Indiana University-Purdue University Indianapolis (IUPUI) / Acute Kidney Injury (AKI) is a prevalent and significant problem whose primary treatment is supportive care. Ischemic preconditioning is a strategy used to protect organs from ischemic injury via a prior injury. Ischemic preconditioning in the kidneys has been shown to confer protection onto kidneys from subsequent ischemic insults with attenuated serum creatinine values in treated rats. In the preconditioned kidneys, the enzyme IDH2 was discovered to be upregulated in the mitochondria. Hydrodynamic fluid delivery to the kidney was found to be a viable technique for delivering this gene to the kidney, resulting in artificially upregulated expression of IDH2. Via a two-pronged effort to discern the functional significance of ischemic preconditioning and hydrodynamic IDH2 fluid injections, we performed mitochondrial oxygen respiration assays on both preconditioned and injected kidneys. We found that renal ischemic preconditioning resulted in no significant difference between sham and preconditioned, subsequently injured kidneys, which is similar to the results from the serum creatinine studies. Hydrodynamically IDH2-injected, and subsequently injured kidneys respire significantly better than vehicle injected, and subsequently injured kidneys, which shows that hydrodynamic injections of IDH2 protects kidneys against injury, and partially mimics the effects of preconditioning.

Malaria remains one of the world's major health problems, especially in developing countries. A better understanding of the pathology and pathophysiology of severe malaria is key to develop new treatments. Different approaches have been used in malaria research including the in vitro co-culture models with endothelial cells and both murine and simian animal models. However these are open to controversy due to disagreement on their representativeness of human disease. Using human post-mortem tissue in malaria research is another important approach but is practically challenging, limiting the availability of post mortem samples from malaria patients. The work in this thesis had two main themes. First I examined the role of the endothelial signalling Angiopoetin-Tie-2 receptor pathway in malaria. Ang-2 has been shown to be a significant biomarker of severe and fatal malaria. I examined the tissue specific expression of proteins from this pathway in post-mortem brain tissues from fatal malaria cases, but found no difference between cerebral malaria and non-cerebral malaria cases. Ang-2 correlated with the severity of malaria in these patients. An attempt to examine the interaction of hypoxia and the Ang-Tie-2 pathway in vitro using a co-culture model of human brain endothelial cells was unsuccessful due to contamination of the cell line. The second part of the thesis aimed to utilise molecular pathology techniques including miRNA and whole-genome microarrays. I have shown for the first time that these can be successfully applied to human post-mortem tissue in malaria. First I used archival tissues to examine the microRNA signature in the kidney of patients with malaria associated renal failure. Second I optimised a protocol to preserve post mortem tissue for molecular pathology, from an autopsy study in Mozambique. Using the subsequent total mRNA transcriptomic data and bioinformatics analysis this work has expanded our knowledge of differential gene expression and the families of genes which are dysregulated in the brain in response to malaria infection.

Pas de résumé français / Pas de résumé anglais

