Dissertations / Theses on the topic 'Diabetes Insipidus'

Background: Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease with symptoms of polydipsia, polyuria and dehydration caused by arginine vasopressin deficiency. Disease onset is within infancy or adolescence. A variety of disease-causing mutations of the arginine vasopressin neurophysin II gene (AVP) on chromosome 20p13 have been described. Methods: Two Polish families with adFNDI were screened for mutations. Processing of wild-type (WT) and mutant AVP was monitored using immunocytochemical methods in stably transfected Neuro2A cells. AVP secretion into the cell culture supernatant was investigated with an enzyme immunoassay. Results: In the first family a heterozygous p.G96D mutation was identified. Some patients additionally carried a novel heterozygous mutation p.A159T. The second family presented with a heterozygous mutation p.C98G. Confocal laser microscopy unveiled accumulation of p.G96D and p.C98G prohormones in the cellular bodies, whereas WT and p.A159T prohormones and/or processed products were located in the tips of cellular processes. Reduced levels of AVP in supernatant culture medium of p.G96D and p.C98G transfected cells in comparison to p.A159T and WT cells were found. Conclusions: We conclude that the p.G96D and p.C98G mutations cause adFNDI in the two reported families. The sequence variant p.A159T does not seem to have disease-causing effects
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Os pacientes que usam lítio (Li) para tratamento do transtorno bipolar frequentemente apresentam poliúria e deficiência de concentração urinária, sintomas do Diabetes Insipidus Nefrogênico (DIN). Animais tratados com Li apresentam baixos níveis de produção de adenosina monofosfato cíclico (AMPc) em resposta ao hormônio antidiurético (HAD). O Sildenafil (Sil), um inibidor da fosfodiesterase 5 (PDE5), eleva os níveis intracelulares de guanosina monofosfato cíclico (GMPc), levando a inserção de aquaporina 2 (AQP2) na membrana plasmática das células do ducto coletor. Portanto, inibidores de PDE podem promover a inserção de AQP2 na membrana plasmática mesmo sem a ativação do receptor de HAD, indicando a participação de uma via alternativa mediada pelo GMPc. Nós investigamos as vias de ação do Sil no tratamento da DIN induzida pelo Li. Ratos Wistar foram divididos nos seguintes grupos: grupo controle, recebendo dieta alimentar normal durante quatro semanas; grupo Li, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas; grupo Li + Sil, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas e 200 mg por quilo de dieta de Sil a partir da segunda semana; grupo Sil, recebendo dieta alimentar normal durante a primeira semana e a partir da segunda semana recebendo dieta normal com 200 mg de Sil por quilo de dieta. Os animais do grupo Li desenvolveram poliúria, diminuição da osmolalidade urinária e diminuição da expressão da AQP2 tanto na fração citoplasmática como de membrana celular e o Sil reverteu essas alterações. Demonstramos ainda que a concentração de GMPc intracelular estava aumentada nos túbulos papilares tratados com Sil. Observamos que a provável via de fosforilação da AQP2 induzida pelo GMPc é pela PKA. Além disso, o tratamento com Sil aumenta a expressão de pCreb, fator de transcrição para ativação do gene da AQP2. Observamos ainda que o Li diminui a expressão de eNOS e o tratamento com Sil normaliza essa diminuição. Assim, concluímos que o tratamento com Sil em ratos com DIN melhora a poliúria aumentando a produção e a inserção de AQP2. O tratamento com Sil pode ser benéfico para pacientes que sofrem com DIN induzido pelo Li
Patients taking lithium to treat bipolar disorder often present polyuria and urinary concentrating defect. In addition, lithium-treated animals present lower cyclic adenosine monophosphate production in response to vasopressin. Sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, elevates intracellular cyclic guanosine monophosphate (cGMP) levels, leading to plasma membrane accumulation of aquaporin 2 (AQP2). Therefore, PDE inhibitors might induce AQP2 membrane insertion even without vasopressin receptor activation by activating a parallel cGMP-mediated signal transduction pathway. We investigated Sil pathways of action in rats with lithium-induced nephrogenic Diabetes Insipidus (NDI). Wistar rats received lithium (40 mmol/kg food) or not for 4 weeks (Li or control), some rats also receiving sildenafil (200 mg/kg food) in weeks 2-4, with or without lithium (Li+Sil orSil). Animals in Li group developed polyuria, decreased urinary osmolality and decreased expression of AQP2 in both the cytoplasmic fraction and the cell membrane and Sil reversed these changes. We also demonstrated that intracellular cGMP concentration was increased in papillary tubules treated with Sil. We found that PKA may be involved in the pathway of cGMP induced AQP2 phosphorylation. In addition, Sil treatment increases Creb phosphorylation. Creb phosphorylation, acts as AQP2 gene transcription factor. We also observed that Li decreases eNOS expression and treatment with Sil normalizes this alteration. We conclude that Sil treatment improves polyuria by increasing production and insertion of AQP2. Sil treatment may be beneficial to patients suffering from induced DIN Li

Os pacientes que usam lítio (Li) para tratamento do transtorno bipolar freqüentemente apresentam poliúria e deficiência de concentração urinária, sintomas do Diabetes Insipidus nefrogênico (DIN). Animais tratados com Li apresentam baixos níveis de produção de adenosina monofosfato cíclico (AMPc) em resposta ao hormônio antidiurético (HAD). O Sildenafil (Sil), um inibidor da fosfodiesterase 5 (PDE5), eleva os níveis intracelulares de guanosina monofosfato cíclico (GMPc), levando a inserção de aquaporina 2 (AQP2) na membrana plasmática das células do ducto coletor. Portanto, inibidores de PDE podem promover a inserção de AQP2 na membrana plasmática mesmo sem a ativação do receptor de HAD, indicando a participação de uma via alternativa mediada pelo GMPc. Nós investigamos o efeito do Sil na expressão renal das proteínas de membrana AQP2, UT-A1, NKCC2, NHE3, P-ENaC em ratos com DIN induzido pelo Li. Ratos Wistar foram divididos nos seguintes grupos: grupo controle, recebendo dieta alimentar normal durante quatro semanas; grupo Li, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas; grupo Li + Sil, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas e 200 mg por quilo de dieta de Sil a partir da segunda semana; grupo Sil, recebendo dieta alimentar normal durante a primeira semana e a partir da segunda semana recebendo dieta normal com 200 mg de Sil por quilo de dieta. Os animais do grupo Li desenvolveram poliúria, diminuição da osmolalidade urinária e diminuição da expressão da AQP2. No grupo Li+Sil, o Sil foi capaz de reverter parcialmente a poliúria, diminuir o clearance de água livre, aumentar a osmolalidade urinária e aumentar a expressão da AQP2. A expressão de UTA1 foi completamente normalizada com o tratamento com Sil. A expressão das proteínas NKCC2 e NHE3 apresentaram-se aumentadas no grupo tratado com Li, e o Sil não foi capaz de reverter tal alteração. Além disso, o tratamento com Sil reverteu completamente o aumento da resistência vascular renal. Assim, concluímos que o tratamento com Sil em ratos com DIN melhora a poliúria, aumenta a smolalidade urinária e diminui o clearance de água livre pelo aumento da expressão de AQP2 e UT-A1. O tratamento com Sil pode ser benéfico para pacientes que sofrem com DIN induzida pelo Li.
Patients taking lithium to treat bipolar disorder often present polyuria and urinary concentrating defect. In addition, lithium-treated animals present lower cyclic adenosine monophosphate production in response to vasopressin. Sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, elevates intracellular cyclic guanosine monophosphate (cGMP) levels, leading to plasma membrane accumulation of aquaporin 2 (AQP2). Therefore, PDE inhibitors might induce AQP2 membrane insertion even without vasopressin receptor activation by activating a parallel cGMP-mediated signal transduction pathway. We investigated the effect of sildenafil on renal expression of AQP2, UT-A1, sodium/hydrogen exchanger (NHE3), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), and the epithelial sodium channel alpha subunit (P-ENaC). Wistar rats received lithium (40 mmol/kg food) or not for 4 weeks (Li or control), some rats also receiving sildenafil (200 mg/kg food) in weeks 2-4, with or without lithium (Li+Sil or Sil). In Li+Sil rats, urine output was markedly lower, as was water free clearance, whereas urine osmolality was higher. Semiquantitative immunoblotting revealed the following: AQP2 expression was partially normalized; UT-A1 expression was completely normalized; expression of NKCC2 and NHE3 was significantly higher in Li rats (although not significantly different between Li+Sil rats and Li rats); and P-ENaC protein expression was unaltered in all groups. Sildenafil treatment completely reversed the lithium-induced increase in renal vascular resistance. In conclusion, sildenafil treatment of lithium-induced nephrogenic diabetes insipidus (NDI) improves polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1. Sildenafil treatment could be beneficial in patients with lithium-induced NDI.

