Avaliação, in vivo e in vitro, da reatividade vascular renal à vasopressina em ratos endotoxêmicos: importância da via Rho-A/Rho-quinase

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em FarmacologiaSepse é uma infecção local, acompanhada de uma resposta inflamatória sistêmica e vinculada à elevada incidência e taxa de mortalidade. Apesar desta patologia ser alvo de inúmeras pesquisas, poucos estudos avaliam o papel do leito vascular renal nessa condição. Neste trabalho, avaliamos as alterações vasculares renais e o envolvimento da via Rho-A/Rho-quinase em ratos endotoxêmicos. Ratos machos Wistar, com peso de 230-280 g provenientes do Biotério Central da Universidade Federal de Santa Catarina, receberam, via intraperitoneal, PBS (1 ml/kg) ou LPS (10 mg/kg), 6 ou 24 horas antes dos protocolos experimentais. Os animais tratados com LPS apresentaram PAM reduzida, aumento da FC, hiporresponsividade à Phe, além do aumento na concentração plasmática de NO, uréia e creatinina, como descrito por vários autores, demonstrando reprodutibilidade do modelo. Na análise da reatividade vascular renal, houve hiporreatividade à Phe nos rins dos animais do grupo LPS 24 horas. No entanto, a administração de AVP no leito renal dos animais tratados com LPS revelou uma hiper-reatividade a este agente, além de uma maior sensibilidade à inibição da enzima Rho-quinase (ROCK) no grupo LPS 6 horas. A análise, in vivo, da pressão arterial e fluxo microvascular renal também revelou hiper-reatividade à AVP nos animais endotoxêmicos. Além disto, a administração de 0,1 mg/kg de Y-27632 (i.v.), um inibidor da enzima ROCK, diminuiu o efeito da AVP na PAM dos animais do grupo controle. O mesmo não foi observado nos grupos endotoxêmicos. A administração de Y-27632 não influenciou a redução do fluxo sanguíneo renal em resposta às menores doses de AVP, mas nos grupos LPS 6 e 24 h, reduziu significativamente a diminuição causada pela maior dose de AVP utilizada (30 nmol/kg). A análise de parâmetros cardíacos revelou que não há correlação entre as alterações cardíacas induzidas pelo LPS e a hiper-reatividade à vasopressina. As expressões de proteínas não se mostraram alteradas nos grupos que receberam LPS quando comparadas ao PBS. Por fim, constatamos que houve diminuição na produção inicial de urina que se reestabelece 24 horas após o choque endotoxêmico, o que pode estar relacionando a variações nos níveis de AVP plasmático durante a endotoxemia. Podemos concluir que o leito renal não apresenta hiporreatividade à AVP durante a endotoxemia, o que pode contribuir para a eficácia desse mediador em elevar a pressão arterial no choque séptico. Alterações na funcionalidade da via Rho-A/Rho-quinase parecem contribuir de forma diferenciada para os efeitos vasculares da AVP durante a sepse. Novos estudos precisam ser desenvolvidos a fim de esclarecer o potencial benéfico ou deletério dessa inter-relação.Sepsis is a local infection, followed by systemic inflammatory response and linked to high incidence and mortality rate. Although very studied, few researches have investigated the role of renal vascular bed in this condition. In this work, we evaluated the changes in the functionality of the renal vascular bed, and the involvement of the Rho-A/Rho-kinase pathway, in endotoxemic rats. Male Wistar rats (230-280 g) received by intraperitoneal route either saline (1 ml/kg) or LPS (10 mg/kg), 6 or 24 hours before the experimental protocols. The endotoxemic animals showed reduced mean arterial pressure (MAP), increased heart rate, hyporesponsiveness to phenylephrine and high levels of plasmatic nitrate+nitrate, urea and creatinine levels, as described by several studies. In the analysis of renal vascular function, kidneys from LPS 24 group were hyporeactivity to Phe. However, the renal bed of the animals treated with LPS showed a hyper-reactivity to vasopressin (AVP). An increased sensitivity to inhibition of Rho-kinase (ROCK) was found in the groups LPS 6 hours. The effects of AVP on systemic blood pressure and renal blood flow (both measured in vivo) were also enhanced in endotoxemic rats. In addition, the administration of 0.1 mg/kg of Y-27632 (i.v.), a ROCK inhibitor, decreased the effect of AVP on MAP of control animals., but not in endotoxemic groups. The administration of Y-27632 partially avoided the reduction in renal blood flow elicited by lower doses of AVP in all experimental groups, but had no effect against the highest dose of AVP (30 nmol/kg) in groups LPS 6 and 24 h. Analysis of cardiac parameters showed no correlation between the LPS-induced cardiac changes and the increased effects of AVP. The protein expression of components of the Rho-A/Rho-kinase pathway had not been significantly impaired in kidneys from LPS-treated animals, when compared to control samples. Finally, we did observe a decreased urinary output in the first 6 h after LPS administration, that was restored between 18-24 hours of endotoxemia. Changes in diuresis may be related to variations in plasma levels of AVP during endotoxemia. We concluded that the renal bed does not present hyporeactivity to AVP in endotoxemic shock, what can contribute to the effectiveness of this mediator in raising blood pressure in septic shock. Differential changes in the functionality of the Rho-A/Rho-kinase pathway may contribute to the increased vascular effects of AVP in renal vessels during a septic state. Further studies need to be carried out to provide more evidence regarding the beneficial or deleterious importance of this relationship