Made available in DSpace on 2016-01-26T12:51:25Z (GMT). No. of bitstreams: 1 joaofernandopicollodeoliveira_dissert.pdf: 2216002 bytes, checksum: 172981ec9e4daa13d8e1e6872337867d (MD5) Previous issue date: 2008-12-10 / Nephrotoxicity is the main adverse effect of aminoglycoside use. There are few information about its prevalence and risk factor in intensive care unit patients. Objectives: To assess the prevalence, mortality and risk factors for aminoglycoside nephrotoxicity in ICU patients. Casuistic and Method: In order to assess the prevalence of, and risk factors for aminoglycoside nephrotoxicity in the ICU, 360 consecutive patients starting aminoglycoside therapy in the ICU with a baseline calculated GFR (cGFR) &#8805;30 ml/min/1.73 m2 were evaluated. Results: Of them, 209 (58%) developed aminoglycoside-induced nephrotoxicity (AKI, decrease in cGFR >20% from baseline), while 151 did not (non-AKI). Both groups had similar baseline cGFR. The AKI group developed a lower cGFR nadir (45 ± 27 vs. 79 ± 39 ml/min/1.73 m2, p<0.001), was older (56 ± 18 y vs. 52 ± 19 y, p=0.033), had a higher prevalence of diabetes (19.6% vs. 9.3%, p=0.007), used other nephrotoxic drugs (51% vs. 38%, p=0.024) and iodinated contrast more frequently (18% vs. 8%, p=0.0054), showed higher prevalence of hypotension (63% vs. 44%, p=0.0003), shock (56% vs. 31%, p<0.0001), and jaundice (19% vs. 8%, p=0.0036). Mortality was 44.5% in the AKI and 29.1% in the non-AKI groups (p=0.0031). A logistic regression model identified as significant (p<0.05) independent factors affecting aminoglycoside-induced nephrotoxicity baseline cGFR<60 ml/min/1.73 m2 (OR 0.42), diabetes (OR 2.13), simultaneous use of other nephrotoxins (OR 1.61) or iodinated contrast (OR 2.13), and hypotension (OR 1.83). Conclusion: The AKI was frequent among ICU patients using aminoglycoside, and it was associated with high mortality. The presence of diabetes, hypotension, simultaneous use of other nephrotoxic drugs, and iodinated contrast were independent risk factors for the development of aminoglycoside-induced nephrotoxicity. / Nefrotoxicidade é a principal complicação do uso de aminoglicosídeos. Existem poucas informações sobre a prevalência e os fatores de risco para nefrotoxicidade por aminoglicosídeos em paciente internados em unidades de terapia intensiva. Objetivos: Avaliar a prevalência, a mortalidade e os fatores de risco para nefrotoxicidade por aminoglicosídeos em pacientes internados em Unidade de Terapia Intensiva geral. Casuística e Métodos: Foram avaliados a prevalência, os fatores de risco e a mortalidade da nefrotoxicidade por aminoglicosídeo de 360 pacientes internados em terapia intensiva com filtração glomerular calculada por fórmula (MDRD, RFG) basal &#8805;30ml/min/ 1,73m2, que iniciaram o uso do antibiótico na terapia intensiva; nefrotoxicidade foi definida como queda >20% na RFG em relação ao RFG basal. Resultados: Entre os pacientes estudados 209 (58%) desenvolveram nefrotoxicidade (IRA) e 151 não alteraram a função renal (não IRA). Ambos os grupos (média±desvio padrão) tinham RFG basal similar (89&#61617;42 ml/min/ 1,73m2 no grupo IRA versus 84±42 ml/min/ 1,73m2 no grupo não IRA). O grupo IRA teve menor nadir de RFG (45±27 ml/min/ 1,73m2 versus 79±39 ml/min/ 1,73m2, p<0,001), idade maior (56&#61617;18 anos versus 52&#61617;19 anos, p=0,033), maior prevalência de diabetes (19,6% versus 9,3%, p=0,007), uso simultâneo mais freqüente de outras drogas nefrotóxicas (51% versus 38%, p=0,024) e contraste (18% versus 8%, p=0,0054), maior prevalência de hipovolemia (44% versus 27%, p=0,001), hipotensão (63% versus 44%, p=0,0003), choque (56% versus 31%, p<0,0001) e icterícia (19% versus 8%, p=0,0036). A mortalidade foi 44,5% no grupo IRA e 29,1% no grupo não IRA (p=0,0031). A análise por regressão logística identificou como fatores de risco independente para a nefrotoxicidade por aminoglicosídeo, RFG basal <60ml/min/1,73m2 [OR 0,42 (IC 95% 0,24-0,72, p=0,02)], diabetes [OR 2,13 (IC 95% 1,01-4,49, p=0,046)], uso simultâneo de outras drogas nefrotóxicas [OR 1,61 (IC 95% 1,00-2,59, p=0,048)], uso de contraste iodado [OR 2,13 (IC 95% 1,02-4,43, p=0,043)] e hipotensão [OR 1,83 (IC 95% 1,14-2,94, p=0,012)]. Conclusões: Nefrotoxicidade por aminoglicosídeo foi freqüente e associada a alta mortalidade em pacientes de UTI. A presença de diabetes, hipotensão, uso simultâneo de outras drogas nefrotóxicas e contraste iodado foram fatores de risco independentes para o desenvolvimento de nefrotoxicidade.

