Fan, Rongrong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 130-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

O peptídeo-1 semelhante ao glucagon (GLP-1) é um hormônio incretina secretado pelas células L do trato gastrointestinal e liberado imediatamente após a ingestão de alimento. O GLP-1 estimula a secreção de insulina pós-prandial moderando a elevação precoce da glicose no sangue. Embora primariamente envolvido na homeostase da glicose, o GLP-1 é capaz de induzir a diurese e natriurese, quando administrado em doses farmacológicas em humanos e em roedores. Estudos prévios do nosso laboratório demonstraram que o mecanismo de ação renal do GLP-1, bem como de agonistas sintéticos do receptor GLP-1R, envolve o aumento do fluxo plasmático renal (FPR) e do ritmo de filtração glomerular (RFG) bem como a diminuição da reabsorção de sódio dependente da isoforma 3 do trocador Na?/H? (NHE3) em túbulo proximal renal. Entretanto, até o momento, nenhum estudo investigou se o GLP-1 endógeno exerce efeitos sobre o manuseio renal de sal e água, nem o seu papel fisiológico sobre a regulação da atividade do NHE3. Portanto, o objetivo deste estudo foi testar a hipótese que o GLP-1 endógeno modula a função renal de ratos, ao menos em parte, via inibição da atividade do NHE3 em túbulo renal. Para este fim, ratos Wistar (2-3 meses de idade) foram devidamente anestesiados, submetidos à traqueostomia e tiveram a veia jugular e a bexiga canuladas para infusão de uma solução contendo 100 ug/kg/min do antagonista do receptor GLP-1R exendin-9 (Ex-9, 40 uL/min) por um período de 30 minutos e para a coleta de urina, respectivamente. A infusão sistêmica de Ex-9 diminuiu a concentração de AMPc urinário e atividade da PKA cortical renal consistente com o bloqueio da sinalização deflagrada pela interação GLP-1/GLP-1R no rim. Além disso, a administração sistêmica de Ex-9 reduziu a diurese, natriurese, RFG, FPR, clearance de lítio e pH urinário. Em experimentos de microperfusão estacionária in vivo, não foram observadas diferenças no fluxo de bicarbonato dependente de NHE3 entre os túbulos proximais perfundidos com exendin-9 (2 uM) e os túbulos perfundidos com solução controle. No entanto, a perfusão tubular proximal com Ex-9 foi capaz de bloquear completamente as ações inibitórias do GLP-1 (20 nM) sobre a atividade do NHE3. Por outro lado, a infusão sistêmica do Ex-9 reduziu os níveis de fosforilação da serina 552, sítio consenso para a fosforilação por PKA localizado na região C-terminal do NHE3, e que está associado à inibição da atividade de troca Na+/H+ mediada por este transportador. Baseando-se nos achados que a infusão sistêmica do Ex-9 aumenta a reabsorção de sódio e secreção de H?, reduz o clearance do lítio e os diminui os níveis de fosforilação do NHE3 na serina 552 são consistentes com um aumento na atividade deste transportador na ausência/redução da sinalização mediada pela interação do GLP-1 endógeno com seu receptor no rim. Por sua vez, o fato do Ex-9 não afetar a atividade do NHE3 sob as condições experimentais da microperfusão estacionária in vivo é condizente com o fato do GLP-1 não ser sintetizado no néfron e sugere fortemente que é o GLP-1 filtrado que se liga ao seu receptor no túbulo proximal renal resultando na diminuição da reabsorção de bicarbonato de sódio mediada pelo NHE3. Em conjunto, estes resultados sugerem que o GLP-1 endógeno exerce efeito tônico sobre o manuseio renal de sódio e água, mediando portanto, uma relação funcional entre a homeostase glicêmica e volêmica / The glucagon like peptide-1 (GLP-1) is an incretin hormone secreted by the L-cells of the gastrointestinal tract and released immediately after ingestion of food. GLP-1 stimulates postprandial insulin secretion moderating early increase in blood glucose. Although primarily involved in glucose homeostasis, GLP-1 is capable of inducing diuresis and natriuresis when administered in pharmacologic doses in humans and rodents. Previous studies from our laboratory have shown that the renal mechanism of action of GLP-1 and synthetic agonists of GLP-1R receptor, involves an increase of renal plasma flow (RPF) and glomerular filtration rate (GFR) as well a decrease in reabsorption of sodium mediated by the Na? / H? exchanger (NHE3) isoform 3 in the renal proximal tubule. However, to date, no study has investigated whether endogenous GLP-1 exerts effects on the renal handling of salt and water, or its physiological role in the regulation of the activity of NHE3. Therefore, the aim of this study was to test the hypothesis that endogenous GLP-1 modulates renal function in rats, at least in part, via inhibition of the NHE3 in renal tubule. To this end, male Wistar rats (2-3 months old) were properly anesthetized, tracheostomized and the jugular vein and the bladder were cannulated to the infusion of a solution containing 100 ug / kg / min GLP-1R antagonist receiver exendin-9 (Ex-9, 40 uL/min) for a period of 30 minutes and to collect urine, respectively. Systemic infusion of Ex-9 reduced the urinary concentration of cAMP and the renal cortical PKA activity, consistent with the blockage of the signal triggered by the interaction of GLP-1 / GLP-1R in the kidney. Furthermore, systemic administration of ex-9 reduced diuresis, natriuresis, GFR, RPF, lithium clearance and urinary pH. In experiments of in vivo stationary microperfusion, no