Psychiatric patients treated with lithium (Li+) may develop nephrogenic diabetes insipidus (NDI). Although the etiology of Li+-induced NDI (Li-NDI) is poorly understood, it occurs partially due to reduced aquaporin-2 (AQP2) expression in the kidney collecting ducts. A mechanism postulated for this is that Li+ inhibits adenylyl cyclase (AC) activity, leading to decreased cAMP, reduced AQP2 abundance, and less membrane targeting. We hypothesized that Li-NDI would not develop in mice lacking AC6. Whole-body AC6 knockout (AC6(-/-)) mice and potentially novel connecting tubule/principal cell-specific AC6 knockout (AC6(loxloxCre)) mice had approximately 50% lower urine osmolality and doubled water intake under baseline conditions compared with controls. Dietary Li+ administration increased water intake and reduced urine osmolality in control, AC6(-/)-, and AC6(loxloxCre) mice. Consistent with AC6(-/-) mice, medullary AQP2 and pS256-AQP2 abundances were lower in AC6(loxloxCre) mice compared with controls under standard conditions, and levels were further reduced after Li+ administration. AC6loxloxCre and control mice had a similar increase in the numbers of proliferating cell nuclear antigen-positive cells in response to Li+. However, AC6(loxloxCre) mice had a higher number of H+-ATPase B1 subunit-positive cells under standard conditions and after Li+ administration. Collectively, AC6 has a minor role in Li-NDI development but may be important for determining the intercalated cell-to-principal cell ratio.

Orientadores: Desanka Dragosavac, Antonio Luis Eiras Falcão, Sebastião Araujo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Introdução - Alterações do sódio plasmático são freqüentemente encontradas no pós-operatório de pacientes submetidos à neurocirurgia e estão associadas à piora do estado neurológico desses pacientes. Essas alterações podem ser explicadas por três diferentes síndromes: Síndrome Perdedora de Sal (SPS), Síndrome da Secreção Inapropriada do Hormônio Antidiurético (SIADH) e Diabetes Insipidus (DI). A SPS e a SIADH apresentam características laboratoriais semelhantes como hiponatremia e natriurese, diferindo apenas quanto à volemia. Evidências recentes têm mostrado que a maioria dos pacientes hiponatrêmicos com doença intracraniana e que eram anteriormente diagnosticado como SIADH, na verdade são hipovolêmicos e apresentam SPS. Considerando que o tratamento adequado da SIADH (restrição volêmica) pode aumentar a incidência de infarto cerebral, piorando o prognóstico de pacientes com SPS (hipovolêmicos), e o tratamento para o DI envolve o uso de desmopressina (hormônio antidiurético sintético), a qual pode piorar a hiponatremia da SIADH e SPS, o correto diagnóstico diferencial entre essas síndromes torna-se essencial para um tratamento adequado. Objetivo - Verificar a incidência de alterações do sódio, correlacionando-as com alterações da AVP plasmática e identificar as síndromes responsáveis por essas alterações em pacientes neurocirúrgicos. Local - Hospital das Clínicas da Universidade Estadual de Campinas. Período - Novembro de 2000 a abril de 2001. Desenho - Estudo prospectivo, aberto, observacional. Metodologia - Foram estudados 30 pacientes submetidos à craniotomia para ressecção de tumor cerebral (grupo A) e clipagem de aneurisma (grupo B) no pré-operatório e durante os primeiros cinco dias de pós-operatório. Foram realizadas dosagens diárias de sódio e osmolaridade séricos e urinários (urina de 12h), além de dosagem da arginina-vasopressina (AVP) plasmática no 1º, 3º e 5º dias pós-operatórios. Resultados - O distúrbio do sódio mais freqüente foi a hiponatremia (sódio sérico < 135mEq/L), encontrada em 63,3% dos pacientes durante o pós-operatório, sendo a maior incidência observada no D1 (40%), estando presente no pré-operatório em 33,3% dos pacientes. A hipernatremia (sódio sérico > 146mEq/L) ocorreu em 3,5% dos pacientes do grupo A no pós-operatório. Natriurese (sódio urinário > 110mEq/12h) foi observada em 93,3% dos pacientes no pós-operatório, tendo sido maior no D1 e D2 e já estando presente no pré-operatório, sem diferença estatisticamente significativa entre os grupos. Poliúria (volume urinário > 30mL/kg/24h) foi observada em 100% dos pacientes, sendo mais freqüente no D2, estando presente também no pré-operatório. A AVP plasmática apresentou níveis elevados (>5,0pg/mL) em 10% e diminuídos (<0,5pg/mL) em 46,7% dos pacientes no pós-operatório, sendo esta mais freqüente no D3 (26,7%), sem diferenças estatisticamente significativas entre os grupos. A SPS foi encontrada em 27/30 pacientes (90%), sendo que 14 (46,7%) apresentaram SPS associada a DI e 13 (43,3%), apenas SPS. A SIADH foi encontrada em 3/30 pacientes (10%). Conclusão - A hiponatremia foi o distúrbio do sódio mais freqüentemente encontrado no pós-operatório de pacientes submetidos a neurocirurgia. A poliúria e a natriurese acompanham a hiponatremia e podem ser encontradas desde o pré-operatório, provavelmente, devido à presença de alterações cerebrais prévias (tumor ou HSA). A SPS foi a síndrome mais freqüente, estando associada com grande freqüência a níveis reduzidos de AVP plasmática. A SIADH foi a menos freqüente, podendo tratar-se apenas de resposta apropriada ao estresse cirúrgico, aumento de pressão intracraniana, dor, medicações ou perda de sangue durante a cirurgia
Abstract: Introduction - Alterations of plasmatic sodium are alterations frequently found in the neurosurgery postoperative period and are associate with the worsening of the these patients¿ neurological state. These alterations can be explained by three different syndromes: Cerebral Salt Wasting Syndrome (CSWS), Syndrome of the Inappropriate Secretion of Antidiuretic Hormone (SIADH) e Diabetes Insipidus (DI). The CSWS and the SIADH present similar laboratorial characteristics as hyponatremia and natriuresis, differing only to the volemy. Recent evidences have shown that the majority of the hyponatremic patients with intracranial disease were firstly diagnoses as SIADH, they are hypovolemic and present CSWS. Considering that the adequate treatment to the SIADH (volemic restriction) can increase the incidence of cerebral infarction, worsening the prognostic of patients with CSWS (hypovolemic) and that the treatment for the DI involves the desmopressin use (synthetic antidiuretic hormone), which can worsen the hyponatremia of the SIADH and CSWS; the differential diagnosis between these syndromes becomes essential for an adequate treatment. Objectives: To verify the occurrence of sodium alterations, correlating them with alterations of the plasmatic AVP and to identify the responsible syndromes for these alterations in neurosurgical patients. Local: Hospital of the Clinics of the State University of Campinas. Period: November of 2000 to April of 2001. Set: prospective, opened, observational study. Methodology: 30 patients submitted to the craniotomy for the cerebral tumor resection (group A) and aneurism correction (group B) were studied in the preoperative period and during the first 5 days of the postoperative period. Daily dosages of sodium and plasmatic and urinary osmolarity were used (12-hour urine); besides the dosage of the plasmatic arginine-vasopressin (AVP) in 1st, 3rd and 5th postoperative days. Results: The most frequent sodium disturbance was the hyponatremia (seric sodium <135meq/l), found in 63,3% patients during the postoperative period, being the major observed incidence in the 1st postoperative day (40%), presented in the preoperative in 33,3% of the patients. The hypernatremia (seric sodium > 146mEq/L) occurred in 3,5% of the group A patients in the postoperative period. Natriuresis (urinary sodium > 110mEq/12h) was observed in 93,3% of the patients in the postoperative day, being higher in the 1st and 2nd postoperative days and has already been presented in the preoperative period, without significant statistically difference between the groups. Poliury (urinary volume > 30ml/Kg/24h) was observed in 100% of the patients, being more frequent in the 2nd postoperative day when all the patients had presented poliury, being also present in the preoperative period. The plasmatic AVP presented high levels (> 5,0pg/ml) in 10% and decreased( <0,5pg/ml) in 46,7% of the postoperative patients, and more frequent in the 3rd postoperative day (26,7%), without significant statistically difference between the groups. The CSWS was found in 27/30 patients (90%), seeing that, 14 (46,7%) related to DI and 13 (43,3%), only CSWS. The SIADH was found in 3/30 patients (10%). Conclusion: Hyponatremia was the most frequently sodium disturbance found in the postoperative period of submitted patients to tumor resection and cerebral artery aneurism correction. The poliury and natriuresis follow the hyponatremia and can be found since the pre-operative period, probably, because of the presence of previous cerebral alterations (tumor or HSA). The CSWS was the most incident syndrome, being associated with high frequency to low plasmatic AVP levels. The SIADH was the least frequent and it could be just an appropriate reply to the surgical stress, increase of intracranial pressure, pain, drugs or loss of blood during the surgery
Mestrado
Pesquisa Experimental
Mestre em Cirurgia

