Orientador: Jose B. Lopes de Faria / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T08:15:20Z (GMT). No. of bitstreams: 1 Ribaldo_PerolaDelBianco_M.pdf: 920213 bytes, checksum: e99a6b84c11f7c94edce6679b6c82c1d (MD5) Previous issue date: 2008 / Resumo: Introdução e Objetivos: Ha. muito se sabe que a hiperglicemia e a hipertensão arterial são fenômenos promotores de estresse oxidativo no tecido renal. O estresse oxidativo, por sua vez, contribui fortemente com a doença renal do diabetes, especialmente pela geração de oxidantes via NAD(P)H oxidase. Em contrapartida, inúmeros pesquisadores tem sugerido que o chá verde (Camellia sinensis) possa reduzir o estresse oxidativo. Desta forma, como objetivos deste estudo, testamos se a administração de chá verde a ratos espontaneamente hipertensos (SHR) diabéticos ameniza o dano renal, verificando ainda se os efeitos se fazem por redução do estresse oxidativo. Métodos: Ratos SHR com 12 semanas de idade foram tornados diabéticos por injeção de estreptozotocina (50 MG/kg) e os grupos controle receberam somente o veiculo (tampão citrato de sódio pH 4.5). Animais diabéticos (SD) (glicemia > 270 MG/dl) e controle (SC) foram randomicamente divididos para receber chá verde (CV) ou água (A) como fonte única de líquidos, totalizando 8 a 10 animais por grupo, sendo eles SCA (SHR controle recebendo água), SCC (SHR controle recebendo chá verde), SDA (SHR diabético recebendo água) e SDC (SHR diabético recebendo chá verde). Peso, glicemia, pressão arterial sistólica (PAS) e albuminuria foram determinados a cada 4 das 12 semanas de acompanhamento. Resultados: Durante as 12 semanas de estudo, as glicemias foram significativamente maiores (p<0.001) nos animais SDA do que nos controles SCA e SCC , não sendo alteradas pela administração de CV. As PAS não foram afetadas pelo diabetes ou pelo tratamento com CV. A albuminuria dos animais não diabéticos SCA e SCC foram semelhantes, aumentando significativamente (p=0.048) nos SDA e reduzindo no tratamento com o CV (SDC) de forma significativa (p=0.048). A atividade da NAD(P)H oxidase, determinada por quimiluminescência foi significativamente maior (p=) no grupo SDA comparado ao SCA. O tratamento com CV reduziu de forma significativa (p=0.01) na ausência e presença do diabetes. A expressão da subunidade NOX-4 da NAD(P)H oxidase, determinada por Western Blot, aumentou de forma significativa (p=0.04) no grupo SDA em relação aos grupos não diabéticos SCA e SCC. Porem, em ratos diabéticos tratados com CV (SDC) esta expressão reduziu significativamente (p=0.04) chegando aos níveis do controle. A nitro tirosina, um marcador de modificação de proteína induzida pelo estresse oxidativo/nitros ativo avaliada por Western Blot, foi significativamente (p=0.04) maior no SDA do que no SCA e SCC, sendo no entanto significativamente reduzida (p=0.04) no grupo SDC . A expressão renal de colágeno IV determinada por Western Blot foi significativamente aumentada (p=0.008) nos ratos SDA comparados aos SCA e SCC. NA presença do diabetes, a administração de CV reduziu de forma significativa (p=0.013) esta variável. Conclusão: Em ratos hipertensos e diabéticos, o chá verde restabeleceu o equilíbrio redox, reduzindo marcadores de nefropatia sem alterar glicemia e pressão arterial. Desta forma, o consumo de chá verde pode ser benéfico a pacientes diabéticos e hipertensos. / Abstract:It has been suggested that green tea (GT) has beneficial properties in the treatment or prevention of human disease. The major biological mechanisms of action of GT are being attributed to its antioxidant properties. Several lines of evidence suggest that oxidative stress contributes to the pathogenesis of diabetic nephropathy. The effect of GT on diabetic nephropathy is unclear. We tested the hypothesis that GT prevents diabetes and hypertension-related renal oxidative stress and attenuates renal injury. Spontaneously hypertensive rats (SHR) with streptozotocin-induced diabetes and control SHR were treated daily with tap water or fresh GT extract. After 12 weeks, the systolic blood pressure was unchanged by diabetes or GT. However, there was a decrease in body weight and a significant (p<0.0001) increase in blood glucose level in diabetic SHR. These parameters were unaffected by GT therapy. GT prevented the increase in 8-hydroxy-2'- deoxyguanosine, an indicator of renal oxidative stress-induced DNA, and