Participation of angiotensin II and MAP-kinase (Mitogenic-Activated Protein Kinase) in the postnatal renal development of Wistar rats

O desenvolvimento renal em ratos e camundongos começa por volta do 11º ou 12º dia de vida intra-uterina e continua até a segunda semana após o nascimento. A formação de matriz extracelular (MEC) representa um dos eventos-chave na diferenciação de glomérulos. Várias linhas de evidências experimentais mostram que o sistema-renina-angiotensina (SRA) participa do desenvolvimento renal. Muitos sinais são transmitidos da superfície celular para o núcleo através da cascata das MAP (Mitogenic-Activated Protein) quinases. Esse estudo visa avaliar a expressão das MAP quinases no córtex renal durante o desenvolvimento renal pós-natal e a participação da AII na ativação dessas vias nos filhotes de mães submetidas à uma dieta com conteúdo elevado de sódio ou à exposição ao losartan durante a gestação e amamentação ou somente durante a amamentação. Analisamos também os efeitos desses tratamentos na estrutura e na função renal. Os filhotes de ratas Wistar de 1, 7, 15 e 30 dias foram divididos em 3 estudos com 2 sub-grupos em cada um: Estudo 1- Controle 1 (C1): filhotes de mães com ingestão normal de NaCl, Experimental 1 (E1): filhotes de mães submetidas à ingestão de solução salina 0,15 M; Estudo 2- Controle 2 (C2): filhotes de mães que receberam solução sacarose 2% durante a gestação e amamentação; Experimental 2 (E2): filhotes de mães que receberam losartan (0,4 g/L) diluído em solução de sacarose 2% durante a gestação e amamentação e Estudo 3- Controle 3 (C3): filhotes de mães que receberam solução sacarose 2% durante a amamentação, Experimental 3 (E3): filhotes de mães que receberam losartan (0,4 g/L) diluído em solução de sacarose 2% durante a amamentação. Os filhotes foram sacrificados em diferentes períodos após o nascimento (1, 7, 15 e 30 dias) e os rins retirados para análise histológica, imunohistoquímica, TUNEL e estudos de Western blot. As avaliações das reações imunohistoquímicas para α-SMA, fibronectina, p-ERK e p-JNK foram feitas através de escores que refletiam a extensão da área com imunorreação. Foram contadas as células PCNA e AII positivas e células apoptóticas por glomérulo e por área de córtex renal de 0,245 mm2. A análise da densitometria das bandas obtidas nos estudos de Western blot para AT 1, AT 2, p-p38 e tubulina foi realizada utilizando-se o programa lmage J. As expressões de α-SMA, fibronectina, PCNA, AII e p-ERK no córtex renal foram maiores no primeiro dia após o nascimento e diminuíram com o desenvolvimento renal. No estudo 1, verificamos redução nas expressões de α-SMA, fibronectina e PCNA no córtex renal dos animais de 1 dia de mães submetidas à dieta com alto conteúdo em sódio quando comparados aos controles de mesma idade. Estas alterações foram acompanhadas por redução no número de células AII positivas no córtex renal desses animais. A expressão de p-ERK foi menor no córtex renal dos animais de 1 dia do grupo E1 comparados aos controles, enquanto que as expressões das MAP quinases p-JNK e p-p38 foram maiores no córtex renal dos animais de 30 dias do grupo E1 quando comparados aos controles. Nestes animais também foi verificado maior número de células apoptóticas. Os animais de 30 dias deste grupo apresentaram redução na taxa de filtração glomerular (TFG) e aumento da pressão arterial quando comparados aos controles de mesma idade. A expressão de receptores AT 1 estava reduzida e a de AT 2 aumentada no córtex renal dos animais de 1 e 30 dias do grupo E1. O tratamento com losartan durante a gestação e amamentação (Estudo 2), bem como durante a amamentação (Estudo 3) provocou aumento das expressões de α-SMA, fibronectina, PCNA e o número de células apoptóticas no córtex renal dos animais de 15 e 30 dias em relação aos respectivos controles. Os animais de 30 dias do grupo E2 apresentaram também redução da expressão de receptores AT 1 e aumento de receptores AT 2, e alterações na estrutura renal caracterizadas pelo alargamento do espaço intersticial do córtex renal, atrofia e dilatação tubulares. A excreção urinária de albumina estava bastante elevada nos animais de 30 dias do grupo E2. Concluindo, as alterações de estrutura e função renal observadas nos filhotes de mães submetidas a tratamentos que interferem com a atividade do SRA, estavam associadas com as modificações nas