damage observed in the progression of CRF.
Assuntos
Expressão Gênica , Falência Renal Crônica/genética , Medula Renal/metabolismo , Proteínas de Membrana Transportadoras/genética , RNA Mensageiro/genética , Animais , Modelos Animais de Doenças , Seguimentos , Humanos , Immunoblotting , Imuno-Histoquímica , Sistema do Grupo Sanguíneo Kidd , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Medula Renal/patologia , Proteínas de Membrana Transportadoras/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportadores de UreiaRESUMO
Hemolytic uremic syndrome (HUS), principally caused by shiga toxins (Stxs), is associated with Shiga toxin-producing Escherichia coli (STEC) infections. We previously reported Stx2 expression by host cells in vitro and in vivo. As the genes encoding the two Stx subunits are located in bacteriophage genomes, the aim of the current study was to evaluate the role of bacteriophage induction in HUS development in absence of an E. coli O157:H7 genomic background. Mice were inoculated with a non-pathogenic E. coli strain carrying the lysogenic bacteriophage 933W (C600Φ933W), and bacteriophage excision was induced by an antibiotic. The mice died 72 h after inoculation, having developed pathogenic damage typical of STEC infection. As well as renal and intestinal damage, markers of central nervous system (CNS) injury were observed, including aberrant immunolocalization of neuronal nuclei (NeuN) and increased expression of glial fibrillary acidic protein (GFAP). These results show that bacteriophage 933W without an E. coli O157:H7 background is capable of inducing the pathogenic damage associated with STEC infection. In addition, a novel mouse model was developed to evaluate therapeutic approaches focused on the bacteriophage as a new target.
RESUMO
Subtilase cytotoxin (SubAB) is a member of the AB5 cytotoxin family and is produced by certain strains of Shiga toxigenic Escherichia coli. The toxin is known to be lethal to mice, but the pathological mechanisms that contribute to Uremic Hemolytic Syndrome (HUS) are poorly understood. In this study we show that intraperitoneal injection of a sublethal dose of SubAB in rats triggers a systemic response, with ascitic fluid accumulation, heart hypertrophy and damage to the liver, colon and kidney. SubAB treated rats presented microalbuminuria 20 days post inoculation. At this time we found disruption of the glomerular filtration barrier and alteration of the protein reabsorption mechanisms of the proximal tubule. In the kidney, SubAB also triggered an epithelial to mesenchymal transition (Wuyts et al., 1996). These findings indicate that apart from direct cytotoxic effects on renal tissues, SubAB causes significant damage to the other organs, with potential consequences for HUS pathogenesis. IMPORTANCE: Uremic Hemolytic Syndrome is an endemic disease in Argentina, with over 400 hundred new cases each year. We have previously described renal effects of Shiga Toxin and its ability to alter renal protein handling. Bearing in mind that Subtilase Cytotoxin is an emerging pathogenic factor, that it is not routinely searched for in patients with HUS, and that to the date its systemic effects have not been fully clarified we decided to study both its systemic effects, and its renal effects to assess whether SubAB could be contributing to pathology seen in children.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga Toxigênica/metabolismo , Subtilisinas/metabolismo , Albuminúria/induzido quimicamente , Animais , Ascite/induzido quimicamente , Cardiomegalia/induzido quimicamente , Colo/efeitos dos fármacos , Colo/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Escherichia coli/toxicidade , Síndrome Hemolítico-Urêmica/etiologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Ratos Sprague-Dawley , Subtilisinas/toxicidadeRESUMO
Previous work done in our laboratory showed that water restriction during 24 and 72h induced changes in cardiovascular NOS activity without altering NOS protein levels in young and adult animals. These findings indicate that the involvement of NO in the regulatory mechanisms during dehydration depends on the magnitude of the water restriction and on age. Our aim was to study whether a controlled water restriction of 1 month affects cardiac function, NO synthase (NOS) activity and NOS, and cav-1 and -3 protein levels in rats during aging. Male Sprague-Dawley rats aged 2 and 16 months were divided into 2 groups: (CR) control restriction (WR) water restriction. Measurements of arterial blood pressure, heart rate, oxidative stress, NOS activity and NOS/cav-1 and -3 protein levels were performed. Cardiac function was evaluated by echocardiography. The results showed that adult rats have greater ESV, EDV and SV than young rats with similar SBP. Decreased atria NOS activity was caused by a reduction in NOS protein levels. Adult animals showed increased cav-1. Water restriction decreased NOS activity in young and adult rats associated to an increased cav-1. TBARS levels increased in adult animals. Higher ventricular NOS activity in adulthood would be caused by a reduction in both cav. Water restriction reduced NOS activity and increased cav in both age groups. In conclusion, our results indicated that dehydration modifies cardiac NO system activity and its regulatory proteins cav in order to maintain physiological cardiac function. Functional alterations are induced by the aging process as well as hypovolemic state.