nitrotyrosine, an indicator of oxidative stress-induced protein modification, that were significantly (p<0.01) elevated in diabetic SHR. Likewise, GT attenuated(p=0.048) ADPH oxidase-dependent superoxide generation and the expression of renal ortex NOX-4, a subunit of NADPH oxidase, that were elevated (p=0.013) in diabetic HR. Treatment with GT significantly (p=0.048) reduced the indices of renal injury, lbuminuria and renal expression of collagen IV in diabetic SHR. GT reestablished he redox state and reduced the indicators of nephropathy without altering blood lucose and blood pressure levels in diabetic SHR. These findingssuggest that the onsumption of GT may ameliorate nephropathy in diabetic hypertensive patients. / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

La néphropathie diabétique (ND), principale cause d’insuffisance rénale chronique, est caractérisée par une dysfonction des podocytes rénaux. Cette dysfonction podocytaire peut être causée par une résistance à l’insuline induite suite à l’exposition des podocytes aux acides gras libres (AGL). L’un des mécanismes par lequel les AGL réduisent les actions de l’insuline serait l’activation de la voie de Mammalian target of rapamycin (mTOR). Les objectifs sont de caractériser les mécanismes de résistance à l’insuline par les AGL dans les podocytes et d’étudier l’implication de la voie du complexe mTORC1 menant à la ND dans un modèle de diabète de type 2. In vivo, la fonction et la pathologie rénale des souris diabétiques de type 2 (db/db) ont été évaluées. Des podocytes murins ont été cultivés pendant 96 h en conditions normales (5,6 mM, NG) ou élevées (25 mM, HG) de glucose avec ou sans palmitate (25 μM) pour les dernières 24 h. In vitro, les podocytes exposés en HG ont montré une diminution de l’activation d’Akt induite par l’insuline. Le palmitate seul a diminué de 50% l’activation d’Akt alors que la combinaison HG + palmitate a accentué cette diminution en la réduisant de 72%. Cette inhibition se ferait via la phosphorylation en sérine d’IRS1. En effet, en présence de palmitate, la phosphorylation d’IRS1 (ser307) est augmentée d’environ 2 fois. De plus, la phosphorylation d’IRS1 par le palmitate est corrélée à une augmentation de la phosphorylation de mTOR (ser2448) et de son substrat S6 (ser240/244). L’inhibition de la voie de signalisation de l’insuline par la voie mTOR serait due à l’activation de la PKC-α suite à une stimulation au palmitate. Pour ce qui est de mTORC2, la phosphorylation inhibitrice de Rictor (thr1135) augmente de 47% en présence de palmitate. In vivo, dans les souris db/db, l’augmentation des marqueurs de la ND (albuminurie, expansion du mésangium, hypertrophie du glomérule et expression de TGF-beta) est associée à une élévation de la p-mTOR, p-Rictor et de p-S6 dans les glomérules rénaux. En conclusion, le phénomène de résistance à l’insuline par les AGL dans les podocytes serait causé par l’activation de PKC-α/mTORC1 menant à la phosphorylation d’IRS1 en sérine 307, un mécanisme complémentaire aux actions de l’hyperglycémie, et contribuant de façon indépendante à la progression de la ND. De plus, l’inhibition du complexe mTORC2 contribue à la diminution de la signalisation de la voie de l’insuline. / Abstract : Diabetic nephropathy (DN) is the leading cause of chronic renal failure in diabetic patients and is characterized by the dysfunction of podocytes. Our laboratory has shown that hyperglycemia caused podocyte insulin unresponsiveness and cell death via the upregulation of PKC- and SHP-1, a tyrosine phosphatase. In contrast, free fatty acids (FFA)-induced insulin resistance in podocytes is not associated with SHP-1 expression. Thus, other signaling pathways could be implicated including the activation of the Mammalian target of rapamycin (mTOR) complexes pathway. The aim of this study was to investigate the insulin resistance mechanisms caused by FFA in podocytes leading to DN in type 2 diabetes. In vitro, cultured podocytes were exposed to normal (5.6 mmol/L; NG) or high glucose (25 mmol/L; HG) levels for 96 h and to palmitate (25 µmol/L) the last 24h with or without insulin stimulation (10 nmol/L). As previously showed, podocytes exposed to HG decreased Akt activation upon insulin stimulation. Palmitate treatment alone reduced insulin-induced Akt phosphorylation by 50% while a combination of palmitate and HG blunted Akt