expressões dos receptores AT1 e AT2 de AII e de MAP quinases durante o desenvolvimento renal pós-natal.Renal development in rats and mice usually begins around the 11st and 12nd day of intrauterine life and continues until postnatal second week. Extracellular matrix (ECM) production has an important role on the glomerular differentiation process. Experimental evidences suggest that angiotensin II (AII) participates on postnatal renal development. Many signals are transmitted from cellular surface to the nucleus through MAPK (Mitogenic-Activated Protein Kinase) cascade. Several effects of AII are related to the activation of these pathways. The aim of this study was evaluate the MAPK expression on the renal cortex during postnatal renal development and the role of AII on the activation of these pathways in rats born from mothers submitted to high salt intake or exposed to losartan during gestation and lactation or during lactation. We also analyzed the effects of those treatments on renal structure and function. Newborn Wistar rats aging 1, 7, 15 and 30 days old were divided into 6 groups for performing of 3 studies: Study 1: Control 1 (C1 ), newborn rats from mothers that received normal salt intake; Experimental 1 (E1), newborn rats from mothers that received increased salt intake; Study 2: Control 2 (C2), newborn rats from mothers exposed to sacarose 2% solution during gestation and lactation; Experimental 2 (E2), newborn rats from mothers exposed to losartan (0,4 g/L) diluted in sacarose 2% solution during gestation and lactation; Study 3: Control 3 (C3), newborn rats from mothers exposed to sacarose 2% solution during lactation; Experimental 3 (E3), newborn rats from mothers exposed to losartan (0,4 g/L) diluted in sacarose 2% solution during lactation. Pups rats were killed and kidneys removed for histological and immunohistochemical studies, evaluation of apoptotic cells by TUNEL and Western blot analysis. The evaluation of α-SMA, fibronectin, p-ERK and p-JNK reactions was performed by scores which reflected mainly the changes in the extent rather than intensity of staining, and number of PCNA, AII and apoptotic positive cells were counted the by glomerulus or grid fields from renal cortex measuring 0.245mm2. Densitometry analysis of bands from Western blot studies for AT 1, AT 2, p-p38 and tubulin was performed using the lmage J computerized program. α-SMA, fibronectin, PCNA, AII and p-ERK expressions in renal cortex were higher on 1 day-old animal and decreased with the renal development. ln study 1, we verified reduced expressions of α-SMA, fibronectin and PCNA on renal cortex of 1 day-old animals from mothers submitted to increased salt intake when compared to the controls of same age. These alterations were associated with the reduction of AII positive cells on renal cortex of these animals. p-ERK expression was lower on renal cortex of 1 day-old animals from E1 group compared to the controls, while p-p38 and p-JNK MAPK expressions were higher on the renal cortex of the 30 day-old animals from E1group when compared to the controls of same age. We also observed higher number of apoptotic cells in renal cortex from these animals. Thirty days-old animals from this group also showed reduction in glomerular filtration rate (GFR) and an increase in blood pressure when compared to the controls of the same age. One and 30 day-old animals from E1 group presented reduction of AT 1 receptor and increase of AT 2 receptor expressions in renal cortex. The treatment with Losartan during gestation and lactation (Study 2) and during lactation (Study 3) provoked increase in fibronectin, PCNA and α-SMA expressions as well in the number of apoptotic cells on renal cortex from 15 and 30 day-old animals compared to the respective contrais. Thirty day-old animals from E2 and E3 groups showed a reduction in AT 1 and an increase in AT 2 expressions, and intense alterations on renal structure characterized by widening of interstitial space of renal cortex, atrophy and tubular lumen dilatation. Moreover, albumin urinary excretion of the 30 days-old animals was noticeably increased. ln conclusion, renal structure and function alterations observed in newborn rats from mothers exposed to treatments that interfere with the activity of RAS was associated with modifications on AT 1 and AT 2 receptors and MAPK expressions during postnatal renal development