differences were observed in the NHE3-mediated net bicarbonate flow between proximal tubules perfused with exendin-9 (2 mM) and perfused tubules with control solution. However, the tubular proximal perfusion with Ex-9 was able to completely block the inhibitory actions of GLP-1 (20 nM) on the activity of NHE3. On the other hand, systemic infusion of Ex-9 reduced phosphorylation levels of serine 552, a consensus site for phosphorylation by PKA located in the C-terminal region of NHE3, which is associated with inhibition of exchange activity of Na+/H+ mediated by this transporter. Collectively, the findings that systemic infusion of Ex-9 increases sodium reabsorption and secretion of H+, reduces the lithium clearance and decreases the NHE3 phosphorylation at serine 552 levels are consistent with the idea that NHE3 activity is upregulated in the absence/reduction of the signaling cascade mediated by the interaction of the endogenous GLP-1 with its receptor in the kidney. In turn, the fact Ex-9 does not affect the activity of NHE3 under the experimental conditions of stationary microperfusion in vivo is consistent with the fact that GLP-1 is not synthesized in the nephron. Besides, it strongly suggests that is the filtrated GLP-1 that binds to its receptor in renal proximal tubule, resulting in a decrease in NHE3-mediated sodium bicarbonate reabsorption. Taken together, these results suggest that endogenous GLP-1 exerts a tonic effect on renal sodium and water handling, mediating therefore a functional relationship between volume and glucose homeostasis

O peptídeo-1 semelhante ao glucagon (GLP-1) é um hormônio incretina intestinal que exerce primariamente ações anti-hiperglicemiantes. Afim de viabilizar o emprego clínico deste peptídeo para o tratamento do diabetes mellitus tipo 2, foram criadas as terapias baseadas em incretinas que incluem as gliptinas, drogas que aumentam a meia-vida circulante do GLP-1 endógeno por meio da inibição da enzima dipeptidil peptidase-4 e agonistas exógenos do receptor de GLP-1 (GLP-1R). Demonstrou-se clínica e experimentalmente que estas classes de fármacos apresentam efeitos cardiorrenais benéficos que vão além do controle glicêmico. Dentre estes efeitos cardiorrenais incluem-se: diurese, natriurese e redução da pressão arterial. Recentemente, demonstramos que o bloqueio agudo da sinalização endógena do GLP-1R, por meio da administração sistêmica do antagonista do GLP-1R, exendin-9, em ratos normotensos causou efeitos antidiuréticos e anti-natriuréticos. Estes efeitos renais encontram-se associados à redução do ritmo de filtração glomerular (RFG) e ao aumento da atividade da isoforma 3 do trocador Na+/H+ (NHE3) em túbulo proximal renal. Entretanto, os efeitos da administração crônica do bloqueador de GLP-1R sobre a função renal e níveis pressóricos ainda permanece obscuro. Assim, o presente estudo teve como objetivo testar a hipótese de que o bloqueio do GLP-1 endógeno é capaz de aumentar a pressão arterial de ratos espontaneamente hipertensos (SHR) e que este efeito está associado ao aumento da atividade do NHE3 em túbulo proximal renal. Além disso, testamos a hipótese que o bloqueio do GLP-1R piora o dano renal de ratos hipertensos ao passo que o agonismo farmacológico este receptor exerce renoproteção. Para tal, ratos espontaneamente hipertensos (SHRs) com 5 semanas de idade foram tratados com exendin-9 (EX-9; 25 ug/rato/dia), com agonista de GLP-1R, o exendin-4 (EX-4, 2,5?g/rato/dia) ou solução salina (controle), através de minibombas osmóticas, por um período de 4 semanas. A pressão arterial foi aferida semanalmente através de pletismografia caudal, a urina e sangue dos ratos foram coletados para avaliação da função renal e, ao término do tratamento, houve a mensuração invasiva da pressão arterial, bem como, morte dos ratos e coleta de amostras biológicas para a realização de análises histológicas, bioquímicas e moleculares. A atividade do NHE3 em túbulo proximal renal foi determinada ao final do tratamento, em 4-5 ratos/grupo, por meio de microperfusão estacionária in vivo. Os valores da pressão arterial caudal ao final do tratamento demonstraram que os ratos tratados com exendin-9 apresentavam valores de pressão arterial maiores do que os controles (182 ± 4 vs. 172 ± 1 mmHg, p < 0,05), enquanto que o tratamento com exendin-4 atenuou a elevação da pressão arterial em relação aos controles (161 ± 4 vs. 172 ± 1 mmHg, p < 0,01). O aumento da pressão arterial em SHR tratados com EX-9 foi associado com maior atividade do NHE3 (1,78 ± 0,08 nmol/cm2/s) no túbulo proximal renal comparados aos controles (1,48 ± 0,10 nmol/cm2/s; p < 0,05) bem como aos SHR tratados com EX-4 (1,19 ± 0,07 nmol/cm2/s, p < 0,01). Além disso, os SHRs tratados com o antagonista de GLP-1R apresentaram níveis de excreção de proteínas urinárias, marcadores de fibrose, inflamação, estresse oxidativo e atividade do sistema renina angiotensina (SRA) intra-renal superiores aos do controle. Por outro lado, a administração sistêmica de EX-4 exerceu efeitos anti-proteinurico, anti-inflamatório e antioxidante. A renoproteção mediada pelo tratamento com EX-4 em SHRs foi acompanhada por redução dos níveis de angiotensina II em córtex renal, sugerindo redução da atividade do SRA intra-renal. Em conjunto, estes resultados demonstram que o bloqueio do GLP-1R intensifica o aumento da pressão arterial e exacerba o dano