Implication du PTP dans la mort cellulaire β pancréatique L'hyperglycémie, l'hyperfructosémie et l'ischémie-reperfusion sont délétères pour la viabilité cellulaire β pancréatique, jouant un rôle majeur dans la perte de la masse cellulaire β. Le pore de transition de perméabilité mitochondriale (PTP) est un canal mitochondrial impliqué dans le déclenchement de la mort cellulaire. Des données récentes montrent l'implication du PTP et du stress oxydant dans la toxicité induite par l'ischémie-reperfusion sur cardiomyocytes et également dans la glucotoxicité induite sur cellules endothéliales. La première partie de notre étude a visé à étudier l'implication de l'ouverture du PTP dans la mort cellulaire des cellules INS-1 et des îlots pancréatiques humains soumis à de fortes concentrations de glucose et de fructose. Nous démontrons que l'incubation des cellules INS-1 et des îlots pancréatiques humains en présence de 30 mM de glucose ou 2,5 mM de fructose déclenche une ouverture du PTP et induit la mort cellulaire. La metformine et la Cyclosporine A (CsA) préviennent l'ouverture du PTP et la mort cellulaire induite par le glucose et le fructose. La deuxième partie de notre travail montre que l'exposition des INS-1 à une heure de carence en substrat concomitante d'une hypoxie, suivie d'une restauration des conditions basales conduit à l'ouverture du PTP et à une majoration drastique de la mort cellulaire. Ces deux évènements sont totalement prévenus par l'incubation préalable par la CsA et la metformine mais aussi par la N-Acétyl-Cystéine (NAC) ou par l'exposition à une anoxie, soulignant ainsi le rôle fondamental du stress oxydant dans le déclenchement de l'ouverture du PTP et de la mort cellulaire. Nous montrons qu'au cours de l'ischémie-reperfusion simulée, la production de superoxide est bi-phasique : nous décrivons un premier pic de production au cours de la carence en substrat, lié à un flux reverse d'électrons au sein du complexe I de la chaîne respiratoire. Ce premier pic est suivi d'un deuxième pic de production après la restauration du niveau de substrats et d'O2, lié à l'ouverture du PTP. La NAC, l'anoxie ou la metformine préviennent les deux pics de production de superoxide tandis que la CsA prévient seulement le second pic. Enfin, nous montrons que l'hypoxie seule n'induit ni stress oxydant, ni ouverture du PTP ni mortalité cellulaire. L'ensemble de notre travail démontre le rôle central du PTP dans la gluco-fructotoxicité et dans la toxicité induite par l'ischémie-reperfusion sur la cellule β pancréatique. Ainsi, prévenir l'ouverture du PTP peut-être une approche intéressante pour préserver la viabilité cellulaire β. / PTP involvement in β pancreatic cell death Hyperglycemia, hyperfructosemia and ischemia-reperfusion play a major role in the progression of β cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. PTP opening and oxidative stress have been shown to be involved in ischemia-reperfusion injury on cardiomyocytes and in hyperglycemia-induced cell death in endothelial cells. In the first part of this work, we have examined the involvement of PTP opening in INS-1 cells and human pancreatic islets cell death induced by high levels of glucose or fructose. We first reported that Metformin and Cyclosporin A (CsA) prevented Ca2+-induced PTP opening in permeabilized and intact INS-1 cells. We then shown that incubation of INS-1 cells and human islets in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. Because both Metformin and CsA prevented glucose and fructose induced PTP opening, and hampered glucose and fructose induced cell death, we conclude that PTP opening is involved in high glucose and high fructose induced INS-1 and human islets cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β cell viability. In the second part of the work, we demonstrate that the incubation of INS-1 cells in the absence of energy substrates in hypoxic condition for 1 hour followed by incubation in normal condition led to PTP opening and to a dramatic increase in cell death. Both events were totally prevented when PTP opening was inhibited by either Cyclosporin A (CsA) or Metformin or when the cells were incubated in the presence of the antioxidant N-acetyl-cystein (NAC), in anoxia, highlighting the implication of oxidative stress is the commitment of PTP opening. Superoxide production increased during the removal of energy substrates, due to reverse electron flux through complex I and again increased when normal energy substrate and O2 were restored, due to PTP opening. NAC, anoxia or Metformin prevented the two phases of oxidative stress, while CsA prevented only the second one. Hypoxia alone did not induce oxidative stress, PTP opening or cell death. Our work demonstrates the implication of PTP opening in ischemia-reperfusion injury and gluco- fructotoxicty in β pancreatic cells. We therefore suggest that preventing PTP opening might be a new approach to preserve β cell viability.