Vasopressina no choque séptico: hiper-reatividade vascular renal associada ao aumento da atividade da via de sensibilização ao cálcio Rho-A/Rho-quinase

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia, Florianópolis, 2016.O uso da vasopressina (AVP), em pequenas doses e associada à noradrenalina, tem sido preconizado durante a sepse. Isso porque a vasopressina tem um potente efeito vasoconstritor e seu uso, em altas concentrações, pode causar constrição disseminada e baixa perfusão tecidual, agravando a falência orgânica causada por esta condição. Neste contexto, faz-se necessário entender o efeito sistêmico e local da administração de AVP em diferentes estágios da sepse. Já foi demonstrado o envolvimento da via da Rho-A/Rho-quinase na reatividade a alguns vasoconstritores, dentre eles a própria vasopressina, a fenilefrina e a noradrenalina. Porém pouco se sabe se essa via é crucial nas alterações de reatividade à AVP descritas durante o choque séptico. Assim, utilizando o modelo de ligadura e perfuração do ceco (CLP), nós avaliamos a reatividade vascular sistêmica e renal frente a exposição à AVP além de investigar a participação da via clássica do cálcio, ativada por receptor acoplado à proteína Gq, e a via Rho-A/Rho-quinase na reatividade à vasopressina na sepse. Os animais submetidos à CLP apresentaram hipotensão, taquicardia, além de alterações na função renal e hematológica. A partir destes resultados preliminares, os animais foram divididos em 4 grupos experimentais: controle; CLP 6 horas; CLP 18 horas; e CLP 48 horas. A avaliação in vivo do efeito pressórico e de alterações do fluxo sanguíneo renal induzidos pela administração de AVP revelou um perfil hiporreativo na fase inicial, retornando à reatividade similar ao controle entre 18 e 48 horas após a indução da sepse por CLP. A avaliação por laser Doppler mostrou que a redução do fluxo sanguíneo renal gerada pela vasopressina é significativamente maior 48 horas quando comparado ao grupo controle após a indução da sepse pelo modelo de CLP. A análise dos níveis plasmáticos de vasopressina circulante durante a sepse mostrou-se elevada somente na 6ª hora após a CLP. Já a reatividade de anéis de aorta mostrou-se significativamente menor em todos os grupos CLP, comparado ao controle. O rim isolado e perfundido obtido de animais dos grupos CLP apresentaram-se hiper-reativos à vasopressina, mas não à fenilefrina e nem à noradrenalina. Essa resposta aumentada e sustentada parece ser uma característica das fases intermediária e tardia da sepse. A avaliação, in vitro e por imunoeletroforese, da atividade de V1aR e das vias intracelulares que regulam o mecanismo contrátil induzido pela AVP revelaram a participação dos canais de cálcio de membrana no aumento da reatividade 18 horas após a CLP, e o aumento da atividade de proteínas da via Rho-A/ROCK na fase tardia (48 h) da sepse. Em resumo, nossos resultados mostram a particularidade do leito vascular renal frente à administração de substâncias vasoativas, em especial à vasopressina, e o envolvimento