renal em ratos espontaneamente hipertensos. Por sua vez, o agonismo do GLP-1R exerce efeitos anti-hipertensivo e renoprotetor / Glucagon-like peptide-1 (GLP-1) is an incretin intestinal hormone that primarily exerts anti-hyperglycemic actions. In order to possibilitate the clinical use of this peptide for the treatment of type 2 diabetes mellitus, the incretin based therapies were created, which include the gliptins, drugs that increase the half-life of endogenous GLP-1 through the inhibition of the enzyme dipeptidil peptidase-4, and exogenous agonists of the receptor of GLP-1 (GLP-1R). It is well established that these classes of drugs exert cardiorenal beneficial effects that go beyond glycemic control. Among these cardiorenal effects are diuresis, natriuresis and reduction of blood pressure. We have recently demonstrated that acute blocking of thebaselineGLP-1Rsignaling, via systemic administration of theGLP-1Rantagonist, Exendin-9, causes anti-diuretic and anti-natriuretic effects in normotensive rats. These renal effects are associated with reduction of the glomerular filtration rate (GFR) and stimulation of proximal tubule Na+/H+ exchanger activity isoform 3 (NHE3). However, the effects of the chronic administration of the GLP-1R blocker on renal function and blood pressure levels remain obscure. Thus, this study aimed to test the hypothesis that GLP-1Rblockade elevates blood pressure in spontaneously hypertensive rats (SHR) and that these effects are associated with upregulation of NHE3 activity. Additionally, we tested the hypothesis that GLP-1R blockade worsens kidney damage in hypertensive rats while pharmacological agonism of GLP-1R exerts renoprotection. To this end, 5-week-oldSHR were treated during 4 weeks with Exendin-9 (EX-9; 25 ug/mouse/day), the agonist of GLP-1R receptor, Exendin-4 (EX-4, 2.5 ?g/rat/day) or saline (control), via osmotic minipumps. Blood pressure was weekly measured by plethysmography and urine and blood samples were collected for renal function evaluation. Direct measurement of blood pressure, collection of biological samples for histological, biochemical and molecular analysis were performed at the end of the treatment. At the end of the treatment, 4-5 rats/group were used for determination of NHE3 proximal tubule activity by in vivo determined by stationary microperfusion. SHRs treated with EX-9 displayed higher blood pressure values than SHRs treated with vehicle (182 ± 4 vs. 172 ± 1 mmHg, p < 0.05), while Exendin-4 treatment attenuated blood pressure compared to controls (161 ± 4 vs. 172 ± 1 mmHg, p < 0.01). Blood pressure increase in SHRs treated with EX-9 was associated with higher NHE3 activity (1.78 ± 0.08 nmol/cm2/s) in proximal renal tubule compared to controls (1.48 ± 0.10 nmol/cm2/s; p < 0.05), whileEX-4-treatedSHR treated displayed lower NHE3 activity (1.19 ± 0.07 nmol/cm2/s, p < 0.01). Additionally, SHRs treated with the GLP-1R antagonist show higher levels of urinary protein excretion, fibrosis, inflammation, oxidative stress markers and intrarenal renin angiotensin system (RAS) activity compared to control. On the other hand, systemic administration of EX-4 exertedanti-proteinuric, anti-inflammatory and antioxidant effects. The renoprotection conferred by EX-4 treatment was accompanied by lower renal cortex angiotensin II levels suggesting that GLP-1R activation reduces intra renal RAS activity. Collectively, these results demonstrate that chronic GLP-1R blockade in hypertensive rats intensifies blood pressure increase and exacerbates renal damage. On the other hand, the GLP-1R agonism exerts anti-hypertensive and renoprotetor effects

O peptídeo-1 semelhante ao glucagon (GLP-1) é um hormônio incretina secretado pelas células L do trato gastrointestinal e liberado imediatamente após a ingestão de alimento. O GLP-1 estimula a secreção de insulina pós-prandial moderando a elevação precoce da glicose no sangue. Embora primariamente envolvido na homeostase da glicose, o GLP-1 é capaz de induzir a diurese e natriurese, quando administrado em doses farmacológicas em humanos e em roedores. Estudos prévios do nosso laboratório demonstraram que o mecanismo de ação renal do GLP-1, bem como de agonistas sintéticos do receptor GLP-1R, envolve o aumento do fluxo plasmático renal (FPR) e do ritmo de filtração glomerular (RFG) bem como a diminuição da reabsorção de sódio dependente da isoforma 3 do trocador Na?/H? (NHE3) em túbulo proximal renal. Entretanto, até o momento, nenhum estudo investigou se o GLP-1 endógeno exerce efeitos sobre o manuseio renal de sal e água, nem o seu papel fisiológico sobre a regulação da atividade do NHE3. Portanto, o objetivo deste estudo foi testar a hipótese que o GLP-1 endógeno modula a função renal de ratos, ao menos em parte, via inibição da atividade do NHE3 em túbulo renal. Para este fim, ratos Wistar (2-3 meses de idade) foram devidamente anestesiados, submetidos à traqueostomia e tiveram a veia jugular e a bexiga canuladas para infusão de uma solução contendo 100 ug/kg/min do antagonista do receptor GLP-1R exendin-9 (Ex-9, 40 uL/min) por um período de 30 minutos e para a coleta de urina, respectivamente. A infusão sistêmica de Ex-9 diminuiu a concentração de AMPc urinário e atividade da PKA cortical renal consistente com o bloqueio da sinalização deflagrada pela interação GLP-1/GLP-1R no rim. Além disso, a administração sistêmica