La progression de l'incidence de l'insuffisance rénale chronique dans le monde nécessite d'améliorer les mesures visant à ralentir sa progression et son évolution vers l'insuffisance rénale terminale. Le régime pauvre en protéines ou la prévention des épisodes d'ischémie- reperfusion appartiennent à ces mesures de néphroprotection mais leur bénéfice n'est qu'incomplètement compris. Le rein étant un des organes les plus vascularisé, rapporté à son poids, l'évaluation concomitante de la perfusion rénale ([15O]H2O), du métabolisme oxydatif ([11C]acetate) puis du contenu tissulaire en oxygène (BOLD-IRM) est fondamentale dans la mesure où ces trois mesures ne sont pas corrélées de manière linéaire. La TEP-IRM, appareil hybride combinant les fonctionnalités de la TEP et de l'IRM, permet une évaluation concomitante de ces trois paramètres de manière non invasive et sur rein isolé ce qui permet de l'utiliser en recherche clinique pour l'évaluation de l'effet de différentes interventions. Les régimes pauvres en protéines ont démontré qu'ils permettaient un ralentissement du déclin de la fonction rénale mais le mécanisme à l'origine de cet effet bénéfique n'est pas connu. Nous avons démontré que le contenu en AGE (Advanced Glycation End Products ou protéines modifiées) d'une charge protéique était responsable de la mobilisation de la réserve fonctionnelle rénale avec une augmentation de la perfusion et du métabolisme oxydatif plus importants après une charge protéique riche en AGE par rapport à une charge protéique pauvre en AGE. Nos résultats permettent de s'interroger sur l'intérêt d'une alimentation pauvre en AGE pour les insuffisants rénaux chroniques au-delà d'une restriction protéique pure. La prévention des épisodes d'ischémie- reperfusion est également particulièrement importante dans le cadre de la néphroprotection alors que la revascularisation d'une sténose de l'artère rénale s'accompagne obligatoirement de lésions de reperfusion. L'utilisation de la ciclosporine avant la dilatation d'une sténose de l'artère rénale dans le cadre d'un pré conditionnement a permis de réduire les lésions rénales chez la souris mais ce bénéfice clinique n'a pas été démontré chez l'homme. L'étude CICLOSAAR qui a pour but d'évaluer le bénéfice d'un pré conditionnement par la ciclosporine A avant dilatation d'une sténose de l'artère rénale sur des paramètres fonctionnels rénaux (perfusion, métabolisme oxydatif et contenu tissulaire en oxygène) est en cours. Dans un second temps et afin d'améliorer l'applicabilité clinique de ces techniques d'imagerie fonctionnelle, nous avons démontré que l'acétate, qui était jusque-là utilisé comme un marqueur de métabolisme oxydatif, pouvait également être utilisé comme un marqueur de perfusion rénale grâce à la modélisation de son uptake par le rein. Au total, la TEP- IRM est un outil fondamental pour l'évaluation des paramètres fonctionnels rénaux de manière non invasive chez l'humain. La mise en évidence de l'impact du contenu en AGE sur la perfusion et le métabolisme rénal devrait permettre d'affiner nos conseils diététiques aux insuffisants rénaux pour prévenir la progression de la maladie vers l'insuffisance rénale terminale. L'étude CicloSAAR actuellement en cours devrait permettre d'améliorer les mesures mises en oeuvre pour protéger le rein des lésions de reperfusion après dilatation d'une sténose de l'artère rénale. Enfin, nos résultats sur la forte corrélation entre les données de perfusion en acétate et en eau devraient permettre d'utiliser ce traceur pour obtenir la perfusion et le métabolisme oxydatif à l'issue d'une seule injection de radio-traceur et renforcer l'applicabilité clinique de ces techniques / The increase in the incidence of chronic renal failure worldwide requires improvements in measures to slow its progression to end-stage renal failure. The low-protein diet or the prevention of episodes of ischemia-reperfusion belong to these measures of nephroprotection but their benefit is only incompletely understood. The kidney is one of the most vascularized organs, compared to its weight, the concomitant evaluation of renal perfusion ([15O] H2O), oxidative metabolism ([11C] acetate) and tissue content in oxygen (BOLD-IRM) is fundamental in that these three measures are not linearly correlated. PETMRI, a hybrid device combining the functionalities of PET and MRI, allows a concomitant evaluation of these three parameters in a non-invasive and isolated kidney way which allows to use it in clinical research for the evaluation of the effect of different interventions. Low-protein diets have been shown to slow down the decline in renal function, but the mechanism behind this beneficial effect is not known. We have demonstrated that the AGE (Advanced Glycation End Products) content of a protein load is responsible for the mobilization of the renal functional reserve with an increase in perfusion and oxidative metabolism after a high- AGE high- protein compared to a low- AGE high- protein load. Our results make it possible to question the interest of a low-AGE diet for chronic renal insufficiency beyond a pure protein restriction. The prevention of episodes of ischemia reperfusion is also particularly important in the context of nephroprotection, whereas the revascularization of a stenosis of the renal artery is necessarily accompanied by reperfusion injury. Use of ciclosporin prior to dilation of renal artery stenosis in pre-conditioning reduced renal damage in mice, but this clinical benefit has not been demonstrated in humans. The CICLOSAAR study, which aims to evaluate the benefit of pre-conditioning with ciclosporin before dilation of renal artery stenosis with renal functional parameters (perfusion, oxidative metabolism and tissue oxygen content) is in progress. In a second step and to improve the clinical applicability of these functional imaging techniques, we have shown that acetate, which was previously used as a marker of oxidative metabolism, could also be used as a marker of renal perfusion through the modeling of its uptake by the kidney. In total, PET-MRI is a fundamental tool for the evaluation of renal functional parameters in a non-invasive way in humans. Demonstrating the impact of the AGE content of a protein load on perfusion and renal metabolism should help refine our dietary advice to patients with chronic kidney disease to prevent progression of the disease to end-stage renal failure. The current CicloSAAR study is expected to improve the measures implemented to protect the kidney from reperfusion injury after dilatation of renal artery stenosis. Finally, our results on the strong correlation between acetate and water perfusion data should allow this tracer to be used to obtain perfusion and oxidative metabolism after a single radio tracer injection and reinforce clinical applicability of these techniques