da via de sensibilização do cálcio, Rho-A/Rho-quinase no aumento da reatividade à AVP no estágio tardio da sepse.Abstract : The administration of low doses of vasopressin (AVP) in association with norepinephrine has been recommended for some septic patients. Vasopressin has a potent vasoconstrictor effect and high amounts of this drug can cause widespread constriction and low tissue perfusion, worsening sepsis-induced organ failure. It has been demonstrated the Rho-A/Rho-kinase pathway plays a role in the effects of some vasoconstrictors, including vasopressin, phenylephrine and norepinephrine. However, it remains unknown whether the Rho-A/Rho-kinase pathway is involved in the changes of vascular reactivity to AVP described during septic shock. Using the cecal ligation and puncture (CLP) model of sepsis, we evaluated the systemic and renal vascular reactivity to AVP. We also evaluated the role of the classical calcium pathway activated by G protein coupled receptor, and the Rho-A/Rho-kinase pathway in the changes of the renal vascular reactivity to AVP. The animals subjected to CLP showed hypotension, tachycardia, hyperthermia followed by hypothermia, impaired blood cell counts and increased serum levels of nitrite (an indicative of nitric oxide production) in the first hours (3 to 6 h) after CLP, and disrupted renal function, with reduced diuresis and impaired excretion of electrolytes and urinary metabolic. The rats were allocated into four experimental groups: control; CLP 6 h; CLP 18 h; and CLP 48 h. The effects of AVP on systemic blood pressure were reduced in initial phases of sepsis, returning to normal values between 18 and 48 h after the CLP. The assessment of the renal blood flow showed that the reduction in the renal blood flow was significantly enhanced at 48 hours after CLP, compared with the control group. The plasma levels of vasopressin were increased only six hours after the CLP surgery. The reactivity of aortic rings to AVP was significantly lower in all CLP groups. The kidneys from the CLP 18 and 48 h groups were hyperreactive to vasopressin, but not to phenylephrine or norepinephrine. There were no changes in the expression of V1a receptors in the kidneys from CLP-subjected rats. However, the expression and activity of the Rho-A/ROCK pathway were increased in the CLP 48 h group, compared with control samples. Our study reveals that the renal vascular reactivity to vasopressin varies accordingly with the time point of sepsis, and that at least in late stages of sepsis the enhanced vascular effects of vasopressin in kidneys involves an augmented activation of the Rho-A/Rho-kinase pathway. Taken together, our results show the particularity of the renal vascular bed following administration of vasoactive substances, in special to vasopressin, and the involvement of calcium sensitization pathway, Rho-A/Rho-kinase, in the increased reactivity to AVP found in late stage of sepsis

A violência obstétrica vivenciada por mulheres de um centro educacional no município de São José - SC