de Ex-9 reduziu a diurese, natriurese, RFG, FPR, clearance de lítio e pH urinário. Em experimentos de microperfusão estacionária in vivo, não foram observadas diferenças no fluxo de bicarbonato dependente de NHE3 entre os túbulos proximais perfundidos com exendin-9 (2 uM) e os túbulos perfundidos com solução controle. No entanto, a perfusão tubular proximal com Ex-9 foi capaz de bloquear completamente as ações inibitórias do GLP-1 (20 nM) sobre a atividade do NHE3. Por outro lado, a infusão sistêmica do Ex-9 reduziu os níveis de fosforilação da serina 552, sítio consenso para a fosforilação por PKA localizado na região C-terminal do NHE3, e que está associado à inibição da atividade de troca Na+/H+ mediada por este transportador. Baseando-se nos achados que a infusão sistêmica do Ex-9 aumenta a reabsorção de sódio e secreção de H?, reduz o clearance do lítio e os diminui os níveis de fosforilação do NHE3 na serina 552 são consistentes com um aumento na atividade deste transportador na ausência/redução da sinalização mediada pela interação do GLP-1 endógeno com seu receptor no rim. Por sua vez, o fato do Ex-9 não afetar a atividade do NHE3 sob as condições experimentais da microperfusão estacionária in vivo é condizente com o fato do GLP-1 não ser sintetizado no néfron e sugere fortemente que é o GLP-1 filtrado que se liga ao seu receptor no túbulo proximal renal resultando na diminuição da reabsorção de bicarbonato de sódio mediada pelo NHE3. Em conjunto, estes resultados sugerem que o GLP-1 endógeno exerce efeito tônico sobre o manuseio renal de sódio e água, mediando portanto, uma relação funcional entre a homeostase glicêmica e volêmica / The glucagon like peptide-1 (GLP-1) is an incretin hormone secreted by the L-cells of the gastrointestinal tract and released immediately after ingestion of food. GLP-1 stimulates postprandial insulin secretion moderating early increase in blood glucose. Although primarily involved in glucose homeostasis, GLP-1 is capable of inducing diuresis and natriuresis when administered in pharmacologic doses in humans and rodents. Previous studies from our laboratory have shown that the renal mechanism of action of GLP-1 and synthetic agonists of GLP-1R receptor, involves an increase of renal plasma flow (RPF) and glomerular filtration rate (GFR) as well a decrease in reabsorption of sodium mediated by the Na? / H? exchanger (NHE3) isoform 3 in the renal proximal tubule. However, to date, no study has investigated whether endogenous GLP-1 exerts effects on the renal handling of salt and water, or its physiological role in the regulation of the activity of NHE3. Therefore, the aim of this study was to test the hypothesis that endogenous GLP-1 modulates renal function in rats, at least in part, via inhibition of the NHE3 in renal tubule. To this end, male Wistar rats (2-3 months old) were properly anesthetized, tracheostomized and the jugular vein and the bladder were cannulated to the infusion of a solution containing 100 ug / kg / min GLP-1R antagonist receiver exendin-9 (Ex-9, 40 uL/min) for a period of 30 minutes and to collect urine, respectively. Systemic infusion of Ex-9 reduced the urinary concentration of cAMP and the renal cortical PKA activity, consistent with the blockage of the signal triggered by the interaction of GLP-1 / GLP-1R in the kidney. Furthermore, systemic administration of ex-9 reduced diuresis, natriuresis, GFR, RPF, lithium clearance and urinary pH. In experiments of in vivo stationary microperfusion, no differences were observed in the NHE3-mediated net bicarbonate flow between proximal tubules perfused with exendin-9 (2 mM) and perfused tubules with control solution. However, the tubular proximal perfusion with Ex-9 was able to completely block the inhibitory actions of GLP-1 (20 nM) on the activity of NHE3. On the other hand, systemic infusion of Ex-9 reduced phosphorylation levels of serine 552, a consensus site for phosphorylation by PKA located in the C-terminal region of NHE3, which is associated with inhibition of exchange activity of Na+/H+ mediated by this transporter. Collectively, the findings that systemic infusion of Ex-9 increases sodium reabsorption and secretion of H+, reduces the lithium clearance and decreases the NHE3 phosphorylation at serine 552 levels are consistent with the idea that NHE3 activity is upregulated in the absence/reduction of the signaling cascade mediated by the interaction of the endogenous GLP-1 with its receptor in the kidney. In turn, the fact Ex-9 does not affect the activity of NHE3 under the experimental conditions of stationary microperfusion in vivo is consistent with the fact that GLP-1 is not synthesized in the nephron. Besides, it strongly suggests that is the filtrated GLP-1 that binds to its receptor in renal proximal tubule, resulting in a decrease in NHE3-mediated sodium bicarbonate reabsorption. Taken together, these results suggest that endogenous GLP-1 exerts a tonic effect on renal sodium and water handling, mediating therefore a functional relationship between volume and glucose homeostasis

