CAPA-related peptides belong to a family of neuropeptides localized to the central nervous system that can function in diverse roles in the regulation of water and salt homeostasis in insects. These peptides are known to stimulate fluid secretion by Malpighian tubules (MTs) in Dipteran species, thus serving a diuretic function. In contrast, this thesis demonstrates that members of this family of peptides in Rhodnius prolixus serve an anti-diuretic role and have multiple tissue targets, whereby they oppose the activity of diuretic hormones such as serotonin (5-Hydroxytryptamine hydrochloride; 5-HT). I have identified two genes each encoding three peptides in R. prolixus, suggesting this insect is capable of producing a greater number of CAPA-peptides compared to other insects that contain only a single CAPA gene. Interestingly, while the second peptide encoded in each R. prolixus gene (RhoprCAPA-α2/-β2) inhibits the stimulatory effects of serotonin on tissues such as the anterior midgut and Malpighian tubules, it appears the other CAPA-related and pyrokinin-related peptides do not play a major role in inhibiting the effects of serotonin on these tissues. More specifically, serotonin-stimulated fluid secretion by MTs and fluid absorption by the anterior midgut are reduced by the anti-diuretic peptide, RhoprCAPA-α2. In addition, I have also identified a G protein-coupled receptor which likely mediates the anti-diuretic effect associated with RhoprCAPA-α2 and have functionally characterized this receptor in Chinese hamster ovary cells. Spatial transcript expression analysis in fifth-instars reveals a wide distribution of the receptor in tissues associated with the rapid post-gorging diuresis. Thus, my findings suggest that numerous tissues are regulated by the CAPA peptides in R. prolixus. Gene structure and phylogenetic analyses demonstrate that this receptor is the orthologue of the D. melanogaster capa receptor (CG14575) with homologs in other insects. Taken together, my thesis demonstrates that the RhoprCAPA peptides play an integral role in the coordination and maintenance of anti-diuresis in R. prolixus. This mechanism is necessary following the rapid diuresis associated with blood-feeding by this medically-important insect.

CAPA-related peptides belong to a family of neuropeptides localized to the central nervous system that can function in diverse roles in the regulation of water and salt homeostasis in insects. These peptides are known to stimulate fluid secretion by Malpighian tubules (MTs) in Dipteran species, thus serving a diuretic function. In contrast, this thesis demonstrates that members of this family of peptides in Rhodnius prolixus serve an anti-diuretic role and have multiple tissue targets, whereby they oppose the activity of diuretic hormones such as serotonin (5-Hydroxytryptamine hydrochloride; 5-HT). I have identified two genes each encoding three peptides in R. prolixus, suggesting this insect is capable of producing a greater number of CAPA-peptides compared to other insects that contain only a single CAPA gene. Interestingly, while the second peptide encoded in each R. prolixus gene (RhoprCAPA-α2/-β2) inhibits the stimulatory effects of serotonin on tissues such as the anterior midgut and Malpighian tubules, it appears the other CAPA-related and pyrokinin-related peptides do not play a major role in inhibiting the effects of serotonin on these tissues. More specifically, serotonin-stimulated fluid secretion by MTs and fluid absorption by the anterior midgut are reduced by the anti-diuretic peptide, RhoprCAPA-α2. In addition, I have also identified a G protein-coupled receptor which likely mediates the anti-diuretic effect associated with RhoprCAPA-α2 and have functionally characterized this receptor in Chinese hamster ovary cells. Spatial transcript expression analysis in fifth-instars reveals a wide distribution of the receptor in tissues associated with the rapid post-gorging diuresis. Thus, my findings suggest that numerous tissues are regulated by the CAPA peptides in R. prolixus. Gene structure and phylogenetic analyses demonstrate that this receptor is the orthologue of the D. melanogaster capa receptor (CG14575) with homologs in other insects. Taken together, my thesis demonstrates that the RhoprCAPA peptides play an integral role in the coordination and maintenance of anti-diuresis in R. prolixus. This mechanism is necessary following the rapid diuresis associated with blood-feeding by this medically-important insect.

