published_or_final_version / Pathology / Master / Master of Medical Sciences

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Le cancer de la vessie représente le 4ième cancer en terme d’incidence. La mortalité de ce cancer est principalement due à la formation de métastases secondaires. Actuellement, aucun des médicaments disponibles sur le marché ne permet d’éviter la rechute ni de contrôler la dissémination métastatique. L’enjeu thérapeutique principal dans ce cancer est donc le contrôle de la dissémination métastatique. Le mécanisme de formation des métastases est connu et implique la transition épithélio-mésenchymateuse (EMT) qui permet aux cellules épithéliales d’acquérir les caractéristiques des cellules mésenchymateuse, ainsi que la capacité d’invasion. Dans ce travail, nous nous sommes posés 2 questions. Twist1 protéine connue comme régulateur central dans l’EMT, peut-elle être une cible pronostique et pharmacologique dans le cancer de la vessie ? L’utilisation d’un nouvel anti-invasif le saracatinib, peut il être un nouveau traitement dans le cancer de la vessie. Pour répondre à ces questions, nous avons caractérisé nos lignées de cancer de vessie, pour la protéine Twist1, mais aussi pour les principaux marqueurs de l’EMT (E-cadhérine, N-cadhérine, vimentine). Puis nous avons testé pharmacologiquement (cytotoxicité et invasivité) nos lignées pour le saracatinib. L’expression de la molécule Twist1 a été décevante, seulement une lignée exprime Twist1, de manière faible, et non homogène. Twist n’est donc pas une cible potentielle dans le cancer de la vessie. La sensibilité d’un point de vue cytotoxique au saracatinib est liée à l’expression de la E-cadhérine. Cependant il n’existe pas de relation entre l’expression de la E-cadhérine et l’action anti-invasive du saracatinib. Toutes les lignées sont sensibles. Dans la lignée SD48 (E-cadhérine positive), il y a une augmentation de la protéine E-cadhérine, ainsi qu’une nette relocalisation à la membrane sous traitement saracatinib. La voie du saracatinib semble passer par la E-cadherine, mais cette dernière n’explique pas en totalité son action. / Bladder cancer is the 4th cancer in the world. The mortality is principally caused by metastatic dissemination. Currently, no medicine can controle the relapse, and the metastatic dissemination. The principal therapeutic stategy is the contrôle of dissemination. Le mecanisme of metastate synthesis is knowed, and implicate the epithelial to mesenchymal transition (EMT) who change epithelial cells in mesenchymental cell and acquisition of invasivity. In this work, 2 questions are asking. The Twist1 protein who‘s a central regulator of EMT, can be a new prognostic, diagnostic, and therapeutic target in bladder cancer ? The use of a new anti-invasif drug saracatinib, can be a new treatment in the bladder cancer. To answer at this questions, we carcaterise your cells lines for Twist1 and main EMT marquers (E-cadhérine, N-cadhérine, vimentine). Than we test pharmacology response (cytotoxicity and anti-invasivity) of cells lines for saracatinib. The Twist1 expression is very disappointing, only one cells line is expressing Twist1, poorly, and non homogenous. Twist1 is not a good target in bladder cancer. The cytotoxic sensibility is linked to E-cadherin expression. However, there is no relation between E-cadherin expression and anti-invasive action for saracatinib. All the cells lines are sensitive. In the cells line SD48 (E-cadherin positive), there is an increase of E-cadherin protein, and a localisation at the membrane under saracatinib treatment. The saracatinib seem to use E-cadherin pathway, but thys way not expliquate all the action.