Introdução/Objetivo: Este estudo teve como objetivo testar a hipótese de que a atividade e/ou expressão da dipeptidil peptidase IV (DPPIV), uma enzima que inativa peptídeos com ações cardioreno protetoras, como o peptídeo-1 semelhante ao glucagon (GLP-1) e o peptídeo natriurético cerebral (BNP), estaria associada a um pior prognóstico na insuficiência cardíaca (HF). Métodos: Injúria do miocárdio foi realizada através da ablação do ventrículo esquerdo (VE) por radiofrequência em ratos Wistar machos (200-250 g). Os ratos foram divididos em três grupos: Sham, HF e HF + inibidor de DPPIV (sitagliptina 200mg/kg/b.i.d). Seis semanas após a cirurgia, os animais foram alojados individualmente em gaiolas metabólicas durante 3 dias para avaliação da função renal. Atividade e expressão da DPPIV no plasma e coração foram medidas por espectrofotometria e por immunoblotting, respectivamente. Para a avaliação da função cardíaca um cateter de pressão-volume foi posicionado dentro da cavidade do VE. A análise histológica foi realizada para os parâmetros morfométricos. A atividade da DPPIV no plasma também foi medida em pacientes com HF (n = 190). Resultados: A atividade DPPIV e sua abundância estavam aumentadas em animais com HF em comparação com Sham. Além disso, a atividade de DPPIV no plasma se correlacionou positivamente com o volume diastólico final (R = 0,517; p < 0,001) e o peso do pulmão/peso corporal (R = 0,492; p < 0,01). Uma correlação negativa entre a atividade DPPIV plasmática e a fração de ejeção também foi observada (R = 0,602; p < 0,001). Curiosamente, os animais HF também exibiram um aumento da expressão/atividade de DPPIV no tecido cardíaco, especialmente em células endoteliais. Seis semanas de tratamento com o inibidor de DPPIV sitagliptina atenuou a disfunção cardíaca, fibrose intersticial, congestão pulmonar e infiltração de macrófagos. O tratamento com sitagliptina também elevou os níveis plasmáticos de GLP-1 ativo, e aumentou a ativação de vias de sinalização cardioprotetoras como PKA e Akt; e reduziu os níveis de apoptose e marcadores pró-inflamatórios em comparação com ratos não tratados. Ratos com HF apresentaram maiores níveis circulantes de BNP, contudo a atividade da PKG renal foi mais baixa nesses animais em comparação com o grupo tratado com sitagliptina, sugerindo uma diminuição da razão BNP ativo/total. A função renal não diferiu entre os grupos, mas o ritmo de filtração glomerular estava ligeiramente aumentado no grupo tratado em comparação com os animais HF. Pacientes com HF apresentaram uma maior atividade plasmática da DPPIV e correlações foram encontradas com a com a fração de ejeção (R = -0,20; p = 0,009) e a quimiocina Ccl2 (R² =0,30; p < 0.01). Conclusões: Em conjunto, nossos resultados demonstram que a atividade plasmática da DPPIV se correlaciona com um pior prognóstico em pacientes e animais com HF e que a DPPIV possui um papel importante na fisiopatologia desta doença / Aim: The present study aimed to test the hypothesis that the activity and/or expression of dipeptidyl peptidase IV (DPPIV), an enzyme that inactivates cardiorenal protective peptides including glucagon-like peptide-1 (GLP-1) and brain natriuretic peptide (BNP), would be associated with poorer outcomes in heart failure (HF). Methods: Experimental HF was induced in male Wistar rats (200-250 g) by left ventricular (LV) myocardial injury after radiofrequency catheter ablation. Rats were divided in three groups: Sham, HF and HF+DPPIV inhibitor (sitagliptin 200mg/kg/b.i.d). Six weeks after surgery, animals were individually housed in metabolic cages during 3 days for assessment of renal function. Plasma and heart DPPIV activity/expression were measured spectrophotometrically and by immunoblotting respectively. For evaluation of cardiac function a pressure-volume catheter was positioned into the LV cavity. Histological analysis was performed for morphometric parameters. Plasma DPPIV activity was also measured in patients (n = 190) with heart failure. Results: Plasma DPPIV activity and abundance were increased in animals with HF compared to Sham. Additionally, plasma DPPIV activity positively correlated with ventricular end diastolic volume (R² =0.517; p < 0.001) and lung/body weight (R² =0.492; p < 0.01). A negative correlation between plasma DPPIV activity and ejection fraction was also observed (R² =0.602; p < 0.001). Interestingly, HF animals also exhibited an increase of expression and activity of DPPIV in heart tissue, especially in endothelial cells. Six-week treatment with the DPPIV inhibitor sitagliptin attenuated cardiac dysfunction, mitigated cardiac hypertrophy, interstitial fibrosis, lung congestion and macrophage infiltration. Sitagliptin also raised the plasma levels of active GLP-1, increased activation of cardioprotective signaling pathways including PKA, and Akt; and reduced the levels of apoptosis and pro-inflammatory biomarkers compared to non-treated HF rats. Despite the higher circulating total BNP, renal PKG activity was lower in HF rats compared with sham and sitagliptin-treated rats, suggesting a decrease in active/total BNP ratio. Renal function did not differ between groups, but glomerular filtration rate was modestly, but significantly increased by Sitagliptin compared to HF. Plasma DPPIV activity in patients was also increased compared to healthy subjects and correlations was found with ejection fraction (R² =-0.20; p=0.009) and the chemokine Ccl2 (R² =0.30; p < 0.01). Conclusions: Taken together, our results demonstrate that circulating DPPIV activity correlates with poorer cardiovascular outcomes in human and experimental HF and might play an important role in the pathophysiology of HF.