Assuntos
Envelhecimento/metabolismo , Caveolinas/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Óxido Nítrico/metabolismo , Pressão Osmótica , Animais , Pressão Sanguínea , Peso Corporal , Eletrocardiografia , Comportamento Alimentar , Fibrose , Frequência Cardíaca , Ventrículos do Coração/metabolismo , Hemodinâmica , Masculino , Miocárdio/enzimologia , Óxido Nítrico Sintase/metabolismo , Ratos Sprague-Dawley , Sístole , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , UrinaRESUMO
El síndrome urémico hemolítico (SUH) está definido por la tríada de anemia hemolítica microangiopática, trombocitopenia e insuficiencia renal aguda. En Argentina constituye la primera causa de insuficiencia renal aguda en pediatría. Aproximadamente, del 2% al 4% de los pacientes mueren durante la fase aguda de la enfermedad, y solo un tercio del 96% restante que sobrevive lo hace con secuelas renales, como la persistencia de la proteinuria. Un individuo adulto sano filtra alrededor de 5000 mg/día de proteínas, si bien la excreción en orina es escasa (150 mg/día). La escasa cantidad de proteínas excretadas indica la presencia de un mecanismo de reabsorción a nivel del túbulo proximal. Por lo tanto, la reabsorción tubular renal desempeña un papel muy importante ya que, ante una función glomerular normal, es el principal mecanismo encargado de evitar la depleción proteica corporal. Desde hace aproximadamente 30 años se sabe que la albúmina es reabsorbida en el túbulo proximal. La reabsorción proteica se produce por un mecanismo de endocitosis mediada por el receptor dependiente de clatrina y por endocitosis de fase líquida. Clásicamente se ha descrito que el mecanismo básico del daño renal en el SUH típico y en el atípico es una microangiopatía trombótica, pero de diferentes causas. Sin embargo, debe tenerse en cuenta que la fisiopatología de esta enfermedad es más compleja de lo que se creía, ya que la alteración tubular que surge va a evolucionar en fallas en el mecanismo de endocitosis de proteínas que se suman a las eliminadas por las alteraciones a nivel de la barrera de filtración glomerular.
Hemolytic uremic syndrome (HUS) is defined by the triad of hemolytic anemia microangiopathic, thrombocytopenia and acute renal failure. In Argentina it constitutes the first cause of acute renal failure in Pediatrics. Approximately 2-4% of patients die during the acute phase of the disease, and only a third of the remaining 96% survive with renal sequelae, such as the persistence of proteinuria. A healthy adult filters around 5000 mg/day of proteins, with an excretion in urine of 150 mg/day. The little quantity of proteins excreted indicates the presence of a reabsorption mechanism at the level of the proximal tubule. Therefore, the tubular reabsorption plays a very important role since it is the main mechanism responsible for preventing the depletion of protein. For approximately 30 years, it has been known that albumin is reabsorbed in the proximal tubule. Protein reabsorption occurs by a clathrin-dependent receptor mediated endocytosis mechanism and by fluid phase endocytosis. The basic mechanism of renal damage in typical and atypical HUS has been described as a thrombotic microangiopathy, but of different causes. However, the pathophysiology of this disease is more complex than what was believed since the emerging tubular alteration will ewvolve into failures of the protein endocytosis mechanism that are added to the alterations at the level of the glomerular filtration barrier.