Thesis (MSc.Med.(Urology))--University of the Witwatersrand, Faculty of Health Sciences, 2014. / Bladder cancer is the second commonest urological malignancy and haematuria is the commonest symptom. Cystoscopy and urine cytology are integral for the investigation of haematuria, while the role of molecular markers such as the NMP22 BladderChek test is still being defined. The BladderChek is a qualitative point of care test developed for the detection of the elevated urinary levels of NMP22 associated with bladder cancer. No studies have been performed in South Africa using the BladderChek nor considered using this test to increase the efficiency of the workup of patients with gross haematuria. The primary aim was to establish the percentage of office cystoscopies done as part of a gross haematuria workup at Charlotte Maxeke Johannesburg Academic Hospital that are unnecessary and may be avoided if the BladderChek is positive under defined conditions. A cross-sectional study of the BladderChek test using prospective consecutive sampling, with special care to limit false positives and negatives, of 64 patients with a history of gross haematuria was conducted. The sensitivity, specificity, positive predictive value and negative predictive value for the BladderChek and the urine cytology were 78.9%, 84.4%, 68.2%, 90.5% and 36.8%, 93.0%, 70.0%, 76.9% respectively. The performance of the BladderChek was not affected by the history of gross haematuria, the stage nor grade of malignancy. Urine cytology detected only one malignancy missed by the BladderChek. Approximately 12.6% of office cystoscopies may be avoided and 78.9% of bladder tumours detected if the BladderChek is selectively applied as in this study. This may “fast-track” patients for transurethral resection of bladder tumour. The BladderChek may be a cost-effective alternative to urine cytology.

Alterations in glucocorticoid (GC) biosynthesis and metabolism are associated with a variety of pathophysiological disorders including cholestasis, diabetes and other metabolic disorders. Bile acids (BA) are also important modulators of metabolic functions and regulate cholesterol, triglyceride and glucose homeostasis as well as being critical for dietary fat digestion, enterohepatic function, and postprandial thermogenesis. In intact cells and in vivo, the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme converts inactive GC precursors (cortisone in humans, and 11-dehydrocorticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively) thereby amplifying local intracellular GC levels. Interconversion by 11β-HSD1 of other sterols has also been described. These include conversions of 7keto-cholesterol to 7β-hydroxycholesterol, 7-oxodehydroepiandrosterone (7-oxo-DHEA) to 7α-hydroxy- and 7β-hydroxy DHEA, 7- oxo-lithocholic acid (LCA, a bile acid; BA) to chenodeoxycholic acid (CDCA, a 7α- hydroxylated BA) and ursodeoxycholic acid (UDCA, a 7β-hydroxylated BA) in human liver microsomes. In the liver, BA inhibit 11β-HSD1 but whether 11β-HSD1 regulates BA homeostasis is unclear. Evidence of molecular regulation of the enterohepatic recycling of bile acids by liver glucocorticoid receptor (GR) in mice does suggest a role for 11β-HSD1. It was therefore hypothesised that disruption of 11β-HSD1 expression in mice would impair BA recycling and might affect the relative concentrations of BA within the enterohepatic circuit. The primary objective of the current work was to investigate the impact of altered 11β-HSD1 on BA homeostasis. This was achieved using genetically modified mouse models with altered 11β-HSD1 expression, either globally or restricted to hepatocytes. BA are stored in the gall bladder and are released postprandially, to aid digestion. It was hypothesised that 11β-HSD1 deficiency might the affect the process of postprandial gall bladder emptying/refilling. Mice with global 11β-HSD1 knockout (Hsd11b1-/-) and age-matched control mice (C57Bl/6) were either fasted for 4h and culled or fasted for 4h and re-fed for another 4h before culling. Their response to fasting and re-feeding was assessed with specific focus on organs associated with BA recycling in the enterohepatic circuit (liver, gall bladder, serum and small intestine). Gall bladders of fasted Hsd11b1-/- and C57Bl/6 mice had similar