Background: Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease with symptoms of polydipsia, polyuria and dehydration caused by arginine vasopressin deficiency. Disease onset is within infancy or adolescence. A variety of disease-causing mutations of the arginine vasopressin neurophysin II gene (AVP) on chromosome 20p13 have been described. Methods: Two Polish families with adFNDI were screened for mutations. Processing of wild-type (WT) and mutant AVP was monitored using immunocytochemical methods in stably transfected Neuro2A cells. AVP secretion into the cell culture supernatant was investigated with an enzyme immunoassay. Results: In the first family a heterozygous p.G96D mutation was identified. Some patients additionally carried a novel heterozygous mutation p.A159T. The second family presented with a heterozygous mutation p.C98G. Confocal laser microscopy unveiled accumulation of p.G96D and p.C98G prohormones in the cellular bodies, whereas WT and p.A159T prohormones and/or processed products were located in the tips of cellular processes. Reduced levels of AVP in supernatant culture medium of p.G96D and p.C98G transfected cells in comparison to p.A159T and WT cells were found. Conclusions: We conclude that the p.G96D and p.C98G mutations cause adFNDI in the two reported families. The sequence variant p.A159T does not seem to have disease-causing effects.
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Carbamazepina (Carba) é um anticonvulsivante, uma droga psicotrópica muito utilizada no tratamento de pacientes com distúrbios intelectuais. Esta droga foi utilizada para diminuir o volume urinário no Diabetes Insípido (DI), pois possui um efeito antidiurético, mas a incidência de hiponatremia é uma ocorrência comum. O lítio é uma das drogas mais importantes para o tratamento do distúrbio bipolar. No entanto, ele tem uma grande capacidade de induzir DI dificultando o seu uso em pacientes debilitados psicologicamente. Atualmente, a associação destas drogas é frequentemente utilizada para o tratamento de pacientes com distúrbios psiquiátricos e neurológicos. O objetivo deste trabalho foi investigar o efeito da Carba no ducto coletor medular interno (DCMI) de ratos normais e elucidar a sua ação no DI induzido pelo lítio: 1) Estudos in vitro- A) Estudos com microperfusão de segmentos isolados do néfron onde a permeabilidade à água (Pf, m/sec) foi determinada em DCMI perfundidos de ratos normais (n=20) na presença de Carba à 10-5 M, e na ausência de HAD. B) estudos com a técnica de Imunoblotting para avaliar a expressão da proteína Aquaporina 2 (AQP2) em suspensão de túbulos de DCMI de ratos normais incubados com 10-5 M de Carba por 30 minutos. 2) Estudos in vivo A) quatro grupos foram formados: a) Controle (n=5); b) Li (40 mmol/Kg/dieta por 3 semanas; n=4); c) Li+Carba (40 mmol Li/Kg/dieta + 400 mg carba/Kg/dia por 3 semanas; n=5); d) Carba (400 mg /Kg/dia por 3 semanas; n=5); - B) estudo da expressão da AQP2 no DCMI destes quatro grupos pela técnica de Western Blot. Resultados: 1) Estudos in vitro A) nos estudos de microperfusão a carba adicionada ao banho aumentou a Pf dos DCMI, na ausência de HAD (n=6) Controle12,3+ 3,6, Carba-62,6+14,8 (p<0,01), Recuperação (Rec)-17,4+5,5 (p<0,01). Com o intuito de estudar o mecanismo pelo qual a Carba ativa a cascata do HAD, foi utilizado o inibidor do receptor V2 do HAD (AV2; n=10): Controle-23,5+5,2, Carba-37,4±4,4 (p<0,01), Carba+AV2-19,6±5,0 (p<0,05), Rec-21,4+5,5; e Controle-18,6+7,0, AV2-27,3+7,2, AV2+Carba-25,3+5,7, Rec-32,6+6,4. O inibidor da PKA (H8; n=4) também foi utilizado: Controle-15,0+9,0, Carba-106,1+12,3 (p<0,01), Carba+H8-60,3+16,4 (p<0,01), Rec- 44,5+13,2. B) a análise densitométrica mostrou um aumento de 38,8% na expressão da AQP2 (Controle-100,0+8,3 vs. Carba-138,8+12,12, p<0,05); 1) Estudos in vivo Volume urinário (UV, mL/24h) Controle-10,7+3,0, Li-62,6+6,0 (p<0,001), Li+Carba-28,5+4,9 (p<0,001), Carba-23,3+3,0 (<0,001). Osmolalidade urinária (mOsm/Kg/H2O) Controle-819,6+175,7, Li-149,4+18,0 (p<0,01), Li+Carba-251,5+39,7 (p<0,05), Carba-396,2+75,5 (p<0,01). FENa+ (%) - Controle-0,15+0,01, Li-0,10+0,02, Li+Carba-0,12+0,02, Carba-0,11+0,02. Expressão da AQP2 (%) Controle-100,0+6,7, Li-55,8+5,4 (p<0,01), Li+Carba-75,8+9,6 (p<0,01), Carba- 99,7+4,7 (p<0,05). Não houve diferenças significantes no Na+, K+ e Osmolalidade plasmáticas. Em resumo, nossos dados revelaram que a Carba diminui o UV, aumenta a osmolalidade urinária e a expressão da AQP2 no DI induzido pelo lítio, e aumenta a permeabilidade à água, provavelmente agindo diretamente no receptor da vasopressina (V2). Estes resultados enfatizam que a hiponatremia encontrada nos pacientes que fazem uso da Carba pode ser explicada, pelo menos em parte, pelo aumento da permeabilidade osmótica no DCMI e que a poliúria do DI ocasionado pelo uso do lítio pode ser diminuída com a associação da Carba.
Carbamazepine (Carba) is an anticonvulsant and a psychotropic medication commonly used in the treatment of patients with intellectual disability (ID). This drug has been used to try to decrease the urinary volume in Diabetes Insipidus (DI) because Carba presents an antidiuretic effect, but the incidence of the hyponatremia in neurological patients is a common ocurrence. Lithium (Li) is one of the most important drugs used to treat bipolar mood disorders. However, Li has the undesirable capacity to induce DI complicating its usage in patients psychologically weakened. Nowadays, the association of these drugs is used in the treatment of patients with psychiatric and neurological problems. Our objective was to investigate the effect of Carba in the Inner Medullary Collecting Duct (IMCD) and elucidate its effect in the lithium-induced DI: 1) In vitro study: A) Microperfusion studies the water permeability (Pf, m/sec) was determined in normal rats IMCD isolated and perfused by the standard methods. Carba 10-5M was added to the bath fluid. B) Immunoblotting studies for AQP2 protein expression in IMCD tubule suspension from normal rats incubated with Carba 10-5M for 30 minutes. 2) In vivo study A) four groups of normal rats were done - a) Control (C, n=5); b) Li (40 mmol/kg/food/3 weeks n=4) c) Li+Carba (40 mmol Li/kg/food/3 weeks and 400 mg Carba/kg/bw/2 last weeks, n=5); and Carba (400 mg Carba/kg/bw/3weeks, n=5); - B) AQP2 expression in IMCD from the four groups, by Western Blot. Results: 1) In vitro study A) in microperfusion, Carba added to the bath in Vasopressin (Vp) absence (n=6) increased Pf Control-12.3+3.6, Carba-62.6+14.8 (p<0.01), Recuperation (Rec)-17.4+5.5 (p<0.01). In order to study the mechanism by which Carba activates the Vp cascade, the antagonist of the Vp receptor 2 (AV2; n=10) was used: Control-23.5+5.2, Carba-37.4±4.4 (p<0.01), Carba+AV2-19.6±5.0 (p<0.05), Rec-21.4+5.5; and Control-18.6+7.0, AV2- 27.3+7.2, AV2+Carba-25.3+5.7, Rec-32.6+6.4. The PKA inhibitor (H8; n=4) was also used: Control-15.0+9.0, Carba-106.1+12.3 (p<0,01), Carba+H8-60.3+16.4 (p<0.01), Rec-44.5+13.2. B) the densitometric analysis showed an increased of 38.8% in AQP2 expression (Control- 100.0+8.3 vs. Carba-138.8+12.12, p<0.05); B) In vivo study Urinary volume (UV, mL/24h) Control-10.7+3.0, Li-62.6+6.0 (p<0.001), Li+Carba-28.5+4.9 (p<0.001), Carba-23.3+3.0 (p<0.001). Urinary Osmolality (mOsm/Kg/H2O) Control-819.6+175.7, Li-149.4+18.0 (p<0.01), Li+Carba-251.5+39.7 (p<0.05), Carba-396.2+75.5 (p<0.01). FENa+ (%) - Control- 0.15+0.01, Li-0.10+0.03, Li+Carba-0.12+0.02, Carba-0.11+0.02. AQP2 expression (%) Control-100.0+6.7, Li-55.8+5,4 (p<0.01), Li+Carba-75.8+9.6 (p<0.01), Carba-99.7+4.7 (p<0.05). There were no significant differences in Na+, K+ and plasma osmolality. In summary, our data showed that Carba decreased the UV, increased the UOsm and the AQP2 expression in Li-induced DI, increased the water permeability, probably acting directly in the Vp V2 receptor-Protein G complex, since its action was blocked by the specific Vp V2 receptor antagonist. These results emphazise that the hyponatremia found in patients using Carba could be explained, at least in part, by increased osmotic permeability of IMCD. In addition, poliuria observed in lithium-induced DI can be decreased with Carba-treatment.