O diabetes mellitus do tipo 1 (DM1) é uma doença autoimune caracterizada pela destruição seletiva de células pancreáticas produtoras de insulina. Existem diversas formas de tratamento do DM1, tais como administração de insulina, imunossupressores, transplantes de pâncreas ou de ilhotas pancreáticas, porém todos se mostram ineficientes em algum aspecto. Recentemente, uma nova classe de medicamentos, os inibidores da enzima dipeptidil peptidase 4 (iDPP-4), demonstrou eficiência terapêutica e segurança no tratamento de pacientes com diabetes mellitus do tipo 2 devido ao aumento do hormônio peptídeo-1 semelhante ao glucagon (GLP-1, do inglês glucagon-like peptide-1). Além disso, o uso de inibidores de DPP-4 em modelos experimentais de DM1 demonstrou proteção das células pancreáticas contra apoptose, estimulação de neogênese de ilhotas pancreáticas e melhora do controle homeostático da glicose. Esse presente projeto teve como objetivo avaliar o perfil imunológico e endocrinológico da administração do inibidor de DPP-4 (MK0431) em DM1 experimental quimicamente induzido por estreptozotocina em camundongos C57Bl/6. Os animais diabéticos foram tratados com ração controle ou ração contendo inibidor de DPP-4 (4g MK0431/Kg de ração) ad libitum durante 30 e 90 dias. Durante o tratamento os animais tiveram glicemia, peso e teste de tolerância oral à glicose avaliados. Ao final do tratamento, os animais foram eutanasiados e o sangue, baço, timo, linfonodos pancreáticos e pâncreas foram coletados. Após 30 dias de tratamento com inibidor, foi observado um aumento do hormônio GLP-1 no soro, além de um padrão imunológico favorável. Dentre os mecanismos imunológicos, foi possível observar um aumento de células T reguladoras (CD4+CD25+Foxp3+) no baço e uma diminuição da citocina IFN- no homogenato pancreático. Após 90 dias de tratamento com inibidor, também foi detectado um aumento de insulina e GLP-1 séricos e uma diminuição nos níveis glicêmicos dos animais tratados. Observou-se uma redução no padrão inflamatório no microambiente pancreático, caracterizado pela diminuição das citocinas TNF- e IFN- no homogenato pancreático e por uma redução da freqüência de macrófagos CD11b+ nos linfonodos pancreáticos. Os resultados obtidos neste projeto contribuíram para validar a eficácia terapêutica da administração de inibidor de DPP-4 no tratamento do DM1 experimental, bem como os mecanismos imunológicos e endocrinológicos envolvidos. Sem a ocorrência de efeitos tóxicos relevantes, o uso de inibidores de DPP-4 pode se tornar uma alternativa terapêutica para o tratamento do DM1 em humanos, que constitui uma doença crônica associada à baixa qualidade de vida em longo prazo e necessidade de tratamento de alto custo. / Davanso, M.R. Immunoendocrinological analyses after administration of dipeptidyl-peptidase-4 inhibitor on experimental type 1 diabetes. 2012. 105p. Thesis (Masters Degree) School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 2012. Type 1 Diabetes Mellitus (DM1) is an autoimmune disease characterized by the selective destruction of the insulin-producing pancreatic cells. Several forms of treatment for DM1 are current known such as insulin administration, immunosuppressors, pancreas or pancreatic islets transplantation, however, they all are inefficient in some aspect. Recently, a new class of drugs, the dipeptidyl-peptidase-4 inhibitors (iDPP-4) showed therapeutic efficacy and safety in the treatment with type 2 diabetes mellitus patients due to an increase in the glucagon-like peptide-1 (GLP-1). In addition, the use of DPP-4 inhibitors in experimental models of DM1 has demonstrated a protection of pancreatic cells against apoptosis, stimulation of pancreatic islets neogenesis and improvement in the glucose homeostatic control. This project evaluated the immunological and endocrinological profile of the DPP-4 (MK0431) inhibitor administration in experimental chemically induced DM1 by streptozotocin in C57BI/6 mice. The diabetic animals were treated with either a normal chow diet or diet containing DPP-4 inhibitor (4g MK0431/Kg of diet) ad libitum during 30 and 90 days. During the treatment the animals were evaluated regarding glycemia, weight, and oral glucose tolerance test. At the end of the treatment, the animals were killed and the blood, spleen, thymus, pancreatic lymph nodes and pancreas were collected. After 30 days of treatment with inhibitor, it was observed an increase in the hormone GLP-1 in the serum, besides a favorable immunological pattern. Among the immunologic mechanisms, it was possible to observe an increase in the regulator T cells (CD4+CD25+Foxp3+) of the spleen and a decrease in the cytokine IFN- in the pancreatic homogenate. After 90 days of treatment with inhibitor, it was also noticed an increase in the insulin and serum GLP-1 levels as well as a decrease in the glycemic levels in the treated animals. It was observed a reduction in the inflammatory pattern in the pancreatic microenvironment characterized by a decrease in the cytokines TNF- and IFN- in the pancreatic homogenate and by a reduction in the frequency of CD11b+ macrophages in the pancreatic lymph nodes. The results obtained in this project contributed to validate the therapeutic efficacy of the DPP-4 inhibitor administration in the treatment of experimental DM1, as well as the immunological and endocrinological mechanisms involved. Without the occurrence of relevant toxic effects, the use of DPP-4 inhibitors may become a therapeutic alternative for the treatment of DM1 in humans, which constitutes a chronic disease associated to low life quality and need for high cost treatment.