volumes of bile but in fasted Hsd11b1-/- mice, BA concentrations were higher in serum and liver. As expected, re-feeding caused gall bladder emptying in C57Bl/6 mice with consequent increased serum and liver bile acid concentrations. In Hsd11b1-/- mice, the gall bladder did not empty and serum and liver BA concentrations were similar to the fasted state. To explore possible reasons for this, levels of mRNA encoding proteins known to be involved in hepatic BA transport were quantified using real-time q-PCR. Levels of mRNA encoding NTCP/ SCL10A1/ SCL10A1, the transporter responsible for most hepatocyte BA uptake, were increased in livers of fasted Hsd11b1-/- mice whereas levels of Slc51b mRNA, encoding the OST- transporter that facilitates BA removal from liver to the systemic circulation, and levels of Mrp2 and Atp8b1/FIC1 mRNAs (both encoding proteins which transport BA from liver into gall bladder) were decreased. This suggests that in fasted Hsd11b1-/- mice, BA transporter expression is altered to increase BA influx into hepatocytes and decrease efflux, to compensate for reduced levels of liver BA. These data together imply that bile acid recycling is controlled by 11β-HSD1 activity which regulates gall bladder emptying, hepatic BA concentration and BA transporter activity to ensure continuity of BA recycling within the enterohepatic circuit compartments. These changes may also affect digestion of lipids and fat-soluble micronutrients. Because 11β-HSD1 can directly metabolise secondary BA, it was predicted that 11β-HSD1 deficiency would lead to changes in the BA profile. Profiling of BA in the gall bladder was performed using mass spectrophotometry. In Hsd11b1-/- mice, 7α-hydroxylated BA predominated (cholic acid [CA]>α-muricholic acid [α- MCA]>CDCA>others), in contrast to C57Bl/6 mice in which 7β-hydroxylated BA predominated (ω-MCA>β-MCA>UDCA>others). The ratio of 7α:7β acids was therefore >100-fold greater in Hsd11b1-/- mice. This suggests that 11β-HSD1 either directly or indirectly controls the epimerisation of 7α- to 7β- hydroxylated BAs. Measurement of mRNAs encoding proteins important for hepatic BA biosynthesis in livers of fasted Hsd11b1-/- mice showed decreased expression of Scarb1/SR-B1, Cyp39a1 and Cyp27a1 (though with no change in levels of CDCA, the product of CYP27A1, in liver or bile fluid), compared to fasted control mice. Hepatic levels of Gpbar1/TGR5/GPBAR1 and Cyp3a11 mRNAs, encoding proteins important in BA detoxification, were increased and decreased, respectively. This suggests that Gpbar1/TGR5/GPBAR1, encoding G-protein coupled bile acid receptor (also called TGR5/GPBAR1) and an FXR target, could be induced to detoxify 7α-hydroxylated BA whereas expression of Cyp3a11, which catalyses the conversion of LCA to hyodeoxycholic acid (HDCA) is decreased; bile fluid of Hsd11b1-/- mice contained lower levels of LCA and little to no HDCA, though LCA and HDCA levels in liver were unaltered. Currently, the functional differences between 7α- and 7β- hydroxylated BA are not clear. However, these findings could have significant implications for bile acid-mediated transcription which, in turn, might affect lipid and sterol metabolism. Also, alterations in BA composition may have other physiological consequences via other pathways. Because cholesterol is the precursor of BA synthesis, it was hypothesised that western diet (WD) (containing cholesterol) would exacerbate and/or alter the phenotype of Hsd11b1-/- mice. Gall bladder weights of fasted Hsd11b1-/- and control C57Bl/6 mice did not change with western diet compared to chow diet. In control C57Bl/6 mice, the total BA concentration in the gall bladder increased in response to WD in comparison to chow diet. In contrast, Hsd11b1-/- mice showed no change in total BA concentration when fed on WD in comparison to chow. These data indicate that 11β-HSD1 is required by mice for the normal increase in total BA concentration in bile in response to dietary cholesterol. BA profiling of bile from control mice fed on WD showed no difference in the relative amounts of 7β-hydroxylated BA and 7α-hydroxylated BA to littermates fed on chow diet with the exception of β–MCA which increased, and α–MCA which decreased. Like chow-fed Hsd11b1-/- mice, BA profiling of bile from WD-fed Hsd11b1-/- mice