Assuntos
Humanos , Proteinúria , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Endocitose , Podócitos , Insuficiência Renal , Síndrome Hemolítico-UrêmicaRESUMO
In Argentina, hemolytic uremic syndrome (HUS) constitutes the most frequent cause of acute renal failure in children. Approximately 2%-4% of patients die during the acute phase, and one-third of the 96% who survive are at risk of chronic renal sequelae. Little information is available about the direct effect of Shiga toxin type 2 (Stx2) on the onset of proteinuria and the evolution of toxin-mediated glomerular or tubular injury. In this work, rats were injected intraperitoneally with recombinant Escherichia coli culture supernatant containing Stx2 (sStx2; 20 µg/kg body weight) to induce HUS. Functional, immunoblotting, and immunohistochemistry studies were carried out to determine alterations in slit diaphragm proteins and the proximal tubule endocytic system at 48 hours post-inoculation. We detected a significant increase in microalbuminuria, without changes in the proteinuria values compared to the control rats. In immunoperoxidase studies, the renal tubules and glomerular mesangium showed an increased expression of transforming growth factor ß(1)(TGF-ß(1)). The expression of megalin was decreased by immunoperoxidase and the cytoplasm showed a granular pattern of megalin expression by immunofluorescence techniques. Western blot analysis performed in the renal cortex from sStx2-treated and control rats using anti-nephrin and anti-podocalyxin antibodies showed a decreased expression of these proteins. We suggest that the alterations in slit diaphragm proteins and megalin expression could be related to the development of microalbuminuria in response to lethal doses of Stx2.
RESUMO
Escherichia coli strains producing Shiga toxins (Stxs) colonize the lower gastrointestinal tract and cause watery diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS). HUS is characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Oliguria associated with acute tubular necrosis and microangiopathic thrombosis has been reported as the most common cause of renal failure in Argentinean children. Our study was undertaken to obtain a model of HUS in rats that was similar to the clinical and renal histopathology findings described in humans. Rats were intraperitoneally inoculated with culture supernatant from recombinant E. coli expressing Stx2. Glomerular filtrate volume evaluated from clearance of creatinine resulted in a progressive reduction (from 53% at 24 h to 90% at 48 h). Urine volume increased significantly at 24 h but returned to normal levels at 48 h. Evidence of thrombocytopenia, anemia and leukocytosis was documented. Macroscopic analysis revealed a hyperemic peritoneal face with intestinal water accumulation. The kidneys were friable and congestive. Histopathological analysis showed glomerular and tubular necrosis as well as microangiopathic thrombosis. Our findings indicated vascular damage and kidney lesions similar to those described in humans with HUS.
Assuntos
Modelos Animais de Doenças , Síndrome Hemolítico-Urêmica/induzido quimicamente , Túbulos Renais/efeitos dos fármacos , Toxina Shiga II/toxicidade , Escherichia coli Shiga Toxigênica/fisiologia , Doença Aguda , Animais , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Colo/efeitos dos fármacos , Colo/patologia , Creatinina/urina , Diarreia/induzido quimicamente , Diarreia/patologia , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/patologia , Técnicas Imunoenzimáticas , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Necrose , Ratos , Ratos Sprague-Dawley , Toxina Shiga II/metabolismo , Células VeroRESUMO
El síndrome urémico hemolítico se caracteriza por presentar anemia hemolítica microangiopática, trombocitopenia e insuficiencia renal aguda. Se clasifica en típico, diarrea positivo, inducido por Escherichia coli 0157-H7 (90%) y atípico, más comúnmente secundario a la desregulación de la vía alternativa del complemento (3-10%). La región cromosómica 1q32 contiene el sistema regulador de la activación del complemento humano (RCA). Se relaciona con mutaciones en factores reguladores de C3 como el factor H (FH, la más común), el factor I, el factor B y la proteína constitutiva de membrana (MCP). Presenta patrones de herencia autosómica tanto dominante como recesiva. El déficit de FH autosómico dominante generalmente ocurre en adultos y la mortalidad y el riesgo de enfermedad renal terminal oscila entre el 50%-90%. El objetivo de nuestro trabajo fue estudiar la función tubular y glomerular por técnicas de inmunohistoquímica para detectar mecanismos de transporte para agua (AQPs), urea (UT-A) y proteínas (nefrina, podocalixina y megalina) en un riñón transplantado de un paciente portador de SUH por déficit de FH que desarrolló una recidiva de SUH y lesiones por nefrotoxicidad. Detectamos un mecanismo de adaptación a la uremia por la expresión de novo de un UT-A2 en corteza renal y la disminución del UT-A1 en médula y alteraciones en el manejo proximal del agua por la disminución de la expresión de AQP1 en túbulo proximal. Las modificaciones a nivel de la expresión de la nefrina y la podocalixina podocitarias y de megalina en el túbulo proximal podrían explicar la presencia de proteinuria.