Objective: To identify the prevalence and the main types of Obstetric Violence suffered by a group of women; in addition to investigating the level of knowledge of these women on the subject and instructing them. Methods: In this study, qualitative, observational and cross-sectional analytical research was used, through interviews and surveys conducted with women linked to the Centro de Educação Alegria e Cia. Results: Through the analysis and reading of the questionnaires answered by women, 65 , 9% confirmed having suffered obstetric violence, even though 87.2% of them stated that they had knowledge about the topic. Most of these women (70%) report having higher education and age between 20- and 29-years during pregnancies. The most frequent obstetric violence was routine serum, direction of pulls, vaginal examination, and routine cesarean delivery. Conclusion: According to the research, despite the high level of education and knowledge on the subject, many women still suffer obstetric violence, even though it is currently being commented on more. Most women were unaware of Obstetric Violence.Objetivo: Identificar a prevalência e os principais tipos de Violência Obstétrica sofrido por um grupo de mulheres; além de investigar o nível de conhecimento destas mulheres a respeito do tema e instruí-las. Métodos: Neste estudo foi utilizada a pesquisa qualitativa, do tipo analítico observacional e transversal, através de entrevistas e pesquisas realizadas com as mulheres vinculadas ao Centro de Educação Alegria e Cia. Resultados: Por meio da análise e leitura dos questionários respondidos pelas mulheres, 65,9% confirmaram terem sofrido violência  obstétrica, mesmo 87,2% delas terem afirmado que tinham conhecimento sobre o tema. A maioria dessas mulheres (70%) relata ter escolaridade a nível superior e idade entre 20 e 29 anos durante as gestações. As violências obstétricas mais incidentes foram soro de rotina, direcionamento de puxos, exame de toque vaginal e parto cesáreo de rotina. Conclusão: De acordo com a pesquisa, apesar do alto nível de escolaridade e conhecimento acerca do assunto, muitas mulheres ainda sofrem violência obstétrica, mesmo esta sendo mais comentada atualmente. A grande maioria das mulheres se mostrou desconhecedora da Violência Obstétrica. &nbsp

Effect of alcoholic beverages on progeny and reproduction of mice

Alcohol is the most commonly consumed substance in the world. The objective of this study was to evaluate the influence of alcoholic beverages on male reproduction and possible alterations in their offspring. The mice were divided into 4 groups: beer, wine, cachaça (a type of sugarcane rum), with ethanol concentrations of 1.9 g/kg, and control group treated with PBS. The treatment period was 35 days. The animals which received cachaça, demonstrated significant weight loss in the testes and epididymis. The alcoholic beverages promoted significant testosterone level and fertilization index diminution, and morphological alterations in the spermatozoa. The beer group presented decreased implantation sites and a high frequency of dominant lethal. The number of reabsorptions in the wine group was increased. The fermented beverages presented higher potential to induce visceral malformations, while the cachaça caused fetal skeletal malformations. The cachaça treated group presented a negative impact on semen quality and fertilization potential. The treatment with different alcoholic beverages, during spermatogenesis, demonstrated contrasting degrees of induction of toxic effects, interfering in a general aspect in male reproductive performance, fetal viability during intrauterine life, and birth defects. From the data, it is possible to infer that the distillated beverage caused more harmful effects to reproduction in this study

Extracellular superoxide dismutase is necessary to maintain renal blood flow during sepsis development

Background: Extracellular superoxide dismutase (ECSOD) protects nitric oxide (NO) bioavailability by decreasing superoxide levels and preventing peroxynitrite generation, which is important in maintaining renal blood flow and in preventing acute kidney injury. However, the profile of ECSOD expression after sepsis is not fully understood. Therefore, we intended to evaluate the content and gene expression of superoxide dismutase (SOD) isoforms in the renal artery and their relation to renal blood flow. Methods: Sepsis was induced in Wistar rats by caecal ligation and perforation. Several times after sepsis induction, renal blood flow (12, 24 and 48 h); the renal arterial content of SOD isoforms, nitrotyrosine, endothelial and inducible nitric oxide synthase (e-NOS and i-NOS), and phosphorylated vasodilator-stimulated phosphoprotein (pVASP); and SOD activity (3, 6 and 12 h) were measured. The influence of a SOD inhibitor was also evaluated. Results: An increase in ECSOD content was associated with decreased 3-nitrotyrosine levels. These events were associated with an increase in pVASP content and maintenance of renal blood flow. Moreover, previous treatment with a SOD inhibitor increased nitrotyrosine content and reduced renal blood flow. Conclusions: ECSOD appears to have a major role in decreasing peroxynitrite formation in the renal artery during the early stages of sepsis development, and its application can be important in renal blood flow control and maintenance during septic insult