L'étude immunocytochimique du développement du greffon a mis en évidence de la vasopressine, de l'ocytocine des neurophysines I et II associées. Examen des paramètres de la fonction rénale. La technique de greffe serait susceptible de recréer une restauration fonctionnelle au niveau du système nerveux diencéphalique

Le contrôle de la transcription constitue le principal niveau de la régulation de l'expression des gènes dans les cellules eucaryotes. Le laboratoire a identifié 6 familles indépendantes avec un diabète insipide néphrogénique (DIN) lié à l'X portant de grandes délétions en amont du gène de l'AVPR2. Dans chacune de ces familles, les gènes AVPR2 et AQP2 sont intacts et les hommes sont atteints de DIN lié à l'X dans sa forme rénale « classique ». Le séquençage et l'analyse de 61 kilobases en amont et en aval de l'AVPR2 ont permis l'identification de 6 zones délétées chez 6 familles indépendantes, dont 5 zones de taille supérieure à 7 kilo bases, et une zone, de 102 paires de bases, commune à l'ensemble des délétions. Chez le patient porteur de cette délétion, les récepteurs V2 ne sont pas exprimés dans le tubule collecteur mais le sont au niveau des cellules endothéliales. Notre travail est de tenter de comprendre les mécanismes régulateurs du locus de l'AVPR2, et d'étudier l'expression « tissu spécifique » de ce gène. Les études réalisées dans le système Hprt, confirment le rôle activateur de la séquence de 102 pb. Les expériences in vitro indiquent que cet effet dépende du contexte extracellulaire, de la nature des cellules, ainsi que du promoteur de l'AVPR2. Les protéines liant potentiellement l'une des extrémités de la délétion a révélé la présence, soit de protéines régulatrices, soit de séquences inconnues, toutes exprimées dans le rein. À terme, ces études, ainsi que celles en découlant, permettront de positionner l'AVPR2 comme une cible de choix dans le traitement des diabètes insipides, centraux et néphrogéniques, par thérapie génique.

Le diagnostic de syndrome d'interruption de la tige pituitaire n'est possible que depuis l'avènement de l'IRM. Il se caractérise par une tige pituitaire interrompue ou hypoplasique, une antéhypophyse souvant de petite taille, et la formation d'une posthypophyse ectopique, qui semble remplacer la neurohypophyse absente. Ce syndrome et ses variantes entraînent le plus souvent un déficit multiple hypophysaire notamment thyréotrope et parfois un diabète insipide (DI). L'azxe somatotrope est le premier atteint. Ce syndrome est plus fréquent chez les garçons. L'hypothèse étiologique congénitale de révélation clinique précoce, associée dans 1/4 des cas avec des malformations de la ligne médiane, semble la plus fréquente. La présentation du siège accompagne souvant l'interruption de la tige pituitaire congénitale. Plus rarement sont évoquées les hypothèses post-traumatiques périnatales ou post-natales (traumatisme cranien), plus rare encore sont les hypothèses toxique ou familiale. Les deux enfants présentés sont complémentaires dans leur présentation, puisqu'ils sont représentatifs de la diversité des hypothèses étiologiques principales, de la diversité des images IRM dans ce syndrome et de la diversité des manifestations cliniques. Cécle 8 ans 1/2, est un cas type IRM d'interruption de la tige pituitaire d'origine congénitale. Elle présente un diabète insipide et un déficit somatotrope ayant entraîné un retrd de croissance jusqu'à -4 DS qu'elle n'a pas encore rattrapé, malgré un traitement par HGH. Johann 10 ans 1/2, présente une section probablement post traumatique de la tige pituitaire (AVP), sans neurohypophyse, avec un nodule isointence à la base de l'infundibumum. Il existe un déficit multiple hypophysaire avec diabète insipide. Le retard de croissance a atteint -2 DS pour se corriger actuellemnt grâce à une polysubstitution hormonale.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2019
A Diabetes Insipidus (DI) central é uma doença rara com múltiplas etiologias possíveis. Descreve-se neste trabalho final de mestrado um caso clínico complexo, seguindo a marcha diagnóstica pediátrica da avaliação da poliúria e polidipsia numa criança de 5 anos, com abordagem do diagnóstico diferencial, benefícios e riscos da terapêutica e procedimentos diagnósticos, englobando também a vertente social e ética. Este caso demonstra como é essencial saber gerir a complexidade clínica presente em pediatria, que passa não só pelos conhecimentos teóricos necessários para o reconhecimento precoce da sintomatologia da DI e para a instituição de uma terapêutica adequada, mas também pelo desafio de lidar com os cuidadores.
Central diabetes insipidus (CDI) is a rare disease with multiple underlying causes. We describe a complex clinical case, with the evaluation of polyuria and polydipsia in a 5 year-old child, approaching of differential diagnosis, benefits and risks of treatments and diagnostic procedures, as well as a social and ethic discussion. This clinical case showcases the clinical complexity in pediatrics, regarding the aknowlegment of CDI symptoms and treatment, but also the challenge of dealing with the parents.

碩士
國立陽明大學
遺傳學研究所
87
Nephrogenic Diabetes Insipidus (NDI) is a rare X-linked recessive renal disorder (MIM No. 304800) due to resistance to antidiuretic action of arginine vasopression (AVP) hormone. NDI is characterized with clinical symptoms of polyuria, polydipsia and dehydration. Previous studies have demonstrated that the NDI is caused by the defects of vasopressin 2 receptor (V2R) in renal tubules. The V2R belongs to the family of G-protein coupled receptors that contain seven distinct transmembrane domains. The gene encoding V2R has been cloned and sequenced, which facilitates the genetic study of NDI at molecular level. Several gene defects of NDI have been identified, including various point mutations, insertion and deletions, suggesting genetic heterogeneity of NDI. We have identified two families with clinical diagnosis of NDI. By using the polymerase chain reaction (PCR)-based sequencing, we investigated three protein-coding exons and their flanking introns of V2R gene of these families. A C-to-T transition at nucleotide 316, a C-to-A transversion at nucleotide 861 and an A-to-G polymorphism site at nucleotide 927 were identified. These variants predict an Arg substitution at codon 106 by Cys, a Phe substitution at codon 287 by Leu and an unchanged Leu at codon 309 of V2R, respectively. The frequences of G and A alleles in normal Chinese population at the nucleotide 927 polymorphism site were found to be 0.71 and 0.29, respectively, which is similar to that of Japanese but different from that of Scandinavian. Although the molecular variant at codon 106 and the polymorphism site at codon 309 have been reported. The variant at codon 287 has not been reported previously, and was not found in 100 normal Chinese alleles, suggesting that this c.861 C>A molecular variant might be a novel mutation of V2R causing NDI. Expression of these variant forms of V2R gene in COS-7 cell also showed that these two molecular mutations at nucleotide 316 and 861 had decreased their ability to accumulate cAMP in response to AVP [10-6M] stimulation significantly.