Einleitung: Der Ischämie/Reperfusionsschaden (I/R) ist die wesentliche Ursache des frühen Transplantatversagens nach Lebertransplantation (LTx). Fettlebern sind dabei besonders betroffen. Freie Sauerstoffradikale spielen bei der Pathogenese eine zentrale Rolle. Die Morphologie der so versagenden Transplantatleber ist charakterisiert durch Entzündung, Nekrose und Apoptose. Endogene Radikalfänger wie die Superoxiddismutase (SOD), nicht aber exogen zugefuhrte, bauen freie Radikale ab. Das Ziel der vorgelegten Studien war es, im Modell der LTx von gesunden und verfetteten Lebern in der Ratte mittels adenoviralem Gentransfer von SOD den I/R zu vermindern, die Überlebensrate zu erhöhen und zugrunde liegende Mechanismen aufzuzeigen. Methoden: Bei Experimenten mit verfetteten Lebern wurde eine ausgeprägte Steatose der Spenderlebern durch Füttern einer Ethanol- und fettreichen Diät (Lieber-DiCarli) erzeugt. Explantierte Lebern wurden für 24 h konserviert und orthotop transplantiert. Einigen Spendern wurde 72 h vor Organentnahme Cu/Zn-SOD enthaltendes Adenovirus (Ad.SOD1) i.v. appliziert. Als Kontrollen dienten Fettlebern, welche mit dem Gen von b-Galaktosidase (Ad.lacZ) transfiziert wurden, oder aber gesunde Lebern. Untersuchungsparameter waren neben Transfektionsparametern die Transaminasen, histopathologische Morphologie, Überlebensraten, sowie die Aktivierung von Transkriptionsfaktoren und deren Kinasen. Freie Radikale wurden in der Galle mittels Elektronenspin-Resonanz-Spektroskopie nachgewiesen. In weiteren Experimenten wurden auch die mitochondriale und die extrazelluläre Isoform hinsichtlich ihrer protektiven Wirkung untersucht. Ebenso wurde die Auswirkung der freien Radikale auf die Regeneration nach Teillebertransplantation untersucht. Ergebnisse: 72 h nach Injektion von Ad.lacZ exprimierten etwa 80% aller Hepatozyten die b-Galaktosidase. In der Ad.SOD1 Gruppe war die Genexpression 3-fach, die Aktivität 12-fach erhöht. Im Vergleich zu den unbehandelten oder Ad.lacZ infizierten Empfängern von Fettlebern, stiegen die Transaminasen um etwa 50% bei der Ad.SOD1 Gruppe an. Alle Empfänger von Ad-SOD1 behandelten Fettlebern überlebten, hingegen nur 10% der Ad.lacZ Gruppe. Etwa 35% der Hepatozyten von Fettlebern waren nekrotisch, jedoch nur 10% in Ad.SOD1-behandelten Fettlebern. Ad.SOD1 halbierte die Freisetzung von freien Radikalen und minimierte die Aktivierung von NF-kB. Die Aktivität der Kinase IKK wurde nicht reduziert, der Anstieg der Aktivität von JNK jedoch komplett inhibiert. Die Freisetzung von TNFa wurde nicht beeinflußt. Als wirksamste Isoform hat sich die zytosolische erwiesen, die extrazelluläre ist nach Überexpression ohne protektive Wirkung. Die Leberregeneration läßt sich nach Transplantation durch SOD-Überexpression massiv anregen und das Organversagen bei kritischer Leberzellmasse vermeiden. Schlußfolgerung: Diese Studie zeigt erstmals die Wirksamkeit einer neuen Strategie zur Organprotektion fur gesunde Lebern und Fettlebern. Die Eliminierung von Sauerstoffradikalen spielt bei der Pathogenese eine Schlüsselrolle. Der adenoviraler Gentransfer von SOD stellt ein gangbares therapeutisches Verfahren für die Zukunft dar, um auch marginale, verfettete Organe vor reperfusionsbedingtem Versagen zu schützen. Dabei ist die zytosolische SOD am effektivsten. Auch bei der Teilleber-Transplantation ist diese Therapieform erfolgversprechend. / Background: Oxygen-derived free radicals play a central role in pathomechanisms of reperfusion injury after organ transplantation, and fatty livers are particularly susceptible. Endogenous radical scavenger systems such as superoxide dismutase (SOD) degrade toxic radicals; however, SOD is degraded rapidly when given exogenously. Therefore, the hypothesis that treatment of the donor liver with an adenoviral vector encoding the Cu/Zn-SOD gene (Ad.SOD1), or the Mn-SOD gene or the ec-SOD gene would lead to permanent gene expression and therefore protect the organ against injury and increase survival in a rat model of liver transplantation including fatty livers was tested. Transplantation of reduced-size livers may lead to a hypermetabolic state and increased production of oxygen radicals. Since oxygen radicals may cause liver injury and impair liver regeneration, we tested the hypothesis that overexpression of superoxide dismutase (SOD) in reduced-size livers (RSL) would accelerate regeneration and reduce injury in a rat model of transplantation of RSL. Methods: Donors received chow diet (untreated), high-fat diet, or ethanol-containing high-fat diet. Some donors were infected with Ad-SOD1, while untreated grafts and livers infected with the indicator gene lacZ encoding bacterial b-galactosidase (Ad.lacZ) served as controls. Some livers were harvested 72 hours later, reduced to 45% of weight, and transplanted. After liver transplantation, SOD activity and protein expression in liver, survival, histopathology, release of transaminases, free radical adducts in bile and activation of NF-kB, IkB kinase (IKK), Jun-N-terminal kinase (JNK) and TNFa were evaluated. Moreover, in transplanted split-livers regeneration was evaluated by Brdu-staining, and measurement of cyclinD1 and p21. Results: Approximately 80% of hepatocytes expressed b-galactosidase 72h after injection of Ad-lacZ. Moreover, SOD1 gene expression and activity were increased 3- and 10-fold in the Ad-SOD1 group, respectively. Following transplantation, 20-25% of rats treated with Ad.lacZ survived. In contrast, all SOD1-treated animals survived. Transaminases measured 8h after transplantation in Ad-SOD1 rats were only 40% of those in controls which increased 40-fold above normal values. Approximately 20% of hepatocytes in untreated and Ad.lacZ-infected organs were necrotic 8h after reperfusion, whereas necrosis was nearly undetectable in grafts from rats treated with Ad.SOD1. Free radical adducts were increased 2-fold in the ethanol group compared to untreated controls. Ad.SOD1 blunted this increase and reduced the activation of NF-kB, which was similar in untreated and ethanol-treated groups. Ad.SOD1 did not affect activity of IKK, but JNK activity was blunted. Release of TNFa was not affected. In recipients of Ad.SOD1-RSL survival was dramatically increased (100% vs. 20% in Ad.lacZ-RSL), and peak levels of AST/ALT and bilirubin levels were reduced by 75% and 87.5%, respectively (p

