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Mutations in the chloride-proton antiporter, ClC-