<p>Therapeutic peptides have become a clinically and commercially important drug class providing novel treatment options in variety of disease areas. Today, more than 80 peptide drugs are marketed worldwide and hundreds more are in development. However, the development of peptide drugs can be hindered by their tendency to self-associate to form fibrils, an impurity that can affect potency and increase the potential for adverse immune responses in patients. Fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. From a pharmaceutical development perspective, early detection of instabilities can inform the development of mitigation strategies to minimize the risk of product failure and avoid costly delays in clinical development. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and formulations.</p> <p>The objective of this dissertation was to develop structurally modified fibrillation-resistant peptides based on a mechanistic understanding of the fibrillation process. The therapeutic peptides studied were human calcitonin (hCT), a glucagon/GLP-1 analog, and human insulin B-chain (INSB). Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) and other biophysical methods were used to provide mechanistic understanding of the intermolecular interactions and structural transitions during peptide fibrillation. Coupled with proteolytic digestion, pulsed HDX-MS of fibrillating peptides enabled identification of the residues involved in the early interactions leading to fibrillation based on their differential deuterium exchange rates. The high-resolution residue level information was used to make site-specific modifications to hCT, with phosphorylation in the central region resulting in complete inhibition of fibrillation for the phospho-Thr-13 hCT analog under the stress conditions employed. Reversible ‘prodrug’ modifications such as phosphorylation can aid the rational design of fibrillation-resistant therapeutic peptides. Furthermore, the effects of structural modifications on peptide fibrillation were evaluated by reducing the Cys1-Cys7 disulfide bond in hCT, and by C-terminal amidation or substitution with a helix-stabilizing residue (α-aminoisobutyric acid, Aib) in the glucagon/GLP-1 analog peptide. Finally, studies of insulin B-chain probed fibrillation mechanisms of this therapeutically important peptide, contributing to our understanding of the mechanisms of insulin fibrillation with the broad goal of developing fibrillation-resistant, rapid-acting, monomeric insulin analogs. Overall, the results demonstrate that small structural changes can have significant effects on peptide fibrillation, that pulsed HDX-MS can be used to probe these effects, and that an understanding of these effects can inform the rational development of fibrillation-resistant peptide drugs. </p>

Indiana University-Purdue University Indianapolis (IUPUI) / Glucagon-like peptide 1 (GLP-1) receptor agonists are a class of incretin based therapeutics which aid in blood glucose management in Type II diabetes mellitus (T2DM). Recent studies have demonstrated direct cardiovascular benefits conferred by these agents including protection in ischemia and heart failure. Despite these observations, human clinical trials fail to support improvements in cardiovascular outcomes independent of glucose lowering effects in the T2DM populations. Prior data from our lab demonstrate that obesity impairs GLP-1 associated increases in myocardial glucose uptake. However, the reasons for this impairment/resistance to cardiac effects of GLP-1 in the setting of obesity remain ill defined. This investigation tested the hypothesis that underlying differences in the cardiac proteome and microRNA (miR) transcriptome could contribute to distinct cardiac responses to ischemia and activation of GLP-1 signaling in the setting of obesity. To identify whether obesity modulated cardiac functional responses to GLP 1 related drugs, we first examined the effects of obesity on cardiac function, miR transcriptome, and proteome in response to short duration ischemia-reperfusion (I/R). We observed divergent physiologic responses (e.g. increased diastolic volume and systolic pressure in lean, decreased diastolic volumes in obese) to regional I/R in obese vs lean hearts that were associated with significant molecular changes as detected by protein mass spectrometry and miR microarray. Molecular changes were related to myocardial calcium handling (SERCA2a, histidine-rich Ca2+ binding protein), myocardial structure and function (titin), and miRs relating to cardiac metabolism, hypertrophy, and cell death, including miR-15, miR-30, miR-199a, miR-214. Importantly, these effects were modified differently by GLP-1 agonism in lean vs obese swine. Additional studies investigated the functional effects of 30 days of treatment with the GLP-1 analogue liraglutide on a model of slowly-developing, unrelieved coronary ischemia. Liraglutide failed to reduce infarct size or collagen deposition. However, analysis of left ventricular pressure-volume relationships support that liraglutide improved diastolic relaxation/filling, load-dependent indices of cardiac function, and cardiac efficiency in response to sympathetic stimulation in obese swine. Taken together, these findings support that miR and proteomic differences underlie distinct changes in functional cardiac responses to I/R and pharmacologic activation of GLP-1 signaling in the setting of obesity.

Ce travail porte sur des approches translationnelles dans les synucléinopathies notamment l’atrophie multisystématisée (AMS). Au-delà de leur rôle dans la régulation du glucose, l’insulin et l’insulin like growth factor-1 (IGF-1) ont des propriétés neurotrophiques. Des études ont montrées que la signalisation de l’insuline/IGF-1 est altérée dans la maladie d'Alzheimer et des données suggèrent l’altération de l’insuline/IGF-1 dans la maladie de Parkinson (MP) et l’AMS. Nous avons mis en évidence une résistance à l’insuline dans les neurones des patients MP et AMS ainsi que dans les oligodendrocytes chez les patients AMS.Mon travail a également consisté à cibler la troncation de l’α-synuclein (α-syn) comme cible thérapeutique. Nous avons démontré dans un modèle murin d’AMS que la diminution de l’α-syn tronquée permettait de réduire l’agrégation d’α-syn et la dégénérescence des neurones dopaminergiques.Enfin, nous avons étudié l’implication dans l’AMS des métalloprotéinases matricielles (MMP), des enzymes impliquées dans remodelage de la matrice, la démyélinisation, la troncation de l’α-syn et la perméabilité de la barrière hémato-encéphalique. Ce travail nous a permis de montrer une augmentation de l’expression et de l’activité de MMPs chez les patients AMS. Nous avons également montré que les cellules gliales sont la source de cette augmentation et que la MMP-2 est retrouvée dans les agrégats des patients AMS.Nous montrons ici de caractéristiques distinctes de l’AMS comme des altérations qui se produisent dans les oligodendrocytes. Nous présentons aussi VX-765 comme un candidat prometteur pour ralentir la progression de la pathologie dans un contexte de synucléinopathie. / This work focused on translational approaches in synucleinopathies and more specifically in multiple system atrophy (MSA). Beyond their role in glucose homeostasis, insulin/IGF-1 are neurotrophic factors in the brain. Studies have shown altered insulin/IGF-1 signalling in Alzheimer’s disease and data suggest impaired insulin signaling/IGF-1 in Parkinson's disease (PD) and MSA. The aim of my work was to characterize insulin/IGF-1 signalling in MSA and PD brain tissue. Both groups showed neuronal insulin resistance. Oligodendrocytes in MSA patients were also insulin resistant.In line with the translational approach, we also targeted α-synuclein (α-syn) truncation pharmacologically in MSA transgenic mice, which led to reduced α-syn aggregation and the protection of dopaminergic neurons.We also assessed the activity and distribution of matrix metalloproteinases (MMPs) in the brain of MSA patients compared to healthy controls. MMPs are involved in the remodelling of the extracellular matrix, demyelination, α-syn truncation and blood brain barrier permeability. We showed altered expression and activity of MMPs in two distinct structures in MSA brains. We were also able to show that glial cells were the source of increased MMPs and show a unique expression of MMPs in α-syn aggregates of MSA patients compared to PD, evidence that might hint at a mechanism that is differently altered between PD and MSA.We here show distinct pathological features of MSA such as key alterations occurring in oligodendrocytes, further supporting MSA as a primary oligodendrogliopathy. We also present VX-765 as a candidate drug for disease modification in synucleinopathies.