A Diabetes Insipidus (DI) é uma doença metabólica, pouco frequente na clínica de animais de companhia, e resulta na alteração do balanço hídrico do organismo. A DI ocorre devido a uma alteração na síntese ou secreção da hormona antidiurética (ADH), neste caso designada de diabetes insipidus central, ou devido a uma insensibilidade renal à ação da mesma hormona, designada de diabetes insipidus nefrogénica. Estas alterações resultam na falha da reabsorção tubular de água e na produção de grandes volumes de urina diluída, ou poliúria. De modo a evitar a desidratação, o animal desenvolve polidipsia compensatória. Os sinais clínicos mais frequentes de DI são poliúria/polidipsia e densidade urinária baixa. A ADH, sintetizada no hipotálamo e posteriormente armazenada na neurohipófise, é libertada com base na osmolalidade plasmática. Quando a osmolalidade plasmática aumenta, a ADH é libertada para a corrente sanguínea e no rim, principalmente nas células dos ductos coletores, liga-se ao seu recetor e promove a reabsorção de água do filtrado tubular através da inserção de aquaporinas. O diagnóstico da DI é desafiante e baseia-se na exclusão de doenças mais frequentes que apresentem poliúria/polidipsia. Para isso, é necessário realizar-se anamnese, exame físico, hemograma completo, painel bioquímico, análise de urina e exames de imagiologia. O teste de privação de água modificado e a resposta à administração de desmopressina constituem os procedimentos de confirmação diagnóstica de DI, sendo que a última permite diferenciar DI central e DI nefrogénica. O tratamento é efetuado com desmopressina, diuréticos tiazídicos, cloropropamida e dieta apropriada.
Diabetes Insipidus (DI) is a metabolic disorder, uncommon in pet animals, which results from a disruption in the fluid balance of the organism. DI occurs due to a modification in the production or secretion of the antidiuretic hormone (ADH), designated as Central Diabetes Insipidus or due to a renal insensitivity to that hormone’s action, designated as Nephrogenic Diabetes Insipidus. These changes result in failure of the tubular reabsorption of water and the production of large volumes of diluted urine, or polyuria. In order to avoid dehydration, the animal develops compensatory polydipsia. The most common signs of DI are polyuria/polydipsia and low urine specific gravity. The ADH, synthetized in the hypothalamus and consequently stored in the neurohypophysis, is released based on plasma osmolality. When there is an increase in plasma osmolality, ADH is released into the bloodstream and, in the kidney, primarily in the collecting duct cells, connects to its receptor and promotes water reabsorption of the tubular filtrate through aquaporin insertion. Diagnosing DI is challenging and is accomplished by exclusion of more frequent conditions which include polyuria/polydipsia. As such, anamnesis, physical examination, complete blood count, biochemical profile, urinalysis and diagnostic imaging must be performed. The Modified water deprivation test and the response to the administration of desmopressin comprise the procedures for diagnostic confirmation of DI, where the latter allows to differentiate between central DI and nephrogenic DI. Treatment is carried out with desmopressin, thiazide diuretics, chlorpropamide and appropriate dieting.

Diabetes insipidus (DI) is associated with defects that involve the secretion and the action of hormone arginine vasopressin (AVP) resulting in the excretion of abnormally large volumes of diluted urine. The most common defect that results in disease development is the deficient secretion of the hormone AVP and the disease is referred to as central or neurohypophyseal DI. The AVP hormone is synthesized in magnocellular neurons, that originate in the supraoptic and paraventricular nuclei of the hypothalamus and are projected to neurohypophysis, and the destruction of these neurons leads to a deficiency of AVP hormone, resulting in neurohypophyseal DI. The familial form of disease represents 1% of all causes of neurohypophyseal DI and the main points of the disease are: it is associated with mutations in one allele of the AVP gene, and it is caused by postnatal development of deficient AVP secretion, proposed to result from selective degeneration of the magnocellular neurons. The aims of this thesis are: to review AVP mutations described in the scientific literature, to expand the spectrum of mutations through the analysis of additional patients with DI and to characterize the functional consequences of identified novel AVP mutations. To achieve these aims a bibliographic research was developed; genetic studies were performed to amplify and to sequence the three exons of the AVP gene in 9 patients; an expression vector containing the desired mutations was constructed by subcloning, site-directed mutagenesis and enzymatic digestion; and the functional studies were initialized by optimization of transfection and immunocytochemistry assays for WT AVP cDNA expression vector. Three mutations were identified: c.154T>C, c.289C>G and c.343G>T. The first two mutations are novel and the last mutation is already described in the scientific literature. The AVP cDNA from the expression vector was subcloned in the pVAX/lacZ vector and the mutations were inserted in the AVP cDNA by site-directed mutagenesis and enzymatic digestion. The mutated AVP cDNAs were sequenced and have been prepared to be inserted in the expression vector. The transfection and immunocytochemistry protocols have been optimized for WT AVP cDNA expression vector. This study allowed the increase in the number of mutations from 70 to 72 different mutations, although further work is necessary in order to understand the molecular mechanisms responsible for the development of the disease and to give help and information to patients affected with this disease.
A diabetes insípida (DI) é uma doença rara, caracterizada principalmente pela excreção de elevados volumes de urina na forma diluída podendo, entre várias causas possíveis, ter origem num defeito genético. O desenvolvimento da doença pode dever-se a quatro causas possíveis. A mais comum deve-se a uma deficiência na secreção da hormona antidiurética arginina vasopressina (AVP), sendo referida como DI central ou neurohipofisária. Outra possível causa da doença deve-se a uma insensibilidade, por parte das células renais, aos efeitos da AVP, sendo neste caso designada como DI nefrogénica. A DI pode também dever-se a uma excessiva ingestão de líquidos, que conduz à supressão da libertação da hormona AVP, sendo referida como polidipsia primária. Por fim, um aumento do metabolismo da hormona AVP durante a gravidez pode também ser uma causa da doença, designada por DI gestacional. A hormona AVP é sintetizada nos neurónios magnocelulares. Estes têm origem no núcleo supra-óptico e para-ventricular do hipotálamo e os seus prolongamentos terminam na neurohipófise. A destruição destes neurónios resulta numa deficiência na produção da hormona, conduzindo à DI central. Esta destruição pode ter inúmeras causas, incluindo acidentes, cirurgias, doenças autoimunes, entre outras. Contudo, a doença também apresenta uma base familiar, correspondendo a 1% de todas as causas de DI central. A DI central apresenta sintomas persistentes de poliúria, polidipsia e sede, que geralmente se começam a manifestar vários meses ou anos após o nascimento. A DI central familiar apresenta duas características principais: está associada a mutações num único alelo do gene que codifica a hormona (gene AVP), apresentando assim uma transmissão autossómica dominante; e é causada por uma deficiência progressiva pós-natal na secreção da hormona AVP, que se pensa resultar da degeneração seletiva dos neurónios magnocelulares. O gene AVP é composto por 3 mil pares de bases e encontra-se localizado no braço curto do cromossoma 20. Este gene contém três exões que codificam para o péptido sinalizador, para a hormona AVP, para a neurofisina II (transportador da hormona) e ainda para um glicopéptido, conhecido como copeptina. Após sintetizados, a hormona, a neurofisina II e o glicopéptido são armazenados em vesiculas secretoras, nos terminais axonais dos neurónios, e são libertados após a ocorrência de estímulos. Após a entrada na corrente sanguínea, a hormona vai atuar a nível das células renais de modo a aumentar a sua permeabilidade para as moléculas de água, favorecendo assim a absorção de água no rim. Até à data do início deste trabalho, a doença estava associada a 70 mutações diferentes no gene AVP localizadas ao longo de todo o precursor proteico. Pensa-se que estas mutações são a causa da doença uma vez que interferem na estabilidade da cadeia de aminoácidos, alterando a sua estrutura primária. Teoricamente, mutações que afetem a conformação de proteínas secretoras resultam no desenvolvimento de patologias devido ao seu impacto na função da proteína não conseguindo alcançar o seu destino, ficando retidas no reticulo endoplasmático. Contudo, a razão dos precursores AVP mutados serem tóxicos para os neurónios produtores de AVP está ainda por esclarecer. Existem, até ao momento, três teorias que tentam explicar o mecanismo da doença. O mecanismo não tóxico defende que há uma expressão simultânea dos precursores “wild-type” e dos precursores mutados resultando numa associação de ambos. Assim, o precursor “wildtype” é alterado, uma vez que ambos ficam retidos no reticulo endoplasmático. Contudo, este mecanismo não explica a morte dos neurónios magnocelulares. O mecanismo tóxico defende que a constante acumulação de precursores com conformações alteradas pode interferir com a produção de proteínas essenciais à sobrevivência celular, resultando assim na morte neuronal. Recentemente, um novo mecanismo foi proposto para explicar a patogénese da doença. Observou-se a formação de vesiculas autofágicas, após acumulação de precursores mutados, que resultam na destruição dos retículos endoplasmáticos danificados, juntamente com os agregados proteicos. Durante este processo, se as células forem expostas a insultos metabólicos e ambientais, pode ocorrer apoptose dependente de autofagia, resultando na destruição dos neurónios magnocelulares. A DI central familiar apresenta uma natureza benigna, contudo é uma doença que apresenta uma intensa pesquiza em torno dos seus mecanismos moleculares uma vez que se trata de um modelo de interesse para o estudo de doenças neuro-endócrinas e de transmissões autossómica dominante. O presente estudo tem por objetivos fazer uma revisão das mutações descritas na literatura científica para o gene AVP, aumentar o número de mutações descritas com a análise de novos pacientes diagnosticados com DI central familiar e caracterizar as consequências funcionais das novas mutações identificadas. Para alcançar os objetivos descritos, utilizou-se a seguinte metodologia: a revisão de todas as mutações descritas até à data, através de pesquisa bibliográfica de artigos científicos; realização de estudos genéticos, baseados na amplificação por PCR e na posterior sequenciação dos três exões do gene AVP de 9 pacientes diagnosticados com DI central familiar; inserção das novas mutações num vector de expressão contendo o cDNA do gene AVP, através de técnicas de clonagem, digestão enzimática e mutagénese dirigida; e finalmente a realização de estudos funcionais, por otimização das técnicas de transfecção e imunocitoquímica com o vector de expressão AVP “wild-type”. Os resultados obtidos mostraram que as 3 famílias apresentam mutações no gene AVP. O paciente III-1, da família A, apresenta a alteração de uma timina para uma citosina na posição 154 do cDNA (c.154T>C) que origina a substituição de uma cisteína por arginina na posição 52 da proteína (p.C52R). O paciente II-1, da família B, apresenta uma alteração de citosina para guanina na posição 289 do cDNA (c.289C>G) que resulta na substituição de uma arginina por glicina, na posição 97 da proteína. O paciente II-4 da família C apresenta a alteração de uma guanina para uma timina na posição 343 do cDNA (c.343G>T) que resulta na substituição de um ácido glutâmico por um codão de terminação na posição 115 da proteína. As três mutações estão em heterozigotia e as duas mutações encontradas no exão 2 correspondem a mutações novas, enquanto a mutação presente no exão 3 já se encontra descrita na literatura. Um vector de expressão contendo o cDNA do gene AVP (pRc/RSV-AVP), foi-nos gentilmente oferecido por investigadores da área. O cDNA do gene AVP contido no vector de expressão (pRc/RSV-AVP) foi sub-clonado no vector pVAX/lacZ e, através de mutagénese dirigida, as mutações desejadas (c.154T>C e c.289C>G) foram introduzidas no cDNA. Assim, o cDNA com as mutações está pronto a ser inserido no plasmídeo de expressão. Os ensaios de transfecção e imunocitoquímica foram otimizados para o vector de expressão “wild-type”, uma vez que foi observada marcação para a neurofisina II nos prolongamentos dos neurónios após transfecção de uma linha celular neuronal (N2A) e marcação com anticorpos específicos. Com este estudo, o número de mutações descritas para o gene AVP aumentou de 70 para 72 e mais três famílias fazem parte do número total de famílias estudadas com DI central familiar. É importante continuar o desenvolvimento de estudos funcionais, de modo a obter respostas sobre os mecanismos moleculares responsáveis pelo desenvolvimento da doença uma vez que estas serão importantes não só para a DI central familiar, mas também para o esclarecimento de outras doenças que apresentem mecanismos moleculares semelhantes.