showed a significant decrease in relative levels of 7β-hydroxylated BA (UDCA < β-MCA < others) and an increase in percentage of 7α-hydroxylated BAs (CA>α-MCA>CDCA>others) compared to C57Bl/6 controls. These data show that Hsd11b1-/- mice fail to show the normal increase in 7β-hydroxylated BA and decrease in 7α-hydroxylated BA observed in control mice in response to a cholesterol containing diet, suggesting 11β-HSD1 deficiency blunts the influence of cholesterol on BA composition. Measurement of hepatic mRNAs encoding BA transporters suggest that hepatocyte uptake of BA is decreased in C57Bl/6 on WD compared to those mice on chow diet, whereas this was not the case in Hsd11b1-/- mice where hepatic expression did not change with diet. Thus, Hsd11b1-/- mice failed to increase expression of Ntcp/ Scl10a1/ Scl10a1 appropriately, suggesting impaired hepatic BA uptake, while Slc51b (encoding OST-β) expression was increased, compared to control mice, possibly to reduce hepatic BA concentration by transporting BA out of hepatocytes into the systemic circulation. Therefore, Hsd11b1-/- mice may adapt to a cholesterol-induced increase in hepatic BA by blunting hepatic BA uptake via NTCP/ SCL10A1/ SCL10A1 and increasing hepatic efflux via OST-β. The effects of 11β-HSD1 deficiency upon BA recycling and BA profile and concentration within the enterohepatic circuit, could reflect 11β-HSD1 action within the liver or could be due to actions in other tissues. / To investigate the role of hepatic 11β-HSD1 specifically, 11β-HSD1 liver-specific knockout (Hsd11b1LKO), 11β- HSD1 liver-specific over-expressors (Hsd11b1LOE) and control mice with exon 3 of the Hsd11b1 gene “floxed” (Hsd11b1F) were studied. Findings from this study indicate a role for 11β-HSD1 in adaption to dietary cholesterol and suggest that hepatic 11β-HSD1 (as opposed to 11β-HSD1 in extra-hepatic tissues) is the main factor regulating BA metabolism. Also, work from this thesis demonstrates 11β-HSD1 is an important regulator of gall bladder emptying and filling, an important component of enterohepatic bile acid recycling. Based on these findings it is anticipated that therapeutic use of 11β-HSD1 inhibitors will result in BA imbalances within the enterohepatic circuit and therefore BA homeostasis. Care must therefore be observed when implementing therapeutic use of 11β-HSD1 inhibitors, with particular focus on patients with cholestasis, Addison’s disease and critically ill patients who already have known BA imbalances in their enterohepatic system.

Orientador: Andrigo Barboza de Nardi / Coorientador: Alexandre Martini de Brum / Banca: Virgínia Tessarine Barbosa / Banca: Tiago Luís Eilers Treichel / Banca: Luís Gustavo Gosuen Gonçalves Dias / Banca: Paola Castro Moraes / Resumo: O aumento da expectativa de vida dos animais de companhia favorece a ocorrência de casos de câncer, como o de bexiga. De forma que, faz-se necessário dispor de alternativas que minimizem o sofrimento e melhorem a qualidade de vida desses pacientes após a remoção cirúrgica da bexiga. O objetivo da presente pesquisa foi avaliar a exequibilidade da técnica de ureterostomia cutânea em suínos após a realização da cistectomia total, quando comparada à técnica de ureterostomia colônica, assim como registrar e avaliar as alterações pós-operatórias. Foram utilizados 20 animais, distribuídos em dois grupos. Todos foram submetidos à cistectomia radical e, em seguida, a ureterostomia cutânea ou colônica. A exequibilidade das técnicas foi avaliada durante os procedimentos. Para tal observou-se o tamanho da incisão, o tempo de diérese, de realização da derivação urinária e o tempo de síntese, estimou-se a perda sanguínea e o grau de dificuldade na realização dos procedimentos de preparo dos ureteres, preparo do sítio de ureteroanastomose e realização da implantação ureteral. As avaliações clínicas foram realizadas durante 14 dias, quando foi realizada a eutanásia dos animais. Após a eutanásia foram realizadas avaliações macroscópicas dos rins, ureteres, dos sítios da derivação urinária e também foram feitas análises microscópicas dos rins e microbiológicas da urina. Os resultados indicam que a ureterostomia cutânea apresentou maior eficiência quando comparada à colônica nos quesitos perda de sangue, tamanho da incisão realizada