L’hyperglycémie de stress et la variabilité glycémique, consécutives à la réaction inflammatoire péri opératoire, sont associées à une morbidité et une mortalité accrues en chirurgie cardiaque. L’insulinothérapie intraveineuse administrée à l’aide de protocoles complexes, dits « dynamiques », constitue à l’heure actuelle le traitement de référence de l’hyperglycémie de stress. L’intérêt du contrôle glycémique péri-opératoire est admis par tous, sans qu’il existe de consensus véritable quant aux objectifs à atteindre, et reste très exigeant en termes de charge de soins. Dans la 1ère partie de ce travail, nous avons voulu vérifier si, 7 ans après sa mise en place, l’observance du protocole d’insulinothérapie utilisé dans notre Unité de Soins Intensifs de Chirurgie Cardiaque était conforme à celle mesurée lors de son implantation. Nous avons constaté des dérives majeures dans l’application du protocole qui ont pu être corrigées par la mise en place de mesures correctrices simples. Dans une 2ème partie du travail, nous avons cherché à évaluer si, à l’instar de la chirurgie cardiaque classique, une variabilité glycémique accrue était associée à une altération du pronostic des patients bénéficiant d’une procédure moins invasive (remplacement valvulaire aortique percutané ou TAVI). Nous avons ainsi analysé les données des patients ayant bénéficié d’un TAVI dans notre centre, et inclus dans les registres multicentriques français France et France-2. Nos résultats suggèrent une association entre une augmentation de la variabilité glycémique et un risque accru de complications cardiovasculaires majeures dans les 30 premiers jours, indépendamment de la qualité du contrôle glycémique obtenu. Enfin, dans une 3ème partie nous avons voulu savoir si exenatide, analogue de synthèse de GLP-1, permettait d’améliorer le contrôle glycémique péri opératoire en chirurgie cardiaque. Nous avons conduit un essai randomisé contrôlé de phase II/III montrant que l’administration intraveineuse (IV) d’exenatide, ne permettait pas d’améliorer la qualité du contrôle glycémique ou de réduire la variabilité glycémique par rapport à l’insuline IV, mais permettait de retarder l’administration d’insuline et de diminuer la quantité d’insuline administrée. Notre étude suggère également une diminution de la charge en soins. Du fait des données rapportées chez l’animal et dans l’infarctus du myocarde, nous avons également conduit une étude ancillaire suggérant l’absence d’effets cardioprotecteurs majeurs d’exenatide sur les lésions d’ischémie-reperfusion myocardiques, ne permettant pas d’améliorer la fonction cardiaque gauche à court et à moyen terme. L’optimisation du contrôle glycémique en chirurgie cardiaque nécessite ainsi la recherche de stratégies visant à améliorer l’observance des protocoles de soins et à réduire la variabilité glycémique. La place des analogues du GLP-1 reste à définir dans cette indication. / Stress hyperglycemia and glycemic variability are associated with increased morbidity and mortality in cardiac surgery patients. Intravenous (IV) insulin therapy using complex dynamic protocols is the gold standard treatment for stress hyperglycemia. If the optimal blood glucose target range remains a matter of debate, blood glucose control using IV insulin therapy protocols has become part of the good clinical practices during the postoperative period, but implies a significant increase in nurse workload. In the 1st part of the thesis, we aimed at checking the nurse-compliance to the insulin therapy protocol used in our Cardiac Surgery Intensive Care Unit 7 years after its implementation. Major deviations have been observed and simple corrective measures have restored a high level of nurse compliance. In the 2nd part of this thesis, we aimed at assessing whether blood glucose variability could be related to poor outcome in transcatheter aortic valve implantation (TAVI) patients, as reported in more invasive cardiac surgery procedures. The analysis of data from patients who undergone TAVI in our institution and included in the multicenter France and France-2 registries suggested that increased glycemic variability is associated with a higher rate of major adverse events occurring between the 3rd and the 30th day after TAVI, regardless of hyperglycemia. In the 3rd part if this thesis, we conducted a randomized controlled phase II/III trial to investigate the clinical effectiveness of IV exenatide in perioperative blood glucose control after coronary artery bypass graft surgery. Intravenous exenatide failed to improve blood glucose control and to decrease glycemic variability, but allowed to delay the start in insulin infusion and to lower the insulin dose required. Moreover, IV exenatide could allow a significant decrease in nurse workload. The ancillary analysis of this trial suggested that IV exenatide did neither provide cardio protective effect against myocardial ischemia-reperfusion injuries nor improve the left ventricular function by using IV exenatide. Strategies aiming at improving nurse compliance to insulin therapy protocols and at reducing blood glucose variability could be suitable to improve blood glucose control in cardiac surgery patients. The use of the analogues of GLP-1 in cardiac surgery patients needs to be investigated otherwise.