Diuretic and hipotensive activity of aqueous extract of parsley seeds (Petroselinum sativum Hoffm.) in rats

PURPOSE: the vegetal specie, Petroselinum sativum Hoff., known as parsley, is widely used in the Brazilian folk medicine as diuretic. The objective of this study is to verify if Brazilian use of parsley aqueous extract has similar effects with investigations that show a diuretic effect of P. sativum in rats. METHODS: 19 rats were anesthetized and we cannulated the trachea, left carotid artery (for arterial pressure measurement) and urinary bladder (to collect urine). After 40 minutes of adaptive surgery conditions, anesthetized rats were administrated as related with their group: control (CON), oral administration with 1.0 mL of filtered water, and treated group (AE), oral administration with aqueous extract of seeds of parsley 20% (AE). Urine was collected three times (30 minutes each) and then this material was used for sodium and potassium determinations, to evaluate the amount excreted of these ions. Blood pressure was measured by mercury manometer for 9 times. All data were statistically evaluated. RESULTS AND CONCLUSION: in the analyzed parameters, CON group did not show any differences; but AE group showed an increased of urinary flow and sodium and potassium amount excreted, and also decreased arterial pressure. All the parameters presented these modifications after 30 minutes of administration of AE (p<0.05). These results show that the treatment with the AE results in natriuretic and hypotensive effects in anesthetized Wistar rats, confirming the use of Brazilian population of this herb as diuretic

MAPK and angiotensin II receptor in kidney of newborn rats from losartan-treated dams

Several lines of evidence suggest that angiotensin II (A-II) participates in the postnatal development of the kidney in rats. Many effects of A-II are mediated by mitogen-activated protein kinase (MAPK) pathways. This study investigated the influence that treatment with losartan during lactation has on MAPKs and on A-II receptor types 1 (AT(1)) and 2 (AT(2)) expression in the renal cortices of the offspring of dams exposed to losartan during lactation. In addition, we evaluated the relationship between such expression and changes in renal function and structure. Rat pups from dams receiving 2% sucrose or losartan diluted in 2% sucrose (40 mg/dl) during lactation were killed 30 days after birth, and the kidneys were removed for histological, immunohistochemical, and Western blot analysis. AT(1) and AT(2) receptors and p-p38, c-Jun N-terminal kinases (p-JNK) and extracellular signal-regulated protein kinases (p-ERK) expression were evaluated using Western blot analysis. The study-group rats presented an increase in AT(2) receptor and MAPK expression. In addition, these rats also presented lower glomerular filtration rate (GFR), greater albuminuria, and changes in renal structure. In conclusion, newborn rats from dams exposed to losartan during lactation presented changes in renal structure and function, which were associated with AT(2) receptor and MAPK expression in the kidneys

"Juntos decidimos o que queremos fazer": a participação da criança em educação de infância (conceções e práticas numa sala de educação pré-escolar)