activation by 72%. The inhibition of Akt by palmitate was associated with the increase of PKC- activation leading to mTOR phosphorylation and its substrate S6. Moreover, the mTORC1 complex activation enhanced the serine 307 phosphorylation of IRS1 known to de-activate IRS1. Furthermore, palmitate also mediated the mTORC2 complex inhibition via the Thr1135 phosphorylation of Rictor. In vivo, the implication of mTORC1 complex in DN development was evaluated using 25 weeks old type 2 diabetes mice (db/db). Mice developed increased albuminuria, mesangial cell expansion and glomerular hypertrophy compared to non-diabetic mice, which correlated with the phosphorylation of mTOR, Rictor and S6. In conclusion, elevated FFA levels caused activation of PKC-/mTORC1 pathway and inhibition of mTORC2 leading to insulin resistance in podocytes and DN progression.

Although the etiology of diabetic nephropathy is still uncertain, proteinuria due to podocyte injury and loss (podocytopenia) are early features of the disease. Significant increases in thromboxane A2 (TXA2) production as well as expression of its receptor in animal models of diabetic nephropathy led to the hypothesis that TXA2 acting via its thromboxane-prostanoid (TP) receptor induces podocytopenia resulting in proteinuria. Systemic infusion of a TP antagonist demonstrated an important role of TXA2/TP signalling in our model of streptozotocin induced type-1 diabetic nephropathy by reducing kidney damage including proteinuria. Podocyte specific TP overexpressing mice did not demonstrate more pathologic or dynamic kidney damage than non-transgenic mice in STZ-induced diabetic nephropathy. Further assessment of the TP transgene functionality in this mice line is necessary to validate those results. Whereas the importance of TXA2/TP signalling is undeniable in diabetic nephropathy, it appears that podocyte TP receptors might not be directly targeted.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12973号 / 論農博第2823号 / 新制||農||1037(附属図書館) / 学位論文||H28||N4955(農学部図書室) / 32411 / 新制||農||1037 / (主査)教授 久米 新一, 教授 今井 裕, 教授 松井 徹 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19396号 / 医博第4047号 / 新制||医||1012(附属図書館) / 32421 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 野田 亮, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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Résumé : La néphropathie diabétique (ND) est une complication microvasculaire du diabète évoluant ultimement en insuffisance rénale et l’hyperglycémie est connue comme étant l’un des facteurs de risques. De larges études cliniques, tel que le DCCT et l’UKPDS, ont montré que si le contrôle intensif de la glycémie se faisait de façon précoce, il serait possible de retarder le développement de la ND. Cependant, les résultats de l'EDIC montrent que si ce contrôle intensif se faisait plus tardivement, suite à une période d’hyperglycémie, il n’empêcherait plus sa progression. Les podocytes ont un rôle critique dans le maintien des fonctions rénales et leur apoptose corrèle de façon très spécifique avec la progression de la ND. Récemment, nous avons rapporté que SHP-1, une protéine tyrosine phosphatase, était augmentée en concentrations élevées de glucose (HG), menant à une inhibition des voies de signalisation de l'insuline. Notre hypothèse est que l’augmentation de l’expression de SHP-1 causée par l’hyperglycémie persiste même après réduction des niveaux de glucose, phénomène de mémoire hyperglycémique, causant une résistance à l'insuline, la mort des podocytes et une absence de réversibilité liée à la progression de ND. Les résultats in vivo montrent que la fonction et la pathologie rénale continuent de progresser et ce en dépit de la normalisation des niveaux de glucose avec implants d’insuline de 5 à 7 mois d’âge La progression de la pathologie corrèle avec le maintien de l’augmentation de l’expression de SHP-1, contribuant au maintien de l’inhibition des voies de l’insuline. En culture, des podocytes murins exposés en HG pendant 96 h et ensuite exposés en condition normale de glucose(NG) pour les dernières 24 h montrent une persistance de l’inhibition des voies de signalisation de l’insuline qui corrèle avec l’augmentation persistante de l’expression et l’activité phosphatase de SHP-1. L’activité des caspases 3/7 dans les podocytes est plus élevée lorsque ceux-ci sont exposés en HG qu’en NG. Le retour en NG pour