Made available in DSpace on 2014-07-29T15:13:51Z (GMT). No. of bitstreams: 1 tese_liliana_memezes2.pdf: 1993828 bytes, checksum: f3f5df5f10fa0d970bf616d0ca324395 (MD5) Previous issue date: 2009-09-19 / Renal ischemia is present in different situations such as vascular or kidney surgery and also in renal transplantation. The aim of this study was to evaluate the renal function in dogs submitted to ischemia and reperfusion after chlorpromazine application. Twelve adult mongrel dogs were distributed into two groups with six animals: group A - ischemia and reperfusion without previous administration of chlorpromazine; group B - ischemia and reperfusion treated previously with chlorpromazine. Blood and urine samples were harvested in four different times: before ischemia, at the beginning of reperfusion, 120 minutes after of reperfusion and every week until postsurgical day 28 in order to check the possible ischemia/reperfusion late effect. Renal function was evaluated by clinical examination, serum urea and creatinine levels and urinary GGT activity. PU/CU and urinary activity of GGT were more sensitive for detecting acute tubular injury then routine urine examination, because these variables showed earlier alterations. It was not possible to verify the chlorpromazine protective action through urinalysis, creatinine and urea serum levels, excretion of GGT urinary and urine protein:creatinine ratio. / O objetivo deste trabalho foi avaliar a função renal e a expressão da E-caderina e &#61538;-catenina de cães submetidos à isquemia e reperfusão após a aplicação de clorpromazina. Para tais objetivos, foram utilizados 12 cães distribuídos aleatoriamente em dois grupos de seis indivíduos: grupo A com isquemia e reperfusão sem tratamento por clorpromazina e o grupo B com isquemia e reperfusão tratados por clorpromazina. Foi realizada uma incisão paracostal esquerda para identificação e isolamento do rim esquerdo e da artéria renal esquerda, que foi isolada e ocluída nos animais de todos os grupos. Os animais do grupo B receberam clorpromazina via endovenosa, na dose de 5 mg/kg, 15 min antes da clampagem do vaso, que durou 1 h. Após este período, as artérias renais foram desobstruídas e os órgãos foram avaliados por 2h após reperfusão. Avaliações da função renal foram feitas por uranálise, concentração sérica de uréia e creatinina e determinação da GGT urinária. Em cada grupo foram extraídas seis amostras de parênquima renal, para avaliação histológica e marcação com anticorpos anti-E-caderina e anti-&#61538;-catenina. Foi possível concluir que, a avaliação da relação PU/CU e atividade da GGT urinária são exames mais sensíveis para detectar lesão tubular aguda que o exame de urina de rotina. Não sendo possível verificar a ação protetora da clorpromazina. A E-caderina e &#61538;-catenina foram diferencialmente expressas em segmentos do córtex e da medula em rim de cães e o uso da clorpromazina não alterou a expressão das duas proteínas.

La prostaglandine E2 est une hormone lipidique produite abondamment dans le corps, incluant dans le rein où elle agit localement pour réguler les fonctions rénales. Un couplage à la protéine Gαs menant à une production d’AMPc a classiquement été attribué au récepteur EP4 de PGE2. La signalisation d’EP4 s’est cependant avérée plus complexe et implique aussi un couplage aux protéines sensibles à la PTX Gαi et des effets reliés aux β-arrestines. Il y a maintenant plusieurs exemples de l’activation sélective de voies de signalisation indépendantes par des ligands des récepteurs couplés aux protéines G (RCPG), et ce concept désigné sélectivité fonctionnelle pourrait être exploité dans le développement de nouveaux médicaments plus spécifiques et efficaces. Dans une première étude, la puissance et l’activité intrinsèque d’une série de ligands d’EP4 pour l’activation de Gαs, Gαi et de la ß-arrestine ont été systématiquement déterminées relativement au ligand endogène PGE2. Dans ce but, trois essais de transfert d’énergie de résonance de bioluminescence (BRET) ont été adaptés pour évaluer les différentes voies dans des cellules vivantes. Nos résultats montrent une sélectivité fonctionnelle importante parmi les agonistes évalués et ont une implication pour l’utilisation d’analogues de la PGE2 dans un contexte expérimental et possiblement clinique, puisque leur spectre d’activité diffère de l’agoniste naturel. La méthodologie basée sur le BRET utilisée lors de cette première évaluation systématique d’une série d’agonistes d’EP4 devrait être applicable à l’étude d’autres RCPG. Dans une deuxième étude, des peptides reproduisant des régions juxtamembranaires extracellulaires du récepteur EP4 ont été conçus selon le raisonnement que des peptides ciblant des régions éloignées du site de liaison du ligand naturel ont le potentiel de ne moduler qu’une partie des activités du récepteur. L’insuffisance rénale aiguë est une complication médicale grave caractérisée par un déclin brusque et soutenu de la fonction rénale et pour laquelle il n’y a pas de traitement efficace à l’heure actuelle. Nos résultats montrent que le peptidomimétique dérivé d’EP4 optimisé (THG213.29) améliore significativement les fonctions rénales et les changements histologiques dans une insuffisance rénale aiguë