A violência obstétrica vivenciada por mulheres de um centro educacional no município de São José - SC

Objective: To identify the prevalence and the main types of Obstetric Violence suffered by a group of women; in addition to investigating the level of knowledge of these women on the subject and instructing them. Methods: In this study, qualitative, observational and cross-sectional analytical research was used, through interviews and surveys conducted with women linked to the Centro de Educação Alegria e Cia. Results: Through the analysis and reading of the questionnaires answered by women, 65 , 9% confirmed having suffered obstetric violence, even though 87.2% of them stated that they had knowledge about the topic. Most of these women (70%) report having higher education and age between 20- and 29-years during pregnancies. The most frequent obstetric violence was routine serum, direction of pulls, vaginal examination, and routine cesarean delivery. Conclusion: According to the research, despite the high level of education and knowledge on the subject, many women still suffer obstetric violence, even though it is currently being commented on more. Most women were unaware of Obstetric Violence.Objetivo: Identificar a prevalência e os principais tipos de Violência Obstétrica sofrido por um grupo de mulheres; além de investigar o nível de conhecimento destas mulheres a respeito do tema e instruí-las. Métodos: Neste estudo foi utilizada a pesquisa qualitativa, do tipo analítico observacional e transversal, através de entrevistas e pesquisas realizadas com as mulheres vinculadas ao Centro de Educação Alegria e Cia. Resultados: Por meio da análise e leitura dos questionários respondidos pelas mulheres, 65,9% confirmaram terem sofrido violência  obstétrica, mesmo 87,2% delas terem afirmado que tinham conhecimento sobre o tema. A maioria dessas mulheres (70%) relata ter escolaridade a nível superior e idade entre 20 e 29 anos durante as gestações. As violências obstétricas mais incidentes foram soro de rotina, direcionamento de puxos, exame de toque vaginal e parto cesáreo de rotina. Conclusão: De acordo com a pesquisa, apesar do alto nível de escolaridade e conhecimento acerca do assunto, muitas mulheres ainda sofrem violência obstétrica, mesmo esta sendo mais comentada atualmente. A grande maioria das mulheres se mostrou desconhecedora da Violência Obstétrica. &nbsp

<b>Damage caused by exposure to propofol during gestation of mice

  Literature shows that surgical procedure could be necessary at any stage of pregnancy and can cause adverse effects on the mother and fetus. One of the most used anesthetics in surgical centers is propofol however; the safety during pregnancy has not been completely established. The objective of this study was to investigate the possible toxic and teratogenic effects on the intrauterine and post-natal development of mice exposed to the dose of 15 mg kg-1 propofol on the caudal vein fifth, tenth and fifteenth day of gestation. A significant reduction in weight gain was observed in female mice who received a 15 mg kg-1 dose of propofol on the fifth gestational day. A higher rate of embryonic loss post implantation and resorption was also observed in this group. In regards to physical development, the anesthetic increased significantly the offspring weight gain, the time in which pinna detachment occurred, and the anogenital distance of pups whose females received propofol on the fifteenth day of gestation. Based on these results, we concluded that administration of propofol in the beginning stages of gestation increases the number of abortions and promotes alterations in the physical development of pups whose mothers were anesthetized in the final stages of gestation.