Relatório da Prática de Ensino Supervisionada apresentado à Escola Superior de Educação de Lisboa para obtenção de grau de mestre em Educação Pré-EscolarO presente relatório apresenta uma análise crítica e reflexiva do processo desenvolvido num contexto de Jardim de Infância, no âmbito da Prática Profissional Supervisionada, módulo II, com crianças de quatro anos, durante um período de observação e de intervenção de, aproximadamente, quatro meses. Nele apresenta-se também a investigação realizada durante o processo. No decorrer da observação tornou-se notório o facto de estar em presença de um contexto valorizador de uma cultura de participação ativa da criança, em que a mesma é chamada a participar em todas as decisões que lhe dizem respeito (planeamento, avaliação do plano do dia, decisão de projetos e de atividades que destes fazem parte ou não, decisão de tarefas semanais, escolha das áreas de brincadeira e alteração do espaço da sala). Deste modo, a investigação, de carácter qualitativo, mais concretamente um estudo de caso, foi desenvolvida tendo em conta os seguintes objetivos: (i) compreender a visão de participação da equipa educativa da sala e do grupo de crianças pertencentes ao contexto socioeducativo (sala de Educação Pré-Escolar) em análise, (ii) compreender as potencialidades de um contexto socioeducativo assente na participação da criança para o grupo e para cada criança, (iii) identificar formas de participação das crianças em contexto de sala de Educação Pré-Escolar e, ainda, (iv) identificar estratégias de promoção de participação da criança. Neste estudo participaram a equipa educativa da sala e as crianças do grupo, tendo recorrido a diversas técnicas e instrumentos de investigação: observação direta participante, realizada através do registo de notas de campo, observação direta naturalista, realizada através do registo de momentos que aconteciam com a participação da criança, observação direta sistemática (estruturada), através do recurso a grelhas de observação previamente construídas para o efeito, observação indireta, através da realização de inquérito por entrevista à educadora de infância cooperante, de inquérito por questionário à ajudante de ação educativa cooperante e de um focus group com o grupo de crianças. Após os dados recolhidos, recorri à análise de conteúdo e à análise estatística descritiva através da distribuição de frequências, com o intuito de compreender a temática estudada e dar resposta às interrogações e aos objetivos iniciais. Os dados reunidos revelaram que a participação das crianças é sobretudo percecionada, tanto pela equipa educativa da sala, como pelo grupo de crianças, como uma tomada de decisão conjunta entre crianças e adultos, isto é, todos os intervenientes da sala de Educação Pré-Escolar são tidos em consideração nas decisões que dizem respeito ao grupo e à sala. Para mais, foi possível perceber que, em contexto de sala, as crianças participam em vários momentos da rotina, como por exemplo no planeamento semanal e diário, bem como na gestão dos vários desafios e projetos que desenvolvem, decidindo o que, como e quando fazer. Procurou-se identificar possíveis contributos de um contexto assente na participação da criança, tendo os dados revelado que, através desta prática, as crianças desenvolvem competências de comunicação e de como estar em grupo (respeito, colaboração e interação entre pares). As crianças, ao participarem, sentem-se valorizadas e desenvolvem o seu sentido crítico. Estas competências podem ser desenvolvidas através do diálogo, da observação, da metodologia de trabalho de projeto, dos instrumentos de pilotagem e do envolvimento das crianças nas rotinas da sala e do grupo.ABSTRACT This report presents a critical and reflective analysis of the process developed in the context of a kindergarten, within the scope of Supervised Professional Practice, module II, with four-year-old children, during an observation and intervention period of approximately four months. It also presents the investigation carried out during the process. Throughout the observation, it became clear that the presence of a context that values a culture of active participation of the child, in which the child is called to participate in all decisions that concern him (planning, evaluation of the child's plan) day, decision of projects and activities that were part of it or not, decision of weekly tasks, choice of play areas and alteration of the space of the room). In this way, the qualitative investigation, more specifically a case study, was developed taking into account the following objectives: (i) understanding the vision of participation of the educational team of the room and the group of children belonging to the socio-educational context (room of Pre-School Education) under analysis, (ii) understand the potential of a socioeducational context based on the child's participation for the group and for each child, (iii) identify ways of children's participation in the context of Pre-School Education and, furthermore, (iv) to identify strategies to promote child participation. The educational team of the classroom and the children of the group participated in this study, having resorted to various techniques and research instruments: direct participant observation, performed through the recording of field notes, direct naturalistic observation, performed through the recording of moments that happened with the child's participation, systematic (structured) direct observation, through the use of observation grids previously constructed for the purpose, indirect observation, by conducting a survey by interviewing the cooperating kindergarten teacher, by questionnaire survey by the educational action helper cooperative and a focus group with the group of children. After the data collected, I resorted to content analysis and descriptive statistical analysis through the distribution of frequencies, in order to understand the theme studied and answer questions and initial objectives. The data gathered revealed that the participation of children is mainly perceived, both by the educational team of the classroom, and by the group of children, as a joint decision-making between children and adults, that is, all the actors in the Pre-School Education room. are taken into account in decisions concerning the group and the room. Furthermore, it was possible to see that, in the classroom, children participate in various moments of the routine, such as weekly and daily planning, as well as managing the various challenges and projects they develop, deciding what, how and when do. We sought to identify possible contributions from a context based on the participation of the child, and the data revealed that, through this practice, children develop communication skills and how to be in a group (respect, collaboration and interaction between peers). Children, when participating, feel valued and develop their critical sense. These skills can be developed through dialogue, observation, project work methodology, piloting instruments and the involvement of children in the classroom and group routines.N/