El presente trabajo de investigación tuvo por objetivo principal: demostrar el efecto diurético y comprobar la seguridad del zumo del fruto del limón (Citrus limon L.). Para evaluar la diuresis producida por el zumo del fruto del limón según el método de Naiv. V., R. et al (1981) con modificaciones, se liofilizó el zumo del fruto y se administró a ratas albinas hembras de Cepa Holtzman en dosis de 180 mg/Kg, 540 mg/Kg y 900 mg/Kg de de peso corporal en un volumen de 25mL/Kg, comparados a un control negativo y 2 fármacos diuréticos de referencia, para luego recolectar el volumen urinario a razón de 30, 60, 90, 180, 270 y 360 minutos y medir el pH y electrolitos urinarios. Para determinar la seguridad se utilizó el método de las clases toxicas agudas correspondiente a la guía para los Ensayos de Sustancias Químicas N° 423 de la Organización para la Cooperación y Desarrollo Económico (OECD). Se encontró que el pH, electrolitos y volúmenes urinarios del zumo liofilizado difirieron significativamente de los del control negativo y los 2 fármacos de referencia. Se obtuvo asimismo que el zumo del fruto del limón posee una DL50 de 5,000 mg/Kg de peso corporal. Con la aplicación del zumo liofilizado y los fármacos en el biomodelo experimental, se concluye que el zumo del fruto del limón producen un efecto de incremento del pH, electrolitos y volúmenes urinarios y que es seguro de administrar hasta una dosis de 5,000 mg/Kg en una sola toma en 24 horas. / -- The present research had as main objective: demonstrate the diuretic effect and check the safety of the juice from the fruit of the lemon (Citrus limon L.). To evaluate the urine output produced by the fruit of the lemon juice depending on Naiv V., R. et al (1981) method with modifications, the fruit juice was freeze-dried and administered to female albino rats of Holtzman strain at doses of 180 mg/kg, 540 mg/kg and 900 mg/kg of body weight on a volume of 25mL/Kg, compared to a negative control and 2 reference diuretic drugs, and then collected the urine volume at a rate of 30, 60, 90, 180, 270 and 360 minutes and measure the pH and urinary electrolytes. To determine the safety, it was used the Acute Toxic Class Method according to guidelines for the testing of chemicals No. 423 of the Organization for Economic Cooperation and Development (OECD). It was found that the pH, electrolytes and urinary volumes of freeze-dried juice differed significantly from the negative control and the 2 reference drugs. It was obtained that the juice from the fruit of the lemon has a LD50 of 5000 mg /kg of body wieght. Wth the implementation of the freeze-dried juice and drugs in the experimental Biomodel, It is concluded that the juice from the fruit of the lemon produce an effect of increasing the pH, electrolytes and urinary volumes and that it is safe to administer at a dose of 5000 mg/kg in a single intake in 24 hours. / Tesis

To see if aggressive diuresis in first twenty four hours is associated with a comparable number of total days in the hospital as compared to non-aggressive diuresis. In this retrospective cohort study, we compared the length of hospital stay of consecutive patients admitted in one year based on their diuresis during the first twenty-four hours of hospitalization: aggressive diuresis (group 1) i.e. > 2400mL versus non-aggressive diuresis (group 2) i.e. ≤ 2400mL urine output. Patients were excluded if in cardiogenic shock, had creatinine level above 3 mg/dL on admission, or on dialysis. A total of 194 patients were enrolled (29 in group 1 and 165 in group 2 respectively). The Kaplan-Meier estimate of the median cumulative proportion of patients still hospitalized for the group 1 was 4 days and in group 2 was 5 days (log-rank test; P=0.67). In univariate analysis, Cox PH regression showed unadjusted hazard rate of discharge from hospital was slightly higher in group 1 than group 2 but was statistically non-significant (HR=1.08; P=0.70). In multivariate Cox model analysis, creatinine at the time of admission when greater than 1.6mg/dL (P=0.75), LVEF (P= 0.14), total twenty-four hours dose of intravenous Furosemide given (P=0.98) and interaction between Furosemide dose and Creatinine level (P=0.79) were not significant predictor of hospital discharge. Adjusted hazard rate for discharge from hospital was 12% higher in group 1 than group 2 but still statistically non-significant (HR=1.12; P=0.60). Since the length of hospital stay is similar between two groups, we suggest the goal of diuresis to be less than 2400mL in first twenty-four hours to prevent excessive dehydration.