e facilidade de realização da anastomose. Os tempos dos procedimentos realizados assim como o grau de dificuldade no preparo dos sítios de ureteroanastomose não diferiram entre si. Observaram-se óbitos espontâneos em 50% dos animais de cada grupo. Complicações clínicas como anorexia, dor à palpação abdominal, vômito... / Abstract: Increased life expectancy of pets favors the occurrence of cancer, such as the urinary bladder ones. In a way that, it's important finding alternatives that minimize the pain and improve the quality of life of patients after surgical removal of the urinary bladder. The aim of this study was to evaluate the feasibility of cutaneous ureterostomy technique in pigs after the total cystectomy compared to the technique of colonic ureterostomy, as well as detect and characterize possible intraoperative complications. For that, 20 pigs were used, divided into two groups. All patients underwent radical cystectomy and then the cutaneous or colonic ureterostomies. The technical feasibility was assessed during the procedures. The procedures compared were: the incision size, dieresis time, realization of urinary diversion and the synthesis time. Also, the estimated blood loss and the degree of difficulty in performing the preparation procedures of the ureters, ureteroanastomose site preparation and ureteroanastomose itself were compared. The results indicate that cutaneous ureterostomy performimg was better than the colonic ureterostomy in relation to blood loss, incision size and anastomosis performing. In contrast, ureterocolonostomy showed better results on the implementation of ureteral preparation. The times of the procedures performed and the degree of difficulty in the preparation of ureteroanastomose sites did not differ. No animals died during surgery. Thus, it was concluded that both techniques can be indicated as viable urinary diversions and the choice will depend on factors intrinsic to the surgeon and the patient / Doutor

Orientador: João Luiz Amaro / Resumo: A instabilidade vesical é uma patologia bastante comum na prática urológica, porém sua etiopatogenia é pouco conhecida. A utilização de seres humanos na investigação de suas causas é restrita, principalmente devido aos problemas éticos. Nosso objetivo foi estudar as alterações histopatológicas e imunohistoquímicas decorrentes da obstrução parcial da bexiga. Foram utilizados 30 coelhos machos, divididos em grupo G1 (controle clínico, n=15) e grupo G2 (obstruído, n=15). No grupo G2 foi realizada obstrução parcial da bexiga usando um bracelete ajustável de polietileno e foram considerados os animais que desenvolveram contrações involuntárias do detrusor (CID). Foram submetidos a dosagem de uréia e creatinina plasmática, cultura de urina e estudo cistométrico em 2 momentos, inicial (M1) e final (M2) após 4 semanas da obstrução. Após este período, os coelhos foram sacrificados e a bexiga retirada para realização de estudo histopatológico e imunohistoquímico. Observou-se um aumento significativo do peso corporal dos coelhos nos diferentes momentos. O peso da bexiga foi 2,5 vezes maior no grupo G2 comparado com o grupo G1 (controle clínico). Não houve diferença estatisticamente significativa na análise da uréia plasmática. A creatinina plasmática foi significativamente maior no grupo G2 (obstruído) em comparação com o grupo G1 no momento final. Detectou-se cultura positiva em 40% dos casos no momento M1 (inicial) e 50% no M2 (final). A análise do estudo urodinâmico demonstrou aumento significativo da capacidade vesical no momento M2 no grupo G2. Não houve diferença estatisticamente significativa na pressão máxima do detrusor e na complacência vesical nos diferentes momentos. Verificou-se persistência das contrações involuntárias do detrusor em todos os animais... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The bladder instability is a very common problem in urology, however its etiology is rather unknown. The use of human beings is limited in the study of detrusor instability, specially because of ethical problems. For this reason we have proposed an experimental study of the bladder instability in rabbits due to partial bladder outlet obstruction. Thirty male rabbits were divided in G1 (control group) and G2. The animals in G2 had their bladder obstructed by using an adjustable polyethylene ring. Both groups were submitted to cystometrical evaluations in two moments: initial (before surgery) and final (after 4 weeks of obstruction). After this period, the rabbits were sacrificed, their bladder was removed and the alterations evaluated in the histologic and immunohistochemical study. The results showed an increase in the body weight of the rabbits and the bladder was 2.5 times heavier than the control group. There was no significative statistical difference in the plasmatic urea analysis. The results showed significative statistical difference between the groups in the final moment (the final moment was superior than the initial moment in G1). There was positive culture in 40% of the cases in M1 and 50% in M2. The analysis of the urodynamics study presented an increase in the bladder capacity in M2, G2. There was no significative statistical difference in the detrusor maximum pressure and in the bladder compliance. We could detect the persistence of the detrusor spontaneous contractions in all the animals in G2 in M2. The histological study showed 13% of epithelium ulceration and 40% of submucosal fibrosis in the obstructed animals. All the rabbits in G2 presented adventitious fibrosis. The inflammatory reaction was significatively bigger in G2. There was no statistical difference in the analysis of the intermuscular fibrosis. The results showed 54% of... (Complete abstract click electronic address below) / Mestre

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / A cistite hemorrÃgica (CH) Ã um evento inflamatÃrio frequentemente associado ao uso das oxazafosforinas. NÃs demonstramos que amifostina (AMF) pode prevenir o dano tecidual provocado pela ifosfamida (IFO). Sabendo-se que IFO tambÃm altera a funÃÃo motora do trato urinÃrio inferior, o presente trabalho foi realizado com o objetivo de investigar se AMF protege os animais contra as alteraÃÃes da funÃÃo vesical provocadas por IFO, e se este efeito ocorre atravÃs de mecanismos dependentes do Ãxido nÃtrico (NO) e dos canais de potÃssio sensÃveis ao ATP (KATP). Camundongos Swiss machos (25-30g, n=8) receberam salina (CTR)ou IFO (400mg/kg, ip) para induÃÃo de CH. Outro grupo recebeu, 30min antes de IFO, AMF (50mg/kg, sc), aminoguanidina (AMG, 50 mg/kg, ip), ODQ (2 mg/kg, vo) ou glibenclamida (GLI, 10 mg/kg, ip). Outros grupos receberam, 30min antes de AMF, L-arginina (L-ARG, 600 mg/kg, ip), ODQ ou diazÃxido (DIAZ, 2mg/kg, ip). Em outra seÃÃo, os grupos que receberam AMG foram prÃ-tratados com L-arginina e os que receberam GLI foram prÃ-tratados com diazÃxido, ambos com intervalos de 30 mim. O peso Ãmido vesical (PUV) e os parÃmetros macroscÃpicos e histopatolÃgicos foram analisados 12h apÃs a injeÃÃo de IFO. Em experimentos ex vivo, preparaÃÃes de mÃsculo liso vesical foram mantidas em soluÃÃo fisiolÃgica aerada com 95% de O2 â 5% CO2, pH 7.4 e a 37&#8304;C para registro isomÃtrico das contraÃÃes musculares a soluÃÃes despolarizantes de cloreto de potÃssio (KCl) e carbacol (CCh). Para o registro de pressÃo intravesical (PIV) por cistometrograma contÃnuo (CC), foi realizada uma laparatomia e um catÃter de polietileno fixado na bexiga e exteriorizado pela regiÃo abdominal foi conectado a um sistema de infusÃo contÃnua de salina (0.04mL/min) e a um transdutor de pressÃo acoplado a um sistema de aquisiÃÃo de sinais biolÃgicos. Nos animais tratados com AMF o aumento PUV provocado por IFO foi inibido em 83%. L-ARG e DIAZ nÃo preveniram tal efeito de IFO no PUV (1.1% e 11.4%, respectivamente). Os escores macro e microscÃpicos foram significativamente menores em AMF, comparados ao grupo IFO. L-ARG e DIAZ inibiram os escores de AMF. IFO diminuiu a contratilidade de tiras de bexiga ao CCh em relaÃÃo ao grupo CTR (1.36 Â 0.24 Vs 0.18 Â 0.02 g forÃa/mg de tecido seco; p<0.01). AMF preveniu a hipocontratilidade provocada por IFO ao estÃmulo com CCh (1.47 Â 0.16 g forÃa/mg de tecido seco; p<0.01). L-ARG e DIAZinibiram o efeito de AMF (0.6 Â 0.08 e 0.79 Â 0.12 g forÃa/mg de tecido seco, respectivamente; p<0.01 em relaÃÃo a AMF). Na anÃlise por CC, AMF reverteu o aumento da frequÃncia miccional (FM) provocada por IFO (18 Vs 5.6 micÃÃes/15 min, IFO e CTR, respectivamente; p<0.01. AMF â 6.5 micÃÃes/15 min; p<0.01 em relaÃÃo a IFO). L-ARG e