induite par cisplatine ou par occlusion des artères rénales dans des rats Sprague-Dawley. Le THG213.29 ne compétitionnait pas la liaison de la PGE2 à EP4, mais modulait la cinétique de dissociation de la PGE2, suggérant une liaison à un site allostérique d’EP4. Le THG213.29 démontrait une sélectivité fonctionnelle, puisqu’il inhibait partiellement la production d’AMPc induite par EP4 mais n’affectait pas l’activation de Gαi ou le recrutement de la ß-arrestine. Nos résultats indiquent que le THG213.29 représente une nouvelle classe d’agent diurétique possédant les propriétés d’un modulateur allostérique non-compétitif des fonctions du récepteur EP4 pour l’amélioration des fonctions rénales suite à une insuffisance rénale aiguë. / Prostaglandin E2 (PGE2) is a lipid hormone mediator widely produced in the body, including in the kidney where it acts locally to regulate renal function. Classically, the PGE2 receptor EP4 has been classified as coupling to the Gαs subunit, leading to intracellular cAMP increases. However EP4 signaling has been revealed to be more complex and also involves coupling to PTX-sensitive Gαi proteins and ß-arrestin mediated effects. There are now many examples of selective activation of independent pathways by G-protein coupled receptor (GPCR) ligands, a concept referred to as functional selectivity that could be exploited for the development of more specific and efficacious drugs. In a first study, the potencies and efficacies of a panel of EP4 ligands were systematically determined for the activation of Gαs, Gαi and ß-arrestin relative to the endogenous ligand PGE2. For this purpose, three bioluminescence resonance energy transfer (BRET) assays were adapted to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists and have implications for the use of PGE2 analogues in experimental and possibly clinical settings, as their activity spectra on EP4 differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP4 agonists should be applicable for the study of other GPCRs. In a second study, peptides were derived from extracellular juxtamembranous regions of the EP4 receptor following the rationale that peptides that target regions of the receptor remote of the ligand-binding site might modulate a subset of the EP4-mediated activities. Acute renal failure is a serious medical complication characterized by an abrupt and sustained decline in renal function and for which there is currently no effective treatment. Our results show that the optimized EP4-derived peptidomimetic THG213.29 significantly improved renal functions and histological changes in acute renal failure induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 did not displace PGE2 binding to EP4, but modulated PGE2 binding dissociation kinetics, indicative of an allosteric binding mode. THG213.29 exhibited functional selectivity, as it partially inhibited EP4-mediated cAMP production but did not affect Gαi activation or ß-arrestin recruitment. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP4 receptor function for improving renal function following acute renal failure.

La prostaglandine E2 est une hormone lipidique produite abondamment dans le corps, incluant dans le rein où elle agit localement pour réguler les fonctions rénales. Un couplage à la protéine Gαs menant à une production d’AMPc a classiquement été attribué au récepteur EP4 de PGE2. La signalisation d’EP4 s’est cependant avérée plus complexe et implique aussi un couplage aux protéines sensibles à la PTX Gαi et des effets reliés aux β-arrestines. Il y a maintenant plusieurs exemples de l’activation sélective de voies de signalisation indépendantes par des ligands des récepteurs couplés aux protéines G (RCPG), et ce concept désigné sélectivité fonctionnelle pourrait être exploité dans le développement de nouveaux médicaments plus spécifiques et efficaces. Dans une première étude, la puissance et l’activité intrinsèque d’une série de ligands d’EP4 pour l’activation de Gαs, Gαi et de la ß-arrestine ont été systématiquement déterminées relativement au ligand endogène PGE2. Dans ce but, trois essais de transfert d’énergie de résonance de bioluminescence (BRET) ont été adaptés pour évaluer les différentes voies dans des cellules vivantes. Nos résultats montrent une sélectivité fonctionnelle importante parmi les agonistes évalués et ont une implication pour l’utilisation d’analogues de la PGE2 dans un contexte expérimental et possiblement clinique, puisque leur spectre d’activité diffère de l’agoniste naturel. La méthodologie basée sur le BRET utilisée lors de cette première évaluation systématique d’une série d’agonistes d’EP4 devrait être applicable à l’étude d’autres RCPG. Dans une deuxième étude, des peptides reproduisant des régions juxtamembranaires extracellulaires du récepteur EP4 ont été conçus selon le raisonnement que des peptides ciblant des régions éloignées du site de liaison du ligand naturel ont le potentiel de ne moduler qu’une partie des activités du récepteur. L’insuffisance rénale aiguë est une complication médicale grave caractérisée par un déclin brusque et soutenu de la fonction rénale et pour laquelle il n’y a pas de traitement efficace à l’heure actuelle. Nos résultats montrent que le peptidomimétique dérivé d’EP4 optimisé (THG213.29) améliore significativement les fonctions rénales et les changements histologiques dans une insuffisance rénale aiguë induite par cisplatine ou par occlusion des artères rénales dans des rats Sprague-Dawley. Le THG213.29 ne compétitionnait pas la liaison de la PGE2 à EP4, mais modulait la cinétique de dissociation de la PGE2, suggérant une liaison à un site allostérique d’EP4. Le THG213.29 démontrait une sélectivité fonctionnelle, puisqu’il inhibait partiellement la production d’AMPc induite par EP4 mais n’affectait pas l’activation de Gαi ou le recrutement de la ß-arrestine. Nos résultats indiquent que le THG213.29 représente une nouvelle classe d’agent diurétique possédant les propriétés d’un modulateur allostérique non-compétitif des fonctions du récepteur EP4 pour l’amélioration des fonctions rénales suite à une insuffisance rénale aiguë. / Prostaglandin E2 (PGE2) is a lipid hormone mediator widely produced in the body, including in the kidney where it acts locally to regulate renal function. Classically, the PGE2 receptor EP4 has been classified as coupling to the Gαs subunit, leading to intracellular cAMP increases. However EP4 signaling has been revealed to be more complex and also involves coupling to PTX-sensitive Gαi proteins and ß-arrestin mediated effects. There are now many examples of selective activation of independent pathways by G-protein coupled receptor (GPCR) ligands, a concept referred to as functional selectivity that could be exploited for the development of more specific and efficacious drugs. In a first study, the potencies and efficacies of a panel of EP4 ligands were systematically determined for the activation of Gαs, Gαi and ß-arrestin relative to the endogenous ligand PGE2. For this purpose, three bioluminescence resonance energy transfer (BRET) assays were adapted to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists and have implications for the use of PGE2 analogues in experimental and possibly clinical settings, as their activity spectra on EP4 differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP4 agonists should be applicable for the study of other GPCRs. In a second study, peptides were derived from extracellular juxtamembranous regions of the EP4 receptor following the rationale that peptides that target regions of the receptor remote of the ligand-binding site might modulate a subset of the EP4-mediated activities. Acute renal failure is a serious medical complication characterized by an abrupt and sustained decline in renal function and for which there is currently no effective treatment. Our results show that the optimized EP4-derived peptidomimetic THG213.29 significantly improved renal functions and histological changes in acute renal failure induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 did not displace PGE2 binding to EP4, but modulated PGE2 binding dissociation kinetics, indicative of an allosteric binding mode. THG213.29 exhibited functional selectivity, as it partially inhibited EP4-mediated cAMP production but did not affect Gαi activation or ß-arrestin recruitment. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP4 receptor function for improving renal function following acute renal failure.

Indiana University-Purdue University Indianapolis (IUPUI) / Advancements in human genomics have simultaneously enhanced our basic understanding of the human body and ability to combat debilitating diseases. Historically, research has shown that there have been many hindrances to realizing this medicinal revolution. One hindrance, with particular regard to the kidney, has been our inability to effectively and routinely delivery genes to various loci, without inducing significant injury. However, we have recently developed a method using hydrodynamic fluid delivery that has shown substantial promise in addressing aforesaid issues. We optimized our approach and designed a method that utilizes retrograde renal vein injections to facilitate widespread and persistent plasmid and adenoviral based transgene expression in rat kidneys. Exogenous gene expression extended throughout the cortex and medulla, lasting over 1 month within comparable expression profiles, in various renal cell types without considerably impacting normal organ function. As a proof of its utility we by attempted to prevent ischemic acute kidney injury (AKI), which is a leading cause of morbidity and mortality across among global populations, by altering the mitochondrial proteome. Specifically, our hydrodynamic delivery process facilitated an upregulated expression of mitochondrial enzymes that have been suggested to provide mediation from renal ischemic injury. Remarkably, this protein upregulation significantly enhanced mitochondrial membrane potential activity, comparable to that observed from ischemic preconditioning, and provided protection against moderate ischemia-reperfusion injury, based on serum creatinine and histology analyses. Strikingly, we also determined that hydrodynamic delivery of isotonic fluid alone, given as long as 24 hours after AKI is induced, is similarly capable of blunting the extent of injury. Altogether, these results indicate the development of novel and exciting platform for the future study and management of renal injury.