Impaired vascular function in sepsis-surviving rats mediated by oxidative stress and Rho-Kinase pathway

We investigated long-lasting changes in endothelial and vascular function in adult rat survivors of severe sepsis induced by cecal ligation and puncture (CLP) model. For this, male Wistar rats (200–350 g) had their cecum punctured once (non-transfixing hole) with a 14-gauge needle. Performed in this way, a mortality rate around 30% was achieved in the first 72 h. The survivors, together with age-matched control rats (not subjected to CLP), were maintained in our holding room for 60 days (S60 group) and had the descending thoracic aorta processed for functional, histological, biochemical or molecular analyses. Endothelium-intact aortic rings obtained from sepsis-surviving S60 group displayed increased angiotensin II-induced contraction, accompanied by decreased activity of the endogenous superoxide dismutase, augmented reactive oxygen species generation, and increased levels of tyrosine nitration compared with vessels from control group. The superoxide scavengers superoxide dismutase and tempol, and the antioxidant apocynin, were able to avoid this enhanced contractility to angiotensin II in aortic rings from the S60 group. In addition, aortic rings from the S60 group presented reduced sensitivity to Y-27632, a Rho-kinase (ROCK) inhibitor. Immunoblot analyses revealed augmented RhoA and ROCK II, and high levels of phosphorylation of myosin phosphatase target subunit 1 in vessels from S60 rats. In conclusion, aortic rings from sepsis-surviving rats display endothelial dysfunction mediated by the increased production of reactive oxygen species, which in turn reduces the bioavailability of nitric oxide and increases the formation of peroxynitrite, and enhances RhoA-ROCK-mediated calcium sensitization, leading to augmented contractile responses to angiotensin II. Notably, this is the first study demonstrating long-term dysfunction in the vasculature of sepsis-surviving rats, which take place or remain beyond the acute septic insult

Effect of alcoholic beverages on progeny and reproduction of mice

ABSTRACT Alcohol is the most commonly consumed substance in the world. The objective of this study was to evaluate the influence of alcoholic beverages on male reproduction and possible alterations in their offspring. The mice were divided into 4 groups: beer, wine, cachaça (a type of sugarcane rum), with ethanol concentrations of 1.9 g/kg, and control group treated with PBS. The treatment period was 35 days. The animals which received cachaça, demonstrated significant weight loss in the testes and epididymis. The alcoholic beverages promoted significant testosterone level and fertilization index diminution, and morphological alterations in the spermatozoa. The beer group presented decreased implantation sites and a high frequency of dominant lethal. The number of reabsorptions in the wine group was increased. The fermented beverages presented higher potential to induce visceral malformations, while the cachaça caused fetal skeletal malformations. The cachaça treated group presented a negative impact on semen quality and fertilization potential. The treatment with different alcoholic beverages, during spermatogenesis, demonstrated contrasting degrees of induction of toxic effects, interfering in a general aspect in male reproductive performance, fetal viability during intrauterine life, and birth defects. From the data, it is possible to infer that the distillated beverage caused more harmful effects to reproduction in this study

Effect of alcoholic beverages on progeny and reproduction of mice

ABSTRACT Alcohol is the most commonly consumed substance in the world. The objective of this study was to evaluate the influence of alcoholic beverages on male reproduction and possible alterations in their offspring. The mice were divided into 4 groups: beer, wine, cachaça (a type of sugarcane rum), with ethanol concentrations of 1.9 g/kg, and control group treated with PBS. The treatment period was 35 days. The animals which received cachaça, demonstrated significant weight loss in the testes and epididymis. The alcoholic beverages promoted significant testosterone level and fertilization index diminution, and morphological alterations in the spermatozoa. The beer group presented decreased implantation sites and a high frequency of dominant lethal. The number of reabsorptions in the wine group was increased. The fermented beverages presented higher potential to induce visceral malformations, while the cachaça caused fetal skeletal malformations. The cachaça treated group presented a negative impact on semen quality and fertilization potential. The treatment with different alcoholic beverages, during spermatogenesis, demonstrated contrasting degrees of induction of toxic effects, interfering in a general aspect in male reproductive performance, fetal viability during intrauterine life, and birth defects. From the data, it is possible to infer that the distillated beverage caused more harmful effects to reproduction in this study