Influence of hyperbaric oxygen on biomechanics and structural bone matrix in type 1 diabetes mellitus rats.

The aim of this study was to evaluate the biomechanics and structural bone matrix in diabetic rats subjected to hyperbaric oxygen therapy (HBO).Twenty-four male rats were divided into the following groups: Control; Control + HBO; Diabetic, and Diabetic + HBO. Diabetes was induced with streptozotocin (STZ) in the diabetic Groups. After 30 days, HBO was performed every 48h in HBO groups and all animals were euthanized 60 days after diabetic induction. The femur was submitted to a biomechanical (maximum strength, energy-to-failure and stiffness) and Attenuated Total Reflectance Fourier transform infrared (ATR-FTIR) analyses (crosslink ratio, crystallinity index, matrix-to-mineral ratio: Amide I + II/Hydroxyapatite (M:MI) and Amide III + Collagen/HA (M:MIII)).In biomechanical analysis, diabetic animals showed lower values of maximum strength, energy and stiffness than non-diabetic animals. However, structural strength and stiffness were increased in groups with HBO compared with non-HBO. ATR-FTIR analysis showed decreased collagen maturity in the ratio of crosslink peaks in diabetic compared with the other groups. The bone from the diabetic groups showed decreased crystallinity compared with non-diabetic groups. M:MI showed no statistical difference between groups. However, M:MIII showed an increased matrix mineral ratio in diabetic+HBO and control+HBO compared with control and diabetic groups. Correlations between mechanical and ATR-FTIR analyses showed significant positive correlation between collagen maturity and stiffness.Diabetes decreased collagen maturation and the mineral deposition process, thus reducing biomechanical properties. Moreover, the study showed that HBO improved crosslink maturation and increased maximum strength and stiffness in the femur of T1DM animals

Energy of biomechanical analyses in different groups (*p<0.05).

<p>Energy of biomechanical analyses in different groups (*p<0.05).</p

Maximum strength of biomechanical analyses in different groups (*p<0.05).

<p>Maximum strength of biomechanical analyses in different groups (*p<0.05).</p

Parameters analyzed by means of ATR-FTIR spectrums using the program OPUS 6.5.

<p>Amide I band (Collagen ratio between the mature pyridinoline crosslink peaks (PYR)– 1660 cm-1 and immature crosslinking dihydroxinorleucina (DHLNL) - 1690 cm-1); Crystallinity Index (The intensity ratio of peaks 551 and 597 cm-1 for 588 cm-1); Matrix-to-mineral ratio: Amide I + II/Hydroxyapatite (HA) (The ratio between integrated areas of amide I + II (1520–1720 cm-1) for HA (916–1180 cm-1)) and Amide III + Collagen/HA (The ratio between integrated areas of amide III (1210–1270 cm-1) with two collagen bands (1269–1296 cm-1 and 1180–1213 cm-1) for HA (916–1180 cm-1).</p

Stiffness of biomechanical analyses in different groups (*p<0.05).

<p>Stiffness of biomechanical analyses in different groups (*p<0.05).</p