Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2007. / Title from first page of PDF file (viewed January 8, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 75-84).

La cystinose est une maladie lysosomale héréditaire due à une mutation du gène CTNS codant pour un transporteur de cystine, cystinosin, et est caractérisée par l’accumulation de cystine dans les organes causant leur dégénération.Si le rein est le premier organe affecté par la maladie, sa pathogénèse n’est pas encore totalement comprise. La recherche de nouveaux partenaires de la cystinosine a révélé une interaction avec un membre de la famille des galactines, galectine-3 (Gal3). L’étude de cette interaction a mis en évidence un nouveau rôle de la cystinosine dans l’inflammation chronique impliquée dans la pathogénèse rénale de la cystinose. Dans les reins du modèle murin de la cystinose (Ctns-/-), l’expression de Gal3 est augmentée et de nombreux infiltrats de cellules inflammatoires sont observés. De plus, l’expression de la cytokine pro-inflammatoire Monocyte Chemoattractant Protein-1 (MCP1) est augmentée dans le sérum des souris Ctns-/-.Au contraire, peu d’infiltrat et un taux normal de MCP1 sont observés dans les souris Ctns-/-Gal3-/-, ainsi qu’une meilleure structure et fonction rénale.Ce travail pourrait permettre la découverte de nouvelles cibles thérapeutiques pour la cystinose.Des études antérieures ont montré que la transplantation de Cellules Souches Hématopoïétiques (CSH) saines peut efficacement traiter la cystinose dans les souris Ctns-/-. Toutefois, dû aux risques liés à une transplantation allogénique, une transplantation autologue de CSH génétiquement modifiées ex vivo pour exprimer une copie fonctionnelle du gène CTNS a été développée.Nous résumons ici les études pharmacologiques et toxicologiques ainsi que le développement du protocole de transduction des cellules humaines qui seront inclus dans une application intitulée« Investigational New Drug » qui sera soumise à la FDA afin de débuter un essai clinique de phase I/II. / Cystinosis is an inherited lysosomal storage disorder caused by mutations in the gene CTNS encoding the cystine transporter cystinosin, and is characterized by accumulation of cystine in the tissues leading to multiorgan degeneration.The kidney is the first organ impacted by cystinosis but the pathogenesis is still not fully understood. The study of new partners of cystinosin revealed an interaction with galectin-3, a member of the galectin’s family. The investigation of this interaction unraveled a new role for cystinosin in chronic inflammation associated with kidney pathology in cystinosis. Indeed, the cystinosis mouse model, Ctns-/ mice, had increased expression of Gal3 and abundant pro-inflammatory infiltrates in their kidney, as well as increased expression of Monocyte Chemoattractant Protein-1 (MCP1), a proinflammatory cytokine, in their serum.In contrast, few infiltrates and normal MCP1 levels were observed in the Ctns-/- Gal3-/- mice, which also demonstrated better kidney structure and function.This study may lead to the discovery of new drug targets for cystinosis treatment.Previous studies showed that wild-type Hematopoietic Stem Cells (HSCs) transplantation had the potential to rescue cystinosis in Ctns-/- mice.However, due to the risks associated with allogeneic transplant, an autologous transplantation of HSCs genetically modified ex vivo to express a functional CTNS gene has been developed in the laboratory. In this work, we summarized the pharmacology and toxicology studies and manufacturing development that will be included in an Investigational New Drug application to be submitted to the FDA to start a phase I/II clinical trial for cystinosis.

A cistinose nefropática (CN) é uma doença autossômica recessiva, sistêmica e grave, caracterizada pelo acúmulo intralisossomal de cistina. A cisteamine (droga depletora de cistina intralisossomal) é essencial para o tratamento. Entretanto, mesmo quando o uso de cisteamine é precoce, os pacientes, geralmente, evoluem para doença renal crônica terminal por volta da segunda ou terceira décadas da vida e, podem apresentar acometimento extrarenal. Existem evidências que o acúmulo de cistina não é responsável por todas as anormalidades observadas na CN. Estudos têm demonstrado metabolismo alterado do trifosfato de adenosina (ATP), aumento na taxa de apoptose, deficiência de glutatione no citosol e aumento na autofagia mitocondrial, levando a um aumento da produção de espécies oxigênio reativas. Portanto, pacientes com CN podem ser mais suscetíveis ao stress oxidativo (SO) e isto pode contribuir para a progressão da doença renal. Os objetivos deste estudo foram: - avaliar um marcador sérico de SO em crianças com CN, comparar os resultados com os observados em controles normais e, correlacioná-los com o ritmo de filtração glomerular (RFG); - estudar a correlação entre os parâmetros empregados para avaliar o RFG nestes pacientes; - administrar uma droga antioxidante (N-acetilcisteína, NAC) para todos os pacientes e avaliar marcadores de RFG e de SO antes e durante o tratamento com esta droga. Foram incluídos pacientes com CN, com idade inferior a 18 anos, com doença renal crônica classe I a IV (KDOQY) e com boa aderência ao tratamento, incluindo cisteamine. SO foi avaliado através da dosagem sérica de substâncias ácido-tiobarbitúricas (TBARS) e, o RFG foi avaliado pela creatinina e cistatina C séricas e, pelo clearance de creatinina (fórmula de Schwartz). NAC, na dose de 25 mg/Kg/dia, dividida em 3 tomadas, foi administrada a todos os pacientes que foram controles de si mesmos. Marcadores de SO e de RFG foram estudados imediatamente antes do início da droga (T0) e após 3 meses do seu uso (T1). Para avaliação do marcador de SO em pacientes com CN, comparação com controles normais e com os parâmetros de RFG, foram selecionados 20 pacientes, idade de 8,0±3,6 anos e, 43 controles normais com idade de 7,4±1,1 anos. Nos pacientes com CN o nível sérico de TBARS encontrado foi 4,03±1,02 nmol/ml, enquanto que no grupo controle foi 1,60±0,04 nmol/ml (p < 0,0001). Não foi detectada correlação significativa entre TBARS sérico e grau de função renal, avaliada através da creatinina sérica e do clearance de creatinina (fórmula de Schwartz). Neste estudo houve correlação significativa entre todos os parâmetros de RFG estudados, creatinina sérica, cistatina C sérica e clearance de creatinina (fórmula de Schwartz). Na avaliação da influência da NAC sobre o SO e função renal, foram incluídos 23 pacientes (16 meninos), idade 8,5±3,0 anos. Em relação ao TBARS sérico detectamos em T0 mediana de 6,92 nmol/mL (3,3-29,0) e em T1 mediana de 1,7 nmol/mL (0,6- 7,2) (p < 0,0001). Em relação à função renal, houve diferença significativa na creatinina sérica entre T0 (1,1±0,5 mg/dL) e T1 (0,9±0,5 mg/dL) (p < 0,0001), no clearance de creatinina entre T0 (69,7±32,2 mL/min/1,73 m2SC) e T1 (78,5±33,9 mL/min/1,73m²SC) (p=0.006) e na cistatina C sérica entre T0 (1,33±0,53 mg/L) e T1 (1,15±0,54 mg/L) (p=0,0057). Também foram incluídos os dados da creatinina e cistatina C séricas, e do clearance de creatinina observados 6 e 3 meses antes do início da NAC e de 3 e 6 meses após a retirada da NAC (na retirada não foi avaliada a cistatina C sérica). Concluindo, níveis elevados de TBARS sérico foram detectados em pacientes com CN e estes não tiveram correlação com o grau de função renal. Em adição, durante um período de três meses, NAC foi administrada a pacientes com CN e em uso de cisteamine e, foi observada redução do SO e melhora significativa da função renal. Nenhum efeito colateral foi detectado. Este é o primeiro relato que demonstra aumento do SO no soro de pacientes com CN e possíveis benefícios sobre a função renal com a adição de uma droga antioxidante ao arsenal terapêutico. Estudos controlados e com maior número de pacientes são necessários para confirmar estes achados / Nephropathic Cystinosis (NC) is an autosomal recessive systemic severe disease characterized by intralysosomal cystine accumulation. Cysteamine (a depleting-drug of cystine storage) is essential for the treatment. Unfortunately, even when cysteamine treatment begins early, the patients usually develop end stage renal disease at approximately the 2nd or 3rd decades of life, and they can also present extrarenal compromise. There is great evidence that cystine accumulation itself is not responsible for all abnormalities observed in NC. Studies have demonstrated altered ATP metabolism, increased apoptosis, deficiency of glutathione in the cytosol and, increased mitochondrial autophagy leading to increased production of reactive oxidative species. Therefore, cystinotic patients can be more susceptible to oxidative stress (OS) and this can contribute to the progression of the renal disease. Our goal was to evaluate a serum marker of OS in NC children, compare the results with those observed in healthy controls and correlate with glomerular filtration rate (eGFR) parameters. We also evaluated the correlation of the different eGFR parameters in these patients, e.g. serum creatinine, serum cystatin C and creatinine clearance by Schwartz formula. Finally, Nacetylcysteine (NAC, an antioxidant drug) was given to all patients and we evaluated renal function and OS status under this treatment. We enrolled cystinosis patients, aged bellow 18 years with Chronic Kidney Disease class I to IV (KDOQY) with good adherence to the treatment, including cysteamine. OS was evaluated through serum TBARS, eGFR was evaluated by serum creatinine, cystatin C, and creatinine clearance (Schwartz formula). NAC (25mg/Kg/day divided in 3 doses per day) was given during three months to all patients (T1) that were control of themselves, considering the evaluation just before NAC iniciation (T0) In the first part of this study (evaluation of a marker of OS, comparison of the results with those observed in healthy controls and correlation with renal function status), we selected 20 patients aged 8.0±3.6 years and observed serum TBARS levels of 4.03±1.02 nmol/ml. Serum TBARS levels in the 43 healthy controls, aged 7.4±1.1 years, were 1.60±0.04 nmol/ml. There was a significant difference between the serum TBARS levels among the 2 groups (p < 0.0001). We detected no significant correlation between serum TBARS and renal function status. We also observed a significant correlation among the eGFR parameters of this study. In the second part of the study, when patients received NAC and renal function and OS were evaluated, we studied 23 patients (16 males) aged 8.5±3.0 years. In relation to serum TBARS levels at T0 we detected median=6.92 nmol/mL (3.3-29.0) and at T1 median=1.7 nmol/mL (0.6-7.2) (p < 0.0001). In relation to renal function, there was a significant difference in serum creatinine between T0=1.1±0.5 mg/dL and T1=0.9±0.5 mg/dL (p < 0.0001), creatinine clearance: T0=69.7±32.2 and T1=78.5±33.9 mL/min/1.73m²BS (p=0.006), and cystatin C: T0=1.33±0.53 and at T1=1.15±0.54 mg/L (p=0.0057). We also included data from serum creatinine, creatinine clearance and cystatin C 6 (T-6) and 3 months (T-3) before NAC initiation and 3 (T+3) and 6 months (T+6) after NAC withdrawn (after NAC withdrawn cystatin C was not evaluated). In conclusion, an increased level of serum TBARS in patients with NC was observed and this abnormality was not correlated with the renal function degree. In addition, over a three-month period, we used cysteamine and NAC in cystinosis patients without renal replacement therapy and observed a reduction in oxidative stress and significantly improvement in renal function. No sideeffects were observed. Larger and controlled studies are needed to confirm these findings. This is the first report that shows increased OS in serum of NC patients

A cistinose nefropática (CN) é uma doença autossômica recessiva, sistêmica e grave, caracterizada pelo acúmulo intralisossomal de cistina. A cisteamine (droga depletora de cistina intralisossomal) é essencial para o tratamento. Entretanto, mesmo quando o uso de cisteamine é precoce, os pacientes, geralmente, evoluem para doença renal crônica terminal por volta da segunda ou terceira décadas da vida e, podem apresentar acometimento extrarenal. Existem evidências que o acúmulo de cistina não é responsável por todas as anormalidades observadas na CN. Estudos têm demonstrado metabolismo alterado do trifosfato de adenosina (ATP), aumento na taxa de apoptose, deficiência de glutatione no citosol e aumento na autofagia mitocondrial, levando a um aumento da produção de espécies oxigênio reativas. Portanto, pacientes com CN podem ser mais suscetíveis ao stress oxidativo (SO) e isto pode contribuir para a progressão da doença renal. Os objetivos deste estudo foram: - avaliar um marcador sérico de SO em crianças com CN, comparar os resultados com os observados em controles normais e, correlacioná-los com o ritmo de filtração glomerular (RFG); - estudar a correlação entre os parâmetros empregados para avaliar o RFG nestes pacientes; - administrar uma droga antioxidante (N-acetilcisteína, NAC) para todos os pacientes e avaliar marcadores de RFG e de SO antes e durante o tratamento com esta droga. Foram incluídos pacientes com CN, com idade inferior a 18 anos, com doença renal crônica classe I a IV (KDOQY) e com boa aderência ao tratamento, incluindo cisteamine. SO foi avaliado através da dosagem sérica de substâncias ácido-tiobarbitúricas (TBARS) e, o RFG foi avaliado pela creatinina e cistatina C séricas e, pelo clearance de creatinina (fórmula de Schwartz). NAC, na dose de 25 mg/Kg/dia, dividida em 3 tomadas, foi administrada a todos os pacientes que foram controles de si mesmos. Marcadores de SO e de RFG foram estudados imediatamente antes do início da droga (T0) e após 3 meses do seu uso (T1). Para avaliação do marcador de SO em pacientes com CN, comparação com controles normais e com os parâmetros de RFG, foram selecionados 20 pacientes, idade de 8,0±3,6 anos e, 43 controles normais com idade de 7,4±1,1 anos. Nos pacientes com CN o nível sérico de TBARS encontrado foi 4,03±1,02 nmol/ml, enquanto que no grupo controle foi 1,60±0,04 nmol/ml (p < 0,0001). Não foi detectada correlação significativa entre TBARS sérico e grau de função renal, avaliada através da creatinina sérica e do clearance de creatinina (fórmula de Schwartz). Neste estudo houve correlação significativa entre todos os parâmetros de RFG estudados, creatinina sérica, cistatina C sérica e clearance de creatinina (fórmula de Schwartz). Na avaliação da influência da NAC sobre o SO e função renal, foram incluídos 23 pacientes (16 meninos), idade 8,5±3,0 anos. Em relação ao TBARS sérico detectamos em T0 mediana de 6,92 nmol/mL (3,3-29,0) e em T1 mediana de 1,7 nmol/mL (0,6- 7,2) (p < 0,0001). Em relação à função renal, houve diferença significativa na creatinina sérica entre T0 (1,1±0,5 mg/dL) e T1 (0,9±0,5 mg/dL) (p < 0,0001), no clearance de creatinina entre T0 (69,7±32,2 mL/min/1,73 m2SC) e T1 (78,5±33,9 mL/min/1,73m²SC) (p=0.006) e na cistatina C sérica entre T0 (1,33±0,53 mg/L) e T1 (1,15±0,54 mg/L) (p=0,0057). Também foram incluídos os dados da creatinina e cistatina C séricas, e do clearance de creatinina observados 6 e 3 meses antes do início da NAC e de 3 e 6 meses após a retirada da NAC (na retirada não foi avaliada a cistatina C sérica). Concluindo, níveis elevados de TBARS sérico foram detectados em pacientes com CN e estes não tiveram correlação com o grau de função renal. Em adição, durante um período de três meses, NAC foi administrada a pacientes com CN e em uso de cisteamine e, foi observada redução do SO e melhora significativa da função renal. Nenhum efeito colateral foi detectado. Este é o primeiro relato que demonstra aumento do SO no soro de pacientes com CN e possíveis benefícios sobre a função renal com a adição de uma droga antioxidante ao arsenal terapêutico. Estudos controlados e com maior número de pacientes são necessários para confirmar estes achados / Nephropathic Cystinosis (NC) is an autosomal recessive systemic severe disease characterized by intralysosomal cystine accumulation. Cysteamine (a depleting-drug of cystine storage) is essential for the treatment. Unfortunately, even when cysteamine treatment begins early, the patients usually develop end stage renal disease at approximately the 2nd or 3rd decades of life, and they can also present extrarenal compromise. There is great evidence that cystine accumulation itself is not responsible for all abnormalities observed in NC. Studies have demonstrated altered ATP metabolism, increased apoptosis, deficiency of glutathione in the cytosol and, increased mitochondrial autophagy leading to increased production of reactive oxidative species. Therefore, cystinotic patients can be more susceptible to oxidative stress (OS) and this can contribute to the progression of the renal disease. Our goal was to evaluate a serum marker of OS in NC children, compare the results with those observed in healthy controls and correlate with glomerular filtration rate (eGFR) parameters. We also evaluated the correlation of the different eGFR parameters in these patients, e.g. serum creatinine, serum cystatin C and creatinine clearance by Schwartz formula. Finally, Nacetylcysteine (NAC, an antioxidant drug) was given to all patients and we evaluated renal function and OS status under this treatment. We enrolled cystinosis patients, aged bellow 18 years with Chronic Kidney Disease class I to IV (KDOQY) with good adherence to the treatment, including cysteamine. OS was evaluated through serum TBARS, eGFR was evaluated by serum creatinine, cystatin C, and creatinine clearance (Schwartz formula). NAC (25mg/Kg/day divided in 3 doses per day) was given during three months to all patients (T1) that were control of themselves, considering the evaluation just before NAC iniciation (T0) In the first part of this study (evaluation of a marker of OS, comparison of the results with those observed in healthy controls and correlation with renal function status), we selected 20 patients aged 8.0±3.6 years and observed serum TBARS levels of 4.03±1.02 nmol/ml. Serum TBARS levels in the 43 healthy controls, aged 7.4±1.1 years, were 1.60±0.04 nmol/ml. There was a significant difference between the serum TBARS levels among the 2 groups (p < 0.0001). We detected no significant correlation between serum TBARS and renal function status. We also observed a significant correlation among the eGFR parameters of this study. In the second part of the study, when patients received NAC and renal function and OS were evaluated, we studied 23 patients (16 males) aged 8.5±3.0 years. In relation to serum TBARS levels at T0 we detected median=6.92 nmol/mL (3.3-29.0) and at T1 median=1.7 nmol/mL (0.6-7.2) (p < 0.0001). In relation to renal function, there was a significant difference in serum creatinine between T0=1.1±0.5 mg/dL and T1=0.9±0.5 mg/dL (p < 0.0001), creatinine clearance: T0=69.7±32.2 and T1=78.5±33.9 mL/min/1.73m²BS (p=0.006), and cystatin C: T0=1.33±0.53 and at T1=1.15±0.54 mg/L (p=0.0057). We also included data from serum creatinine, creatinine clearance and cystatin C 6 (T-6) and 3 months (T-3) before NAC initiation and 3 (T+3) and 6 months (T+6) after NAC withdrawn (after NAC withdrawn cystatin C was not evaluated). In conclusion, an increased level of serum TBARS in patients with NC was observed and this abnormality was not correlated with the renal function degree. In addition, over a three-month period, we used cysteamine and NAC in cystinosis patients without renal replacement therapy and observed a reduction in oxidative stress and significantly improvement in renal function. No sideeffects were observed. Larger and controlled studies are needed to confirm these findings. This is the first report that shows increased OS in serum of NC patients

Le transport de composés à travers les membranes biologiques est crucial pour la physiologie des cellules eucaryotes. Cependant la fonction de nombreux transporteurs putatifs reste inconnue. C’est notamment le cas de nombreux transporteurs intracellulaires exportant les catabolites du lysosome. Le transporteur lysosomal de cystine, baptisé cystinosine, se caractérise par la présence d’un motif dupliqué appelé " boucle PQ ". Sa dysfonction entraîne une maladie lysosomale, la cystinose, caractérisée par l'accumulation de cystine dans les lysosomes. Les protéines possédant un motif PQ sont retrouvées plus souvent dans les cellules eucaryotes et, à l'exception de la cystinosine, leur fonction reste inconnue. Dans cette thèse, nous démontrons qu'une autre protéine à motif PQ, PQLC2 est le transporteur responsable de l'efflux lysosomal des acides aminés cationiques et qu'il est impliqué dans le traitement de la cystinose.L'hypothèse de départ était basée, d'une part, par sur des prédictions par analyse protéomique de la localisation lysosomale de PQLC2 et, d'autre part, sur des résultats chez S.cerevisiae impliquant les orthologues putatifs de PQLC2, situés à la membrane de la vacuole, dans l'homéostasie des acides aminés cationiques. En utilisant une approche consistant à délocaliser PQLC2 à la membrane plasmique et à acidifier le pH extracellulaire pour mimer la lumière acide du lysosome, nous avons pu, par mesure d'accumulation intracellulaire de composés radiomarqués et par mesure électrophysiologique sur cellule entière, faire la preuve du transport sélectif, actif à bas pH et de faible affinité des acides aminés cationiques par PQLC2. Dans une seconde partie, nous avons mis en évidence l'implication de ce transporteur dans l'efflux lysosomal du produit de réaction entre la cystine accumulée dans les lysosomes de cellules de patients cystinotiques et le principe actif (cystéamine) du traitement pharmacologique de la cystinose.Enfin, dans une dernière partie, nous avons effectué une analyse bioinformatique préliminaire des protéines à motif PQ qui exploitait la pseudo-symétrie de ces protéines pour identifier des résidus potentiellement impliqués dans l'activité de transport. / Transport of solutes across biological membranes is crucial to eukaryotic cell physiology. However, the function of many putative transporters remains unknown, such as the proteins responsible for lysosomal export of metabolites. Cystinosin, the lysosomal cystine exporter defective in cystinosis, is characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes, and, except for cystinosin, their molecular function remains unknown. Here we show that another PQ-loop protein, PQLC2, is a lysosomal transporter for cationic amino acids and that it is required for the treatment of cystinosis. The hypothesis that PQLC2 is a lysosomal metabolite transporter was based on a proteomic study predicting that PQLC2 is located at the lysosomal membrane and on a genetic study that linked putative yeast orthologues with cationic amino acid homeostasis. Using an approach that consisted in misrouting PQLC2 to the plasma membrane of frog oocytes and in acidifying the extracellular medium to mimic the acidic lysosomal lumen, we showed an accumulation of radiolabelled cationic amino acids into mRNA-injected oocytes and an electrogenic, inward current due to a selective, pH-dependent, low-affinity transport of cationic amino acids by PQLC2. Moreoever, we showed that PQLC2 exports a key chemical intermediate (cysteamine-cysteine mixed disulfide) from cystinotic lysosomes treated with the aminothiol drug cysteamine, thus explaining the mechanism underlying the current drug therapy of cystinosis. Finally, in a last chapter, we performed a preliminary bioinformatic study of the family of PQ-loop proteins that took advantage of the pseudo-symmetric structure of these proteins to identify residues potentially important for the transport activity.