Diese Arbeit befasst sich tierexperimentell und klinisch mit Aspekten der Nierenfunktion bei der Behandlung angeborener Herzfehler. Von Patientenseite sind das Neonatal- und Säuglingsalter und ab der Adoleszenz eine chronische Zyanose, von Behandlungsseite Röntgenkontrastmittelgaben, Dauer, Blutviskositätsänderungen und Kreislaufstillstand am kardiopulmonalen Bypass Risikofaktoren. Cortikosteroidgaben, Optimierung von Blutviskosität und Hydratation sowie eine prophylaktische Peritonealdialyse sind Ansätze zur Behandlung eines Nierenschadens. Die Ergebnisse zeigen, dass Verbesserungen der Plasmaviskosität Nierenschäden am hypothermen kardiopulmonalen Bypass vermindern während eine Cortikosteroidgabe vor Kreislaufstillstand bei Ferkeln nicht nephroprotektiv wirkt. Bei Risikopatienten erweist sich der prophylaktische Einsatz einer Peritonealdialyse als günstig. Bei chronisch zyanotischen Patienten mit einer Glomerulopathie und einem erhöhtem Risiko für Röntgenkontrastmittelexposition und kardiopulmonale Byppassoperationen muss der Nierenstatus die Operationsplanung und postoperative Therapie beeinflussen. Nephroprotektion und Verbesserungsmöglichkeiten der Blutviskosität am kardiopulmonalen Bypass müssen weiter untersucht werden. / This work focusses on clinical aspects of kidney function in the treatment of congenital heart disease. Neonates and infants as well as adolescents with cyanosis may be especially at risk. Contrast agents, duration, blood viscosity changes, and circulatory arrest in cardiopulmonary bypass may be risk factors. Corticosteroids, optimized blood viscosity and hydration, and early onset of peritoneal dialysis are considerations of treatment. Our results demonstrate a reduction of renal damage with optimized plasma viscosity during hypothermia in cardiopulmonary bypass, while corticosteroids have no advantage in young pigs after circulatory arrest. Prophylactic treatment with peritoneal dialysis has advantages in patients at risk. In chronicly cyanotic patients with glomerulopathy the risk of contrast agents and cardiopulmonary bypass is elevated. Thus, renal status should influence operative procedures and postoperative treatment. The possibilities of nephroprotection and improvement of blood viscosity should be further evaluated.

In der vorliegenden Arbeit wurde die Bedeutung von intraepithelialen Lymphozyten (IEL), oxidativem Streß und endogenen Schutzmechanismen bei GvHR, Dünndarmtransplantation, Sepsis, Morbus Crohn sowie intestinalem Ischämie/Reperfusionsschaden (I/RS) analysiert. Die Bestimmung der phänotypischen und funktionellen Charaktistika der IEL im Rahmen der o. g. Erkrankungen wies auf eine Selektion bestimmter T-Zell Subpopulationen in der Darmschleimhaut hin. Zusätzlich konnte gezeigt werden, daß IEL nicht nur als Effektorzellen zur mukosalen Barrierefunktion beitragen, sondern auch regulierende Funktionen bei weiteren Abwehrmechanismen der Darmschleimhaut, wie z.B. der NOS-2 Expression besitzen. Die Untersuchungen zum intestinalen I/RS zeigten eine Gewebeschädigung nicht nur im Darm sondern auch der Leber nach selektiver intestinaler Ischämie. Dabei konnte in beiden Organen oxidativer Streß als ein Faktor der Gewebeschädigung nachgewiesen werden. Bei der Modulation des I/RS durch Gabe von Zytokinen konnte eine Zunahme des I/RS durch Gabe von IL-10 und eine Abnahme des I/RS durch IL-2 erreicht werden. Der positive Effekt der IL-2 Gabe war von einer verstärkten und verlängerten NOS-2 mRNA Expression sowie einer gesteigerten NO-Freisetzung begleitet. Im Gegensatz dazu fehlte nach IL-10 Gabe die Zunahme der NOS-2 Epxression ebenso wie ein Anstieg der NO-Metabolite im Serum. Die verminderte NO-Produktion könnte somit den negativen Effekt des anti-inflammatorischen IL-10 auf den I/RS erklären. / In this study, the role of intraepithelial lymphocytes (IEL) was analyzed in Graft-versus-Host disease, small bowel transplantation, sepsis and inflammatory bowel disease. Furthermore, the influence of oxidative stress and endogenous protective mechanisms on the development of intestinal ischemia/reperfusion injury was determined. The phenotypic and functional characteristics of IEL in these diseases indicated that only specific T-cell subsets selectively migrate and/or survive in the intestinal mucosa. In addition, it was demonstrated that IEL display several functions in the intestinal barrier system: they are cytolytic effector cells, but do also exert regulatory functions on the expression of mucosal host defense mechanisms such as NOS-2 expression. The investigations on intestinal ischemia / reperfusion injury revealed that selective intestinal ischemia induces tissue injury not only in the intestine, but in the liver as well. In both organs, oxidative stress plays a predominant role in the development of tissue destruction. Modulation of I/RS by administration of cytokines lead to increased tissue damage after IL-10 administration and reduced tissue injury after IL-2 administration. The beneficial effect of IL-2 may have been due to an increased NOS-2 mRNA expression and the subsequently increased NO production. In contrast, IL-10 administration failed to induce an increased NOS-2 mRNA expression or NO production in the intestine and liver.