DIAZ reverteram as FMs de AMF (p<0,01) (L-ARG: 14.5 e DIAZ: 11.2 micÃÃes/15 min). Os traÃados cistometrogrÃficos de CTR e AMF mostraram ciclos miccionais regulares com contraÃÃes evidentes associadas ao esvaziamento da bexiga. Nas anÃlises de CC de animais tratados com IFO, os traÃados mostraram ciclos irregulares de micÃÃo e nÃo foram observadas contraÃÃes evidentes associadas ao evento da micÃÃo. DIAZ tambÃm apresentou traÃados com este padrÃo. O tratamento com ODQ nÃo alterou o efeito sobre a disfunÃÃo vesical in vitro e in vivo promovida por IFO e nem sobre a proteÃÃo pela AMF. AMF inibe as alteraÃÃes motoras vesicais promovidas por IFO atravÃs de processos mÃltiplos que provavelmente incluem o NO e KATPs, sem envolver, no entanto, a geraÃÃo de GMPc. / Hemorrhagic cystitis (HC) is an inflammatory event often associated with the use of oxazafosforins. We have demonstrated that amifostine (AMF) may prevent tissue damage caused by ifosfamide (IFO). Considering that IFO also changes the motor function of the lower urinary tract, the present study aimed to investigate whether AMF protects animals against IFO-related bladder dysfunction, and if this effect occurs through anitric oxide (NO) and ATP-sensitive potassium channels (KATP) dependent mechanism. Male Swiss mice (25-30g, n = 8) were given saline or IFO (400mg/kg, ip) to induce HC. Another group received, 30min before IFO, AMF (50mg/kg, sc), aminoguanidine (AMG, 50 mg/kg, ip), ODQ (2 mg/kg, po) or glibenclamide (GLI, 10 mg/kg, ip ). Other groups received 30min before AMF, L-arginine (L-ARG, 600 mg/kg, ip), ODQ or diazoxide (DIAZ, 2mg/kg ip). In another experimental setting, the groups that received AMG were pretreated with L-arginine and those receiving GLI were pretreated with diazoxide each drug administered at intervals of 30 min. Bladder wet weight (BWW) and macroscopic and histopathological parameterswere analyzed 12h after IFO injection. Inin vitro assays, bladder smooth muscle preparations were kept in saline solution aerated with 95% O2 - 5% CO2, pH 7.4 and at 37 &#8304; C for isometric muscle contractions record the depolarizing solutions of KCl and carbachol (CCh). For the record of intravesical pressure (IVP) by continuous cystometrogram (CC), laparotomy was performed and a polyethylene catheter attached to the bladder and exteriorized through the abdominal region was connected to a system of continuous infusion of saline (0.04mL/min) and a pressure transducer coupled to an acquisition system biological signals. In animals treated with AMF,BWW was reduced by 83% when compared with IFO-injected mice. L-ARG and DIAZ did not preventIFO-induced BWW effect (1.1% and 11.4%, respectively). Macroscopic and microscopic criteria were significantly reduced in AMF injected mice versus IFO group. L-ARG and DIAZ failed to prevent such criteria in comparison to IFO group. IFO decreased bladder strips contractility response to CCh versus control group (1.36 Â 0.24 Vs 0.18 Â 0.02 g force / mg of dry tissue, p <0.01). AMF prevented the IFO-related decrease in contractility response (1.47 Â 0.16 g force / mg of dry tissue, p <0.01). L-ARG and DIAZ did not alter IFO effect bladder contractility (0.6 Â 0.08 and 0.79 Â 0.12 g force / mg dry tissue, respectively, p <0.01 compared to saline control). In CC analysis, AMF reversed the increased voiding frequency (VF) caused by IFO (18 Vs 5.6 micturition/15 min, IFO and CTR, respectively, p <0.01. AMF â 6.5 micturition/15min; p <0.01 compared to IFO). The VFs in L-ARG and DIAZ (14.5 and 11.2 micturition/15min, respectively) were significantly different from AMF group, p <0.01. Cystometrography recordings of control and AMF groups showed regular micturition cycles with evident contractions associated with bladder emptying, which as markedly different from IFO-injected animals that presented an irregular trace concerning micturition cycles and no evident contractions associated with the urination event. DIAZ also showed similar patterns to that of IFO. Treatment with ODQ did not alter either the in vitro and in vivo effects on bladder dysfunction promoted by IFO or upon protection by AMF. AMF inhibited IFO-related functional alterations in bladders through multiple processes that probably include NO and KATPs without involvingthe generation of cGMP.