碩士
國立陽明大學
生物化學研究所
93
To study kidney-specific gene targeting, it is necessary to generate a mouse strain that expresses Cre recombinase under the control of a tissue-specific gene promoter. To direct the expression of Cre, we used the promoter of the kidney-specific cadherin gene (Ksp-cadherin, cadherin 16). This gene is uniquely expressed in the basolateral membrane of tubular epithelial cells in the kidney. Transgenic mice carrying the Ksp promoter linked to the Cre recombinase gene were produced by pronuclear microinjection. RT-PCR and immunoblot analysis showed that Ksp-Cre mice expressed Cre only in the kidney and not in any other tissues examined. We observed the mice to have polyuria and polydipsia that are the primary symptoms of diabetes insipidus (DI). This disorder is characterized by a urinary concentrating defect resulting from resistance of the collecting duct to the antidiuretic action of vasopressin (AVP). In Ksp-Cre transgenic mice, water intake increased about two-fold and urine volume increased about eight-fold compared with wild-type mice. An analysis of urine and serum information in the Ksp-Cre transgenic mice showed that urine osmolality decreased to approximately 500 mOsm/l. In contrast, the osmolality of urine in wild-type mice was about 2000 mOsm/l. The urine gravity was lower in the Ksp-Cre transgenic mice than in the wild-type mice. Furthermore, BUN , creatinine, calcium, potassium, and magnesium were a little higher in Ksp-Cre transgenic mice than in wild-type mice. Moreover, histology showed that the epithelial cells of the collecting ducts had degenerated. Taken together, these results demonstrate that Ksp-Cre transgenic mice had problems in their collecting ducts. Therefore, Ksp-Cre transgenic mice may be yet another mouse model for nephrogenic diabetes insipidus.

Trabalho final do 6º ano médico com vista à atribuição do grau de mestre (área científica de neurocirurgia) no âmbito do ciclo de estudos de Mestrado Integrado em Medicina .
A diabetes insipidus (DI) é uma das complicações mais frequentes após manipulação cirúrgica da sela turca, nomeadamente devido a adenomas da hipófise. Este estudo retrospetivo a 5 anos avaliou a incidência desta complicação após cirurgia de adenomas da hipófise, no Centro Hospitalar Universitário de Coimbra (CHUC), e procurou a sua associação com sexo, idade, tamanho do adenoma, recidiva, apresentação clínica e complicações cirúrgicas. O trabalho foi feito com base na consulta de processos clínicos, tendo sido selecionados apenas doentes sem DI prévia e abordados por via transesfenoidal. A incidência de DI de curta duração foi de 13,3%, e a de DI definitiva de 3,1%. Idade mais jovem e apresentação com síndrome de Cushing foram associados a maior incidência desta patologia. Apesar de sem significância estatística, verificou-se ainda uma tendência para maior incidência quando houve fístula intraoperatória de liquido cefalorraquidiano (LCR), e menor incidência nos doentes com acromegalia/ gigantismo. A quantidade de doentes que recebeu terapêutica com desmopressina (DDAVP) foi superior à incidência de DI, o que aponta para uma necessidade de critérios mais estreitos para aplicação desta terapêutica de substituição. DI is one of the most common complications after surgical manipulation on the sella turcica, including pituitary adenomas. This 5-year retrospective study evaluates the incidence of this complication after transsphenoidal surgical removal of pituitary adenomas, as well as its’ association with gender, age, size, re-operation, clinical presentation and surgical complications. The study was based on the consult of clinical processes from the CHUC. Patients with previous DI or transcranial approach have been excluded. The incidence of transient DI was 13,3%, whereas definitive DI was established at 3,1%. Both younger and patients presenting with Cushing syndrome showed to have a higher incidence of this pathology. Although below the level of statistical significance, patients with an intraoperative LCR fistula showed a tendency to higher rates of DI. On the other hand, patients with acromegaly/ gigantism seem to have lower rates of this complication. The amount of patients receiving DDAVP therapy was higher than the incidence of DI, which probably reveals the need for stricter criteria for the application of this replacement therapy.