In the yellow fever mosquito, Aedes aegypti (L.), hemolymph-circulating diuretic hormones act upon the renal organs (Malpighian tubules) to regulate primary urine composition and secretion rate; however, the molecular endocrine mechanisms underlying rapid water elimination upon adult eclosion and blood feeding are not fully understood. Bioinformatic analysis of the current Aedes aegypti genome assembly reveals only a single predicted corticotropin releasing factor (CRF)-like diuretic hormone 44 (DH44) gene, but two DH44 receptor genes. The tissue expression profiles of the DH44 receptor(s), and specifically the identity of the DH44 receptor(s) in the Malpighian tubule, are undetermined in any mosquito species. This dissertation shows that Vectorbase gene ID AAEL008292 encodes a DH44 receptor (AaegGPRdih1) transcribed in Malpighian tubules. Sequence analysis and transcript localization indicate that AaegGPRdih1 is the co-ortholog of the Drosophila melanogaster DH44 receptor (CG12370-PA). The presence of conserved amino acid residues between AaegGPRdih1 and vertebrate CRF receptors suggests this mosquito receptor modulates multiple G protein-dependent intracellular signaling pathways. Quantitative PCR analysis of a time course of Malpighian tubule cDNA reveals AaegGPRdih1 abundance increases paralleling periods of observed urination. This suggests that target tissue receptor biology is linked to the known periods of release of diuretic hormones from the nervous system, pointing to a common up-stream regulatory mechanism. Higher relative abundance of AaegGPRdih1 transcript in female Malpighian tubules 24 hours after blood feeding suggests a role for AaegGPRdih1 in the excretion of nitrogen waste. RNA-mediated silencing to establish the significance of AaegGPRdih1 to mosquito Malpighian tubule physiology was inconclusive.

El presente trabajo de investigación tuvo por objetivo principal: demostrar el efecto diurético y comprobar la seguridad del zumo del fruto del limón (Citrus limon L.). Para evaluar la diuresis producida por el zumo del fruto del limón según el método de Naiv. V., R. et al (1981) con modificaciones, se liofilizó el zumo del fruto y se administró a ratas albinas hembras de Cepa Holtzman en dosis de 180 mg/Kg, 540 mg/Kg y 900 mg/Kg de de peso corporal en un volumen de 25mL/Kg, comparados a un control negativo y 2 fármacos diuréticos de referencia, para luego recolectar el volumen urinario a razón de 30, 60, 90, 180, 270 y 360 minutos y medir el pH y electrolitos urinarios. Para determinar la seguridad se utilizó el método de las clases toxicas agudas correspondiente a la guía para los Ensayos de Sustancias Químicas N° 423 de la Organización para la Cooperación y Desarrollo Económico (OECD). Se encontró que el pH, electrolitos y volúmenes urinarios del zumo liofilizado difirieron significativamente de los del control negativo y los 2 fármacos de referencia. Se obtuvo asimismo que el zumo del fruto del limón posee una DL50 de 5,000 mg/Kg de peso corporal. Con la aplicación del zumo liofilizado y los fármacos en el biomodelo experimental, se concluye que el zumo del fruto del limón producen un efecto de incremento del pH, electrolitos y volúmenes urinarios y que es seguro de administrar hasta una dosis de 5,000 mg/Kg en una sola toma en 24 horas. / The present research had as main objective: demonstrate the diuretic effect and check the safety of the juice from the fruit of the lemon (Citrus limon L.). To evaluate the urine output produced by the fruit of the lemon juice depending on Naiv V., R. et al (1981) method with modifications, the fruit juice was freeze-dried and administered to female albino rats of Holtzman strain at doses of 180 mg/kg, 540 mg/kg and 900 mg/kg of body weight on a volume of 25mL/Kg, compared to a negative control and 2 reference diuretic drugs, and then collected the urine volume at a rate of 30, 60, 90, 180, 270 and 360 minutes and measure the pH and urinary electrolytes. To determine the safety, it was used the Acute Toxic Class Method according to guidelines for the testing of chemicals No. 423 of the Organization for Economic Cooperation and Development (OECD). It was found that the pH, electrolytes and urinary volumes of freeze-dried juice differed significantly from the negative control and the 2 reference drugs. It was obtained that the juice from the fruit of the lemon has a LD50 of 5000 mg /kg of body wieght. Wth the implementation of the freeze-dried juice and drugs in the experimental Biomodel, It is concluded that the juice from the fruit of the lemon produce an effect of increasing the pH, electrolytes and urinary volumes and that it is safe to administer at a dose of 5000 mg/kg in a single intake in 24 hours.