Made available in DSpace on 2016-01-26T12:51:46Z (GMT). No. of bitstreams: 1 idamariamfernandescharpiot_tese.pdf: 2769759 bytes, checksum: f77da7f002a249ac9d78cbaceddbde38 (MD5) Previous issue date: 2011-12-12 / Introduction: Renal ischemia is the most important cause of acute kidney injury (AKI). Methylprednisolone (MP) has been shown to give protection against ischemia/reperfusion injury (I/R) in the liver and the heart. Objective: To examine a possible protective role of MP in renal I/R. Methods: Male Wistar rats were treated with 30mg/kg of intravenous MP or saline 1 hour before unilateral renal ischemia (RI), which lasted for 30 minutes. The animals were divided into 3 groups (8 in each group): Sham (sham surgery without RI), Vehicle (Veic)-I/R (saline infusion followed by RI), and the MP-I/R group (MP infusion followed by RI). The glomerular filtration rate (GFR) - which is inulin clearance in ml/min/100g, sodium fractional excretion (FENa), urinary osmolality, and histological analysis were assessed 2 days after RI. Additionally, immunohistochemical staining (2 days after RI) was performed to measure macrophages (ED-1 positive cells), neutrophils (No), and lymphocytes (Lo) and the nuclear factor-&#954;B (NF&#954;-B). Results are expressed as mean ± SD, and were compared by ANOVA, followed by Bonferroni test, with p < 0.05. Results: GFR was 0.92 ± 0.30 ml/min/100g in the MP-I/R group, 0.90 ± 0.27 ml/min/100g in the Sham group, and 0.47 ± 0.24 ml/min/100g in the Veic-I/R group (p < 0.05 vs. MP-I/R and Sham). The FENa was similar in the MP-I/R (0.19%) and Sham groups (0.35%, NS), and higher in the Veic-I/R group (0.62%, p < 0.05 vs. MP-I/R). Urinary osmolality was similar between the 3 groups. Acute epithelial degenerative changes and tubular dilatation were significantly more intense in the Veic-I/R group than the MP-I/R and Sham groups. Only the Veic-I/R group presented with focal acute tubular necrosis. In the cortex, the number of Lo was significantly greater in the Veic-I/R group when compared with the Sham and MP-I/R groups (14.36 ± 3.32 vs. 6.75 ± 1.18 and 5.31 ± 1.63, respectively, p < 0.05 Veic-I/R vs. Sham and MP-I/R) and in the outer medulla (OM) areas (10.58 ± 3.04 vs. 4.51 ± 1.29 and 3.70 ± 0.62, p < 0.05 Veic-I/R vs. Sham and MP-I/R). The number of macrophages was also significantly greater in the Veic-I/R group (9.84 ± 3.18) when compared with Sham (4.65 ± 1.12, p < 0.05) and MP-I/R groups (4.06 ± 1.84, p < 0.05). Similarly, the number of No in the OM was 3.13 ± 2.09 in Veic-I/R vs. 0.74 ± 0.51 in Sham group, and 1.44 ± 1.11 in the MP-I/R group (p < 0.05). The NF&#954;-B expression was more intense in the OM in the Veic-I/R group compared with the Sham and in the MP-I/R groups (0.61 ± 0.33 vs. 0.03 ± 0.03 and 0.12 ± 0.11 respectively, p < 0.05). Conclusion: The pretreatment with high doses of MP conferred striking protection against renal I/R. This protection effect was related to the modulation of I/R-induced inflammatory mechanisms and to inflammatory cell infiltration triggered by I/R. / Introdução: A isquemia renal é a causa mais importante de injúria renal aguda (IRA) hospitalar. A metilprednisolona (MP) tem se mostrado protetora contra a lesão de isquemia/reperfusão (I/R) em fígado e coração. Objetivo: Estudar o possível efeito protetor da MP na lesão de I/R renal. Métodos: Ratos machos Wistar foram tratados com 30mg/kg, por via intravenosa, de MP ou solução de NaCl 0,9%, 1 hora antes de isquemia renal (IR) unilateral de 30 min. Os animais foram divididos em três grupos (n de 8 em cada grupo): Sham (cirurgia sham sem IR), Veículo (Veic)-I/R (infusão de solução de NaCl 0,9% seguida por IR), e MP-I/R (infusão de MP seguida por IR). O RFG (depuração de inulina, ml/min/100g), a fração de excreção de sódio (FENa), a osmolalidade urinária e a análise histológica foram analisados dois dias após IR. Realizou-se também imuno-histoquímica (dois dias após IR) para quantificação de macrófagos (ED-1), neutrófilos (No), linfócitos (Lo) e fator-kapa-B nuclear (NF&#954;-B). Os resultados são expressos como média ± DP e foram comparados por ANOVA, seguido pelo teste de Bonferroni, com p < 0,05. Resultados: O RFG foi 0,92 ± 0,30 ml/min/100g no grupo MP-I/R, 0,90 ± 0,27 ml/min/100g no grupo Sham e 0,47 ± 0,24 ml/min/100g no grupo Veic-IR, (p < 0,05 vs. MP-I/R e Sham). A FENa foi semelhante nos grupos MP-I/R (0,19%) e Sham (0,35%, NS), e maior no grupo Veic-I/R (0,62%, p < 0,05 vs. MP-I/R). Volume e osmolalidade urinária foram similares entre os três grupos. Constataram-se alterações epiteliais degenerativas agudas e dilatação tubular significativamente mais intensas no grupo Veic-I/R em relação aos grupos MP-I/R e Sham. Apenas o grupo Veic-I/R apresentou focos de necrose tubular aguda. O número de Lo foi significativamente maior no grupo Veic-I/R comparado aos grupos Sham e MP-I/R no córtex (14,36 ± 3,32 vs. 6,75 ± 1,18 e 5,31 ± 1,63, respectivamente, p < 0,05 Veic-I/R vs. Sham e I/R-MP) e medula externa (ME) (10,58 ± 3,04 vs. 4,51 ± 1,29 e 3,70 ± 0,62; p < 0,05 Veic-I/R vs. Sham e MP-I/R). O número de macrófagos também foi significativamente maior no grupo Veic-I/R (9,84 ± 3,18) comparado com Sham (4,65 ± 1,12; p < 0,05) e MP-/IR (4,06 ± 1,84; p < 0,05). Da mesma forma, o número de No na medula externa foi 3,13 ± 2,09 em Veic-I/R vs. 0,74± 0,51 em Sham e 1,44 ±1,11 em MP-I/R (p < 0,05 Veic-I/R vs. Sham). A expressão de NF&#954;-B foi significativamente mais intensa na medula externa do grupo Veic-I/R comparada com os grupos Sham e MP-I/R (0,61 ± 0,33 vs. 0,03 ± 0,03 e 0,12 ± 0,11, respectivamente, p < 0,05). Conclusão: O pré-tratamento com doses elevadas de MP protegeu intensamente os animais contra a lesão de I/R renal. Este efeito protetor foi relacionado à modulação de mecanismos de inflamação e infiltração por células inflamatórias desencadeados pela I/R.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-18T19:02:52Z No. of bitstreams: 1 PRISCILA MARQUES SOSA.pdf: 4777732 bytes, checksum: 5e904d610fde40315a7aeacae32f9d55 (MD5) / Made available in DSpace on 2016-04-18T19:02:52Z (GMT). No. of bitstreams: 1 PRISCILA MARQUES SOSA.pdf: 4777732 bytes, checksum: 5e904d610fde40315a7aeacae32f9d55 (MD5) Previous issue date: 2016-03-15 / O AVE é uma das principais causas de morte e incapacidade funcional em todo mundo, sendo dividido em dois subtipos: isquêmico, causado pela diminuição do fluxo sanguíneo; e hemorrágico, caracterizado pelo extravasamento de sangue nos tecidos encefálicos. Considerando a alta taxa de mortalidade e a gravidade das sequelas pós AVE, torna-se de extrema importância a busca por alvos terapêuticos que visem diminuir as sequelas causadas pelos quadros isquêmico e hemorrágico. Sendo assim, este estudo investigou os efeitos neuroprotetores do exercício físico (8 semanas previamente à lesão) em um quadro de AVE isquêmico (através da oclusão bilateral das artérias carótidas comuns) e os efeitos neuroprotetores da apocinina (posteriormente à lesão – 2, 6 e 24h – na dose 0,5mg/kg) em um quadro de AVE hemorrágico (através da infusão de colagenase no corpo estriado) em ratos Wistar. Para avaliar a função motora dos animais, foram utilizados os testes de Campo Aberto (CA), Rotarod (RR) e Escala de Déficit Neurológico (NDS), e, para avaliar o balanço redox estriatal, avaliamos a presença de EROs, TBARS (espécies reativas ao ácido tiobarbitúrico) e capacidade antioxidante total (FRAP). Nossos resultados mostraram que o exercício físico é uma estratégia parcialmente eficaz de proteção em um modelo de AVE isquêmico. No entanto, a apocinina não se mostrou uma estratégia de neuroproteção eficaz em um modelo de AVE hemorrágico. Estes resultados revelam a possibilidade da utilização do exercício físico como estratégia de neuroproteção. A apocinina, por sua vez, precisa ser melhor estudada em casos de AVE hemorrágico, considerando a investigação do seu mecanismo, doses e tempos de administração. / The stroke is one of the leading causes of death and disability worldwide, and is divided into two subtypes: ischemic, caused by a decreased on blood flow; and hemorrhagic, characterized by leakage of blood in brain tissue. Considering the high mortality rate and severity of post stroke sequelae, it is extremely important to search for therapeutic targets aimed at reducing the consequences caused by ischemic and hemorrhagic frames. Thus, this study investigated the neuroprotective effects of physical exercise (8 weeks prior to injury) in an ischemic stroke modle (by bilateral occlusion of the common carotid arteries) and the neuroprotective effects of apocynin (after the injury - 2, 6 and 24 hours - at a dose 0.5 mg/kg) in a hemorrhagic stroke model (by collagenase infusion into the striatum) in Wistar rats. Open Field (OF), Rotarod (RR) and Neurologic Disabilities Scale (NDS) were used to evaluate the motor function of the animals. To the striatal redox balance evaluation we assessed the presence of ROS, TBARS (reactive species to thiobarbituric acid) and total antioxidant capacity (FRAP). Our results showed that physical exercise is a partially effective strategy to protect against ischemic stroke. However, apocynin was not an effective neuroprotective strategy in a experimental model of hemorrhagic stroke. These results show the possibility of using exercise as a neuroprotective strategy. The apocynin need to be better studied in cases of hemorrhagic stroke, whereas the investigation of its mechanism, dosages and times of administration.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH. In the myocardium, NHE1 has been implicated in ischemia/reperfusion (I/R) and cardiac hypertrophy (CH). Hormonal, autocrine and paracrine stimuli, acidosis, cardiotoxic metabolites released during I/R and CH increases NHE1 protein expression and activity. The involvement of NHE1 in CH and I/R has been further supported with the use of NHE1 inhibitors, which have been beneficial in the prevention/regression of several models of CH and I/R injury. Despite the fact that elevation of NHE1 expression and activity have been demonstrated in several models of heart disease, it was unclear whether elevation of NHE1 protein expression was sufficient to induce a specific cardiac pathology, or whether activation of the protein was required. To understand the direct role of NHE1 in CH and I/R, an in vivo and in vitro gain-of-function model, expressing varying levels and activities of NHE1 were examined. In vivo, our N-line mice expressed wild type NHE1 and our K-line mice expressed constitutively active NHE1. In vitro, neonatal rat ventricular cardiomyocytes were infected with the IRM adenovirus containing wild type NHE1 or the K-IRM adenovirus containing active NHE1. We demonstrated that expression of constitutively active NHE1 promotes CH to a much greater degree than expression of wild type NHE1 alone, both in vivo and in vitro. This NHE1-dependent hypertrophic response occurred independent of signaling pathways involved in CH including, mitogen activated protein kinases, p90 ribosomal S6 kinase, calcineurin and glycogen synthase kinase. The NHE1-dependent hypertrophic effect also occurred independent of gender. In addition, the expression of active NHE1 increased the susceptibility of intact mice to neurohormonal stimulation and progressed the hypertrophic response. When these hearts expressing active NHE1 were subjected to I/R using the ex vivo working heart perfusion model, fatty acid (FA) oxidation and glycolysis rates increased, thus generating greater ATP production rates. This was associated with cardioprotective effects in the myocardium, as well as a more energetically efficient myocardium. Expression of the endoplasmic reticulum (ER) stress response proteins, calreticulin and PDI were also shown to be increased relative to controls, and may contribute to the cardioprotection observed. We demonstrate that active NHE1 induces cardioprotection and alters cardiac metabolism in working hearts subjected to I/R. Overall, our results suggest that expression of active NHE1 has a double edged sword effect, on one side it induces CH while on the other side, it protects the heart against I/R injury.

In dieser Dissertation wurden am Ferkelmodell (8-15 kg, drei Gruppen: „Kontrolle“ n=7, „Herz-Lungen-Maschine (HLM)“ n=10, „(-)-Epigallocatechin-3-Gallat (EGCG)“ n=6, die Kontrollgruppe wurde thorakotomiert, die HLM- und die EGCG-Gruppe wurden thorakotomiert und für 90 Minuten an eine HLM angeschlossen, die EGCG-Gruppe erhielt vor und nach der HLM-Zeit EGCG) drei Fragestellungen behandelt: Erstens wurde untersucht, ob die Verwendung einer HLM während eines kardiochirurgischen Eingriffes unter hypothermen Bedingungen mit nicht-pulsatilem Blutfluss und Kardioplegie die Niere schädigte. Dafür wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe mit Hämatoxylin-Eosin (HE) angefärbt und unterschiedliche Strukturen betrachtet, wobei histopathologische Veränderungen in der HLM-Gruppe auffielen. Paraklinisch fanden sich erhöhte nierenspezifische Blutwerte (Serumkreatinin und -harnstoff) in der HLM-Gruppe. Diese Ergebnisse waren hinweisend für eine funktionell relevante Schädigung der Niere durch die HLM. Unterstützend kam ein Absinken des Gesamteiweißes im Serum der HLM-Gruppe hinzu, was auf eine generelle Schädigung des Organismus durch die HLM hindeutete. Zweitens wurde betrachtet, ob die gesetzten Schäden die Merkmale eines Ischämie-Reperfusionsschadens aufwiesen. Hierzu wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe immunhistochemisch (Hypoxie-induzierter-Faktor-1-alpha-Tyramide- Signal-Amplification (HIF-1-alpha-TSA)-, Nitrotyrosin-3-Amino-9-Ethylcarbazol (Nitrotyrosin-AEC)- und Apoptose-induzierender-Faktor-Tyramide-Signal-Amplification (AIF-TSA)-Färbung) angefärbt. Dabei zeigte sich, dass sich die HLM-Gruppe in einer hypoxischen Situation befand (HIF-1-alpha Akkumulation in den Zellkernen), nitrosativem Stress ausgesetzt war (Nitrotyrosin in den Tubuli) und dass sie teilweise so stark geschädigt wurde, dass Apoptose induziert wurde (AIF in Zellkernen) – alle drei Färbungsergebnisse waren hinweisend für einen ischämischen Zustand, in dem sich die HLM-Gruppe befunden hat. Auch die Ergebnisse der durchgeführten renalen Reversed Phase High Performance Liquid Chromatography (RP-HPLC) deuteten auf ebendies hin. Unterstützend wirkten die Ergebnisse des arteriellen Laktats – die HLM-Gruppe zeigte eine Hyperlaktämie – und die Tatsache, dass einige der histologischen Merkmale für eine frühe Schockniere (welche ischämischen Ursprungs sein kann) in der HLM-Gruppe gefunden wurden. Dies alles zeigte, dass der HLM-assoziierte Nierenschaden vorrangig die Natur eines Ischämie-Reperfusionsschadens aufwies. Drittens wurde untersucht, ob EGCG diese HLM-assoziierte Schädigung abmildern konnte. Dafür wurden bei der EGCG-Gruppe alle oben genannten Untersuchungen durchgeführt. Die Ergebnisse zeigten, dass EGCG in der Dosierung 10 mg/kg eine renoprotektive Wirkung gegen die HLM-assoziierten Schäden hatte, und diese abmildern bzw. ihnen entgegenwirken konnte. Diese Ergebnisse sind für die pädiatrische Kardiochirurgie interessant, welche zum Beispiel bei der Korrektur angeborener Herzdefekte auf die Verwendung der HLM angewiesen ist. Komplikationen wie eine Nierenschädigung post operationem sind nicht selten und verkomplizieren den Verlauf. Die vorliegende Dissertation zeigt das renoprotektive Potential des in grünem Tee vorkommenden Katechins EGCG im Umfeld eines kardiochirurgischen Eingriffes mit Verwendung einer HLM. Die Wirksamkeit dieser Substanz ist wahrscheinlich darin begründet, dass sie mehr als ein Antioxidans ist. Neben seiner Radikalfänger- und Stickstoffmonoxidscavenger-Fähigkeiten ist EGCG außerdem antiapoptotisch wirksam. Derzeit wird die Kardiochirurgie mit Verwendung einer HLM in der Veterinärmedizin nur in wenigen Zentren angewendet. Es besteht für die Zukunft jedoch die Hoffnung, dass gerade für Kleintierbesitzer, die ihre Tiere als Familienmitglied betrachten, und auch für zoologische Einrichtungen bei der Diagnose eines Herzfehlers die Kardiochirurgie mit Verwendung einer HLM als Therapiemöglichkeit eine interessante und realistische Alternative zur bislang angewandten palliativen medikamentösen Therapie darstellen kann. / In this dissertation a piglet model (8-15 kg, three groups: “control” n=7, “extracorporeal circulation (EC)” n=10, “EGCG” n=6, the control-group was thoracotomized, the EC- and the EGCG-group were thoracotomized and underwent cardiopulmonary bypass (CPB) for 90 minutes, and the EGCG-group received EGCG before and after the CPB) is presented. Three questions were raised and answered: Firstly, it was investigated if the use of a CPB during cardiac surgery with hypothermia, non-pulsatile blood flow and cardioplegia caused damage to the kidney. In order to answer this question, paraffin slices of the kidney of the control- and the EC-group were stained with hematoxylin-eosin (HE), and different structures were evaluated – this staining showed histopathological changes in the EC-group. Paraclinical, the EC-group showed elevated kidney-specific blood parameters (serumcreatinine and -urea). These findings indicated a functionally relevant impairment of the kidney caused by the CPB. Supporting this, the EC-group also showed a decline of the total amount of proteins in the serum, which was suggestive of a generalized injury of the body by the CPB. Secondly, it was investigated whether the injury of the kidney might have been caused by an ischemia/reperfusion injury. Therefore, paraffin slices of the kidney of the control- and the EC-group were immunhistochemically stained (hypoxia-induced-factor-1-alpha-tyramidesignal-amplification (HIF-1-alpha-TSA)-, nitrotyrosine-3-amino-9-ethylcarbazole (nitrotyrosine-AEC)- and apoptosis-inducing-factor-tyramide-signal-amplification (AIF-TSA)-staining). These stainings revealed, that the EC-group had suffered from a hypoxemic situation (accumulation of HIF-1-alpha in the nuclei), from nitrosative stress (presence of nitrotyrosine in the tubuli), and that the kidney was partly damaged to the point of an induction of apoptosis (presence of AIF in the nuclei) – all three of these findings indicated, that the kidneys of the EC-group were put into an ischemic situation. The findings of the renal reversed phase high performance liquid chromatography (RP-HPLC) indicated the same thing. This was also supported by the blood parameter of lactate – the EC-group showed a hyperlactemia – and by some histological findings in the EC-group, which were characteristical for an early shock-kidney (which may be caused by ischemia). Taken together, these findings showed that the CPB-associated kidney injury was primarily caused by an ischemia/reperfusion injury. Thirdly, it was investigated, whether EGCG might attenuate the CPB-associated kidney injury. For that purpose, all of the investigation methods mentioned above were carried out with the samples of the EGCG-group. The findings showed that EGCG (dose: 10 mg/kg) had a protective effect on the kidney, protecting it against the damage caused by the CPB, and was able to partly attenuate this damage and partly even fully counteract it. These findings are of interest for pediatric cardiac surgery, which for example for the correction of innate heart defects depends on the use of CPB. Complications – like acute renal injury post operationem – occur frequently and complicate the recovery. This dissertation demonstrates the renoprotective potential of the natural compound EGCG in the setting of cardiac surgery with the use of CPB. The reason for the effectiveness of EGCG in this situation probably is that EGCG is more than an antioxidant. EGCG not only works as a radical- and nitric-oxide-scavenger, but also is antiapoptotic. In veterinary medicine cardiac surgery with CPB is done by few centers only. However for the future there is hope that people – especially pet owners who view their companion animals as family members, and zoos – become more and more willing to and interested in having an animal diagnosed with a heart defect treated with cardiac surgery including the use of an CPB, instead of – like its usually done nowadays – only giving palliative medication to the animal.

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