les dernières 24 h n’a aucun effet bénéfique à réduire l’activité des caspases 3/7. Finalement, l’analyse épigénétique a été suggérée comme étant une explication du phénomène de mémoire hyperglycémique. La monométhylation de la lysine 4 de l’histone 3 (H3K4me1), un marqueur d’activation génique, est augmentée sur le promoteur de SHP-1 en HG et demeure élevée malgré le retour en NG pendant les dernières 24 h. En conclusion, l’hyperglycémie engendre une augmentation persistante de SHP-1 due possiblement à des modifications épigénétiques, causant le maintien de l’inhibition les voies de signalisation de l’insuline même après un retour à des niveaux normaux de glucose, contribuant à la progression de la ND. / Abstract : Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Renal podocytes apoptosis induced by hyperglycemia is an early event of DN. Clinical studies have shown that intensive blood glucose control reduced the development of DN but is not sufficient, if started late, to prevent its progression, introducing the concept of “hyperglycemic memory”. We have recently published that the tyrosine phosphatase SHP-1 is elevated in renal cortex of type 1 diabetic mice (Akita), contributing to insulin unresponsiveness and DN. We hypothesized that SHP-1 expression remains elevated regardless of systemic blood glucose normalization, and is responsible for hyperglycemic memory in podocytes leading to DN progression. In vivo contribution of SHP-1 in hyperglycemic memory was evaluated using Akita mice treated with insulin implants after 4 months of diabetes. Both urinary albuminuria and glomerular filtration rate were significantly increased in diabetic mice compared to non-diabetic mice and remained elevated despite normalization of blood glucose levels. Renal dysfunction was associated with a persistent increase of SHP-1 expression in renal cortex and inhibition of insulin action that were not normalized following insulin implants. Mouse podocytes were cultured in normal (5.6mM; NG), high glucose concentrations (25mM; HG) for 120 h or HG (96 h) followed by NG for an additional 24 h (HG+NG). We observed that Akt and ERK phosphorylation induced by insulin was inhibited in HG and were not restored despite returning glucose level to 5.6 mM after the HG period. This inhibition was associated with persistent increase of SHP-1 expression and phosphatase activity, leading to insulin signaling pathway inhibition. Moreover, caspase 3/7 activity in podocytes exposed to HG was higher than in podocytes cultured in NG and returning glucose concentrations to normal range for the last 24 h after the 96 h HG exposure had no effect on reducing caspase 3/7 activity. Epigenetic changes were studied to explain the hyperglycemic memory effect. On SHP-1 promoter, H3K4me1 levels, an activation mark, tended to be more elevated in podocytes exposed to HG and were maintained despite returning to NG levels after the HG conditions. In conclusion, hyperglycemia induces persistent and epigenetic changes of SHP-1 causing insulin unresponsiveness in the podocytes contributing to DN progression.

Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medical Sciences The University of Alcalá, Faculty of Medicine, Department of biomedicine and biotechnology Student: Zuzana Chvojanová Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Consultant: María Isabel Arenas Jimenéz Title of the diploma thesis: Study of the variations in the expression of different adhesion and cytoskeletal proteins of podocytes (E-Cadherin, Podocin, Vimentin) due to Bisphenol A Bisphenol A (BPA) is one of the most widespread compounds in the world, producing over 6 billion metric tons per year. It is widely used as part of polycarbonate plastics and epoxy resins, from which reusable plastic bottles, food boxes and some medical equipment are made. It is also used to coat the inner layer of the cans. Previous studies have shown that BPA contributes to many chronic diseases in the human body, such as kidney disease - diabetic nephropathy. Podocytes - terminally differentiated cells of the Bowman's capsule in glomerulus - are an integral part of the filtration barrier, where they play an important role in preventing the plasmatic proteins from penetrating to the urine. Therefore, in this study, we looked at the effect of BPA on these cells and their particular proteins, using both in vivo and...