The evolution of the blood feeding adaptation has required precise coordination of multiple physiological processes in the insect, such as reproduction, behavior, digestion and diuresis. These processes are under careful synchronous hormonal control. For rapid excretion, multiple diuretic hormones are known. Although originally described based on their ability to stimulate hindgut contractions, the Aedes kinins have been shown to stimulate fluid secretion in female mosquitoes of Aedes aegypti. Aedes kinins are leucokinin-like neuropeptides released from neurosecretory cells in the brain and abdominal ganglia. They act by binding to the Aedes kinin receptor, a G proteincoupled receptor (GPCR). The Aedes kinin receptor has been cloned, sequenced, functionally characterized, and immunolocalized to stellate cells in the Malpighian tubules of Ae. aegypti. In addition to their myotropic and diuretic roles, leucokinin-like peptides and/or their receptors have been also been discovered in the nervous, digestive, and reproductive systems of other arthropod species. Therefore, the Aedes kinins have the potential to function in several simultaneous physiological processes that are stimulated by blood feeding. This thesis aims to understand better their role in the whole mosquito by investigating the Aedes kinin receptor's global expression as well as its in vivo contribution to post-prandial diuresis. Presence of the Aedes kinin receptor was investigated in the head, posterior midgut (stomach), hindgut, ovaries, and Malpighian tubules of both non blood-fed and blood-fed females by western blot using anti-receptor antibodies. The receptor was then immunolocalized in the posterior midgut and rectum. Finally, RNAi was employed to knock down kinin receptor expression, followed by measurement of in vivo urine excretion post blood feeding in a precision humidity chamber. Transcript and protein knockdown were confirmed by qPCR and immunohistochemistry, respectively. Results indicate widespread expression of the Aedes kinin receptor protein in organs novel for hematophagous insects and demonstrate the receptor's fundamental role in rapid diuresis. These findings strongly point to the Aedes kinins as integrative signaling molecules that could coordinate multiple physiological systems. The Aedes kinins could therefore have contributed to the success of the blood feeding adapation in mosquitoes.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-27T13:35:23Z No. of bitstreams: 2 Dissertação - Emmeline Flor Ribeiro - 2014.pdf: 1787776 bytes, checksum: c8a4106ef03ff49855997007460d4558 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-27T14:47:39Z (GMT) No. of bitstreams: 2 Dissertação - Emmeline Flor Ribeiro - 2014.pdf: 1787776 bytes, checksum: c8a4106ef03ff49855997007460d4558 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-27T14:47:39Z (GMT). No. of bitstreams: 2 Dissertação - Emmeline Flor Ribeiro - 2014.pdf: 1787776 bytes, checksum: c8a4106ef03ff49855997007460d4558 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-12-09 / Aspidosperma subincanum Mart. is a medicinal herb used for the treatment of hypercholesterolemia, diabetes and digestive illnesses. The bitter tonic of its bark is known by the indigenous population to stimulate circulatory functions. Although a previous study showed that EEAS induces hypotension associated with bradycardia and vasodilation, no scientific data have been described to evaluate the diuretic effects of this Brazilian medicine plant. The aim of the present study was to evaluate the diuretic activity of an ethanol extract of Aspidosperma subincanum (EEAS), and possible mechanisms of action, using Wistar rats. EEAS, 60, 120 and 300 mg/Kg, or furosemide (20 mg/Kg) were orally administered and the rats were kept individually in metabolic cages for 24h for urine collection 1, 2, 4, 6, 8, 12 and 24h after treatments. To evaluate the involvement of prostanoids in the diuretic action of EEAS, the animals received piroxicam (5 mg/Kg, i.p.), a nonselective inhibitor of cyclooxygenase, before treatment with EEAS 120 mg/Kg. The control groups received only saline (NaCl, 0,9%), or saline and piroxicam. The urinary volume, the water consumption, electrolyte excretion and pH were measured. The oral administration of EEAS 60 and 120mg/Kg increased significantly the urine and electrolyte excretion of Na+ and K+ continuously throughout the study period, wherein increased the urinary output from the first hour after treatment, and increased the electrolytes from the second hour onwards after treatment. EEAS 60 and 120 mg/Kg caused a relative increase in cumulative diuresis around of 77% and 142,95%, respectively, compared with the control group. EEAS 300 mg/Kg increased the urinary excretion from 8 h after treatment. From 4 h until the end of the experiment, the group treated with EEAS 120 mg/kg provided a major excretion of Na+ than the furosemide group, while the group treated with EEAS 60 mg/Kg provided a similar excretion of Na+ when compared to the group that received furosemide. The furosemide group showed significantly higher amounts of K+ in the urine when compared with the others group. The urinary excretion of EEAS was reduced by piroxicam 2 h, 4 h and 8 h after treatments by around 54.2%, 50.3% and 38.9%, respectively. Piroxicam reduced Na+ excretion between 4 and 8 h after treatments by 38.4 and 39.0%, respectively. And still inhibited the K+ excretion by around 28.2% at 4 h and 47.1% at 8 h. The results suggest that EEAS could present compound(s) responsible for diuretic activities, and the mechanism could involve prostanoids system. / O Aspidosperma subincanum Mart. é uma planta medicinal conhecida popularmente por ser utilizada no tratamento de hipercolesterolemia, diabetes e doenças digestivas. O tônico amargo de sua casca é conhecido pela população indígena por estimular as funções circulatórias. Embora, um estudo prévio tenha demonstrado que o extrato etanólico de Aspidosperma subincanum (EEAS) induz hipotensão associada à bradicardia e vasodilatação, nenhum estudo científico avaliou os efeitos diuréticos dessa planta medicinal brasileira. O objetivo do presente estudo foi avaliar a atividade diurética do EEAS e possíveis mecanismos de ação, utilizando ratos Wistar. EEAS, 60, 120 e 300 mg/Kg, ou furosemida (20 mg/Kg) foram administrados, via oral, e os ratos foram colocados individualmente em gaiolas semi-metabólicas durante 24 h para a coleta de urina, nos períodos de 1, 2, 4, 6, 8, 12 e 24 h após os tratamentos. Para avaliar o envolvimento da via dos prostanoides no possível efeito diurético do EEAS, os animais receberam piroxicam (5 mg/Kg, i.p.), um inibidor não seletivo das cicloxigenases, previamente ao tratamento com EEAS 120 mg/Kg. Os grupos controle receberam apenas salina (NaCl, 0,9%), ou salina e piroxicam. O volume urinário, o consumo de água, a excreção de eletrólitos e o pH foram medidos. A administração oral de EEAS 60 e 120 mg/Kg aumentou significativamente a excreção urinária e de eletrólitos Na+ e K+ durante o período de estudo, sendo que houve aumento do volume urinário a partir da primeira hora após os tratamentos, e aumento da excreção de eletrólitos a partir de 2 h após os tratamentos. EEAS 60 e 120 mg/Kg causaram aumento relativo na diurese cumulativa de 24 h em aproximadamente 77% e 142,95%, respectivamente, comparado com grupo controle. EEAS 300 mg/Kg causou aumento da excreção urinária a partir de 8 h após tratamento. De 4 h até o final do experimento, o grupo tratado com EEAS 120 mg/kg proporcionou maior excreção de Na+ do que o grupo tratado com furosemida, enquanto o grupo tratado com EEAS 60 mg/Kg proporcionou excreção similar ao grupo furosemida, sendo que neste grupo ocorreu maior excreção de K+ na urina quando comparado com os outros grupos. A excreção urinária do EEAS foi reduzida por piroxicam nos períodos de 2 h, 4 h e 8 h após os tratamentos na proporção de 54.2%, 50.3% e 38.9%, respectivamente. O piroxicam também inibiu a excreção de Na+ entre 4 e 8 h após os tratamentos, na proporção de 38.4 e 39.0%, respectivamente. Ainda inibiu excreção de K+ na ordem de 28.2% em 4 h e 47.1% em 8 h. Os resultados mostram que o EEAS apresenta compostos ativos responsáveis pela atividade diurética em ratos, e o mecanismo de ação parece envolver a via dos prostanoides.

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