Le diabète insipide néphrogénique (DIN) autosomal peut être causé par les mutations du gène codant pour le canal à eau aquaporine-2 (AQP2). Un modèle couramment utilisé pour l’étude des protéines membranaires telle l’AQP2 est l’expression hétérologue dans les ovocytes de Xenopus laevis. Malheureusement, les techniques déjà existantes de purification de membranes plasmiques sont soit trop longues, trop difficiles ou demandent trop de matériel, ne permettent pas l’analyse adéquate du ciblage des formes sauvage comme mutantes, un élément crucial de ce type d’étude. Nous avons donc dans un premier temps mis au point une technique rapide et efficace de purification de membranes plasmiques qui combine la digestion partielle de la membrane vitelline, sa polymérisation à la membrane plasmique suivi de centrifugations à basse vitesse pour récolter les membranes purifiées. Nous avons utilisé cette technique dans l’étude de deux nouveaux cas familiaux de patients hétérozygotes possédant les mutations V24A et R187C dans un cas et K228E et R187C dans le second cas. Pour chaque mutation, nous avons analysé autant les éléments de fonctionnalité que les paramètres d’expression des protéines mutantes. Les expériences de perméabilité membranaire démontrent que les ovocytes exprimant AQP2-V24A (Pf = 16.3 ± 3.5 x 10-4 cm/s, 10 ng) et AQP2- K228E (Pf = 19.9 ± 7.0 x 10-4 cm/s, 10 ng) ont des activités similaires à celle exprimant la forme native (Pf = 14.4 ± 5.5 x 10-4 cm/s, 1 ng), tandis que AQP2- R187C (Pf = 2.6 ± 0.6 x 10-4 cm/s, 10 ng) ne semble avoir aucune activité comme ce qui est observé chez les ovocytes non-injectés (Pf = 2.8 ± 1.0 x 10-4 cm/s). Les études de co-expression ont démontré un effet d’additivité lorsque AQP2-V24A et -K228E sont injectées avec la forme native et un effet s’apparentant à la dominance négative lorsque AQP2-R187C est injecté avec la forme native, avec AQP2-V24A ou avec –K228E. Les résultats obtenus par immunobuvardage représente bien ce qui a été démontré précédemment, on remarque la présence des mutations K228E, V24A et la forme sauvage à la membrane plasmique, contrairement à la mutation R187C. Cependant, lorsque les mutations sont exprimées dans des cellules mIMCD-3, il n’y a qu’une faible expression à la membrane de la forme –K228E et une absence totale des formes –V24A et –R187C à la membrane plasmique, contrairement à la forme native. Les résultats de nos études démontrent que tout dépendant du système d’expression les formes –K228E et –V24A peuvent être utiles dans l’étude des problèmes d’adressage à la membrane à l’aide de chaperonne chimique. De plus, la forme –R187C démontre des difficultés d’adressage qui devront être étudiées afin de mieux comprendre la synthèse des formes natives.
The autosomal nephrogenic diabetes insipidus (NDI) is caused by mutations of the gene coding for the water channel aquaporine-2 (AQP2). An oftenly used model for the study of membrane proteins such as AQP2 is the heterogenous expression in Xenopus laevis oocytes. Unfortunately, the existing techniques of plasma membranes purification are either too long, too difficult or require too much material, which does not allow adequate analysis of targeting of the native and mutants forms, which is crucial for this type of study. We developed a fast and effective plasma membrane purification technique which combines partial digestion of the vitellin membrane, its polymerization with the plasma membrane followed by a serie of low speed centrifugations to collect the purified membranes. We used this technique to study of two new family cases of heterozygote patients carrying the V24A and R187C mutations in a case and K228E and R187C in the second case. For each mutation, we analyzed the functionality and the parameters of expression of the mutant proteins. The membrane permeability experiments show that the oocytes expressing AQP2- V24A (Pf = 16.3 ± 3.5 x 10-4 cm/s, 10 ng) and AQP2-K228E (Pf = 19.9 ± 7.0 x 10-4 cm/s, 10 ng) have similar activities to the oocytes expressing the native form (Pf = 14.4 ± 5.5 x 10-4 cm/s, 1 ng), while AQP2-R187C (Pf = 2.6 ± 0.6 x 10-4 cm/s, 10 ng) doesn’t seem to have any activity like the un-injected oocytes (Pf = 2.8 ± 1.0 x 10-4 cm/s). The coexpression studies showed an additive effect when AQP2-V24A and -K228E are injected with the native form and an effect being associated with negative dominance when AQP2-R187C was injected with AQP2-V24A, -K228E and the native form. Western blot results confirmed what was observed in the functionality studies. However, when the mutations were expressed in mIMCD-3 cells, there was a slight expression of the K228E mutation to the plasma membrane and a total absence of the mutations –V24A and R187C at the plasma membrane. The results of our studies showed that depending on the expression system the mutations –K228E and -V24A can be used in targeting studies using chemical chaperones.

L’aquaporine-2 (AQP2) est le canal responsable de la réabsorption finale d’eau au niveau du tubule collecteur du rein. À la base, contenue dans des vésicules internes, l’AQP2 est acheminée à la membrane apicale des cellules principales du tubule collecteur suite à une stimulation par l’hormone antidiurétique (ADH). L’incapacité à accomplir cette fonction entraîne le diabète insipide néphrogénique (DIN), une maladie caractérisée par l’inhabileté du rein à concentrer l’urine, entraînant une production de volumes urinaires élevés. Alors que les mutations récessives génèrent des protéines mal structurées et incapables de former des tétramères, les mutations dominantes sont capables de s’associer à leurs homologues sauvages, engendrant ainsi un DIN même chez les patients hétérozygotes. Ce mémoire présente l’analyse biochimique et fonctionnelle d’une nouvelle mutation naturelle de l’AQP2, la mutation T179N, aussi responsable du DIN. Cette dernière est particulièrement intéressante de par son génotype qui implique un caractère dominant, et sa position extracellulaire habituellement réservée aux mutations récessives. Les études comparatives de T179N à deux modèles de mutation récessive et dominante démontrent, tant en ovocytes de Xenopus laevis qu’en lignée cellulaire mpkCCDc14, le caractère récessif de cette nouvelle mutation. Les tests d’immunobuvardage de lysats d’ovocytes en membranes totales et membranes plasmiques purifiées ont révélé que seule la forme sauvage atteint la membrane plasmique alors que le mutant T179N est séquestré dans la cellule. En accord avec ce résultat, les analyses de perméabilité fonctionnelle démontrent aussi une absence d’activité pour T179N. En cellule mpkCCDc14, le mutant T179N exprimé seul n’atteint pas la membrane plasmique suite à l’action de la forskoline, contrairement à la forme sauvage. Cependant, ce mutant peut s’associer à son homologue sauvage en coexpression tant dans les ovocytes qu’en lignée mpkCCDc14 sans toutefois engendrer l’effet typique de dominance négative. En fait, dans ce contexte de coexpression, on remarque une augmentation de la Pf de 83±7 % et une récupération d’adressage à la membrane plasmique en cellule (immunofluorescence). En conclusion, T179N serait un mutant récessif fonctionnellement récupérable lorsqu’en présence de l’AQP2 sauvage.
Aquaporin-2 (AQP2) is the channel responsible for the final reabsorption of water in the collecting duct of the kidney. Basically, contained in internal vesicles, the AQP2 is delivered to the apical membrane of the principal cells of the collecting tubule after stimulation by the antidiuretic hormone (ADH). The failure to perform this function causes nephrogenic diabetes insipidus (NDI); a disease characterized by the inability of the kidney to concentrate urine and induces the production of high urinary volumes. While recessive mutations generate poorly structured proteins unable to form tetramers, dominant mutations are capable of associating with their wild counterparts, thus generating a NDI even in heterozygous patients. This paper presents the biochemical and functional analysis of T179N, a new NDI-causing mutation of the human AQP2. The mutant is particularly interesting because of its dominant genotype, despite its extracellular position usually restricted to recessive mutations. Here, we compare T179N against archetypal recessive and dominant mutations using both Xenopus laevis oocytes and in mpkCCDc14 cell model, and show the recessive nature of the mutation. The immunoblot tests on oocytes lysates in purified total and plasma membranes revealed that only the wild type protein reaches the plasma membrane while the T179N mutant is sequestered within cellular stores. Accordingly, functional analyzes indicate that T179N is inactive. In mpkCCDc14 cells, T179N expressed alone does not reach the plasma membrane in response to forskolin stimulation, unlike the wild-type. However, T179N does show the capacity to associate with its wild-type counterpart in both oocytes and in mpkCCDc14 cells, although without displaying the typical dominant negative effect. In fact, when coexpressed along wild-type, T179N gains back the functionality, with a Pf increase of 83±7 % and adequate plasma membrane targeting in cells (immunofluorescence). In conclusion, the mutant T179N is a mild recessive mutation that is susceptible to functional recovery when in presence of wild type AQP2.

Le récepteur de la vasopressine de type 2 (V2R) joue un rôle crucial dans l’homéostasie hydrique. Exprimé principalement au niveau du rein, son activation par l’hormone antidiurétique arginine-vasopressine (AVP) favorise la réabsorption d’eau, participant ainsi à diminuer la diurèse. Plus de 200 mutations dans le gène du V2R ont été associées au diabète néphrogénique insipide congénital (DINc), une maladie causée par une perte de fonction du récepteur. À l’opposé, trois mutations découvertes récemment induisent un gain de fonction du V2R, et sont la cause du syndrome néphrogénique de l’anti-diurèse inappropriée (NSIAD). Les travaux de cette thèse visent à mieux comprendre les bases moléculaires responsables de la perte ou du gain de fonction des récepteurs mutants associés à ces deux maladies. Dans plus de 50% des cas, les mutations faux-sens affectent négativement l’adoption d’une conformation native par le V2R, provoquant la reconnaissance et la rétention intracellulaire des mutants par le système de contrôle de qualité du réticulum endoplasmique. Nos résultats ont démontré que l’interaction entre les récepteurs mutants et le chaperon moléculaire calnexine est dépendante de N-glycosylation et que sa durée varie en fonction de la mutation. De plus, l’importance de cette modification co-traductionnelle et des interactions lectines-sucres dans le processus de maturation d’un mutant donné s’est avérée une caractéristique intrinsèque, puisque l’absence de N-glycosylation n’a pas affecté le mutant Y128S (phénotype léger) tandis que la maturation du mutant W164S (phénotype sévère) a été totalement abolie. Nos résultats suggèrent aussi que l’action des chaperons pharmacologiques (CP), molécules favorisant la maturation des mutants du V2R, peut survenir à différentes étapes au cours du processus de maturation, selon le mutant réchappé. Ces différences entre muta nts suggèrent des processus biosynthétiques ‘personnalisés’ dictés par la nature de la mutation impliquée et pourraient expliquer la différence de sévérité des manifestations cliniques chez les patients porteurs de ces mutations. Bien qu’une récupération de fonction ait été obtenue pour les mutants Y128S et W164S par un traitement au CP, il n’en est pas de même pour toutes les mutations occasionnant un défaut conformationnel. C’est ce que nous avons démontré pour le mutant V88M, affligé de deux défauts, soit une faible efficacité de maturation combinée à une basse affinité pour l’AVP. Dans ce cas, et malgré une augmentation du nombre de récepteurs mutants la surface cellulaire, la diminution de l’affinité apparente du récepteur mutant pour l’AVP a été exacerbée par la présence résiduelle de CP à son site de liaison, rendant impossible l’activation du récepteur aux concentrations physiologiques d’AVP. Les mutants R137C et R137L ont une activité constitutive élevée et mènent au NSIAD tandis que la substitution de cette même arginine par une histidine (R137H) mène au DINc. Ces trois mutants se sont avéré partager plusieurs caractéristiques, dont une efficacité de maturation réduite et une désensibilisation spontanée élevée. La seule différence iden tifiée entre ces mutants est leur niveau d’activité constitutive. Le CP utilisé dans nos études possède aussi la propriété d’agoniste inverse, mais n’a pourtant pas diminué l’activité constitutive des mutants R137C/L, suggérant une conformation active ‘figée’. Seul l’effet chaperon a été observé, entraînant la hausse de récepteurs à la surface cellulaire, qui se traduit par une augmentation de la production de second messager. Nous avons par contre suggéré l’utilisation d’AVP puisqu’il favorise l’endocytose des récepteurs R137/L sans promouvoir leur activation, diminuant ainsi le nombre de récepteurs actifs à la surface cellulaire. Nous avons identifié la première mutation occasionnant un gain de fonction du V2R qui n’implique pas l’arginine 137. Le mutant F229V a une activité constitutive élevée et, contrairement aux R137C et R137L, il n’est pas sujet à une désensibilisation spontanée accrue. L’observation que des agonistes inverses sont aptes à inhiber l’activité constitutive de ce nouveau mutant est une découverte importante puisque l’insuccès obtenu avec les mutations précédentes suggérait que ces molécules n’étaient pas utiles pour le traitement du NSIAD. Considérés globalement, ces travaux illustrent le caractère particulier des formes mutantes du V2R et l’importance de bien cerner les conséquences fonctionnelles des mutations afin d’apporter aux patients atteints de DINc ou NSIAD une thérapie personnalisée, et de développer de nouveaux agents thérapeutiques adaptés aux besoins.
The vasopressin type 2 receptor (V2R) plays an important role in water homeostasis. Mainly expressed in the collecting ducts of the kidney, V2R activation by the antidiuretic hormone arginine-vasopressin (AVP) leads to water reabsorption, resulting in a decrease urine output. More than 200 mutations in the V2R gene have been link to the aetiology of the congenital form of nephrogenic diabetes insipidus (cNDI), resulting from a receptor loss-of-function. In contrast, three recently identified mutations have been shown to cause a gain-of-function of the V2R leading to the nephrogenic syndrome of inappropriate antidiuresis (NSIAD). The work presented herein is focussed on a better understanding of the molecular determinants leading to the loss- or gain -of-function of V2R mutants. More than 50% of missense mutations affecting the V2R were shown to hamper the receptor’s ability to adopt its native conformation and to cause its intracellular retention by the endoplasmic reticulum quality control system. We thus looked at the role of N-glycosylation and calnexin (Cnx) in the maturation process of mutant V2R, and their importance for receptor rescue by pharmacological chaperones (PC). Our results have shown that N-glycosylation is required for Cnx binding to the receptors and that the duration of this interaction is correlated to the severity of the misfolded state of the mutant. The importance of N-glycosylation and to sugar-mediated interactions in the maturation process of a given V2R mutant was found to be an intrinsic property, as it had no significant repercussion on the mild phenotype-associated Y128S mutant, while it completely abolished maturation of the W164S mutant, associated with a severe phenotype. Moreover, we have shown that pharmacological chaperoning can occur at different steps during the maturation process, according to the mutant studied. These mutant-specific differences indicate that the biosynthetic processing of mutant V2R is highly influenced by the nature of the mutation itself and could partially explain the variations in the clinical outcome severity among NDI-causing mutant V2Rs. Although a functionality rescue of W164S and Y128S mutants was obtained upon exposure to PC, it is not the case for all V2R mutants with a maturation defect. The V88M-V2R was found affected both in its maturation and its affinity toward AVP. In this case, and despite a significant increase in maturation and cell surface expression, the PC treatment led to a further loss in the receptor’s affinity for AVP, preventing its activation at physiological AVP concentrations. The R137C and R137L mutants are endowed with a high constitutive activity leading to NSIAD. Stunningly, substitution of this arginine by histidine (R137H) was associated with cNDI. These three mutant V2R were found to share many characteristics, of which a compromised maturation and elevated spontaneous desensitization. The only difference between these mutants relies on their constitutive activity levels. The PC used in our studies is also an inverse agonist, but failed to reduce the constitutive activity of the R137C/L mutants, entailing a ‘locked’ active conformation. Instead, the chaperoning property of the compound led to an increase in the number of constitutively active receptor at the cell surface. We have thus proposed the use of AVP as a treatment, as it was shown to cause receptor’s endocytosis without promoting their activation, leading to a reduced active receptor number at the cell surface. We have identified a new gain-of-function mutation affecting the V2R, the first not involving arginine 137. The F229V substitution was shown to confer high constitutive activity to the receptor, but unlike the two other NSIAD-causing mutants, it does not undergo elevated spontaneous desensitization. The observation that inverse agonists are efficient at inhibiting the constitutive activity of the F229V mutant is an important discovery since the unfruitful attempts obtained with the other constitutively active mutants led some investigators to the erroneous conclusion that inverse agonists were not useful for the treatment of NSIAD. Taken together, these findings underline the ‘individuality’ of V2R mutants and the importance of their functional characterization in order to bring personalized therapeutic strategies for patients with cNDI or NSIAD, and to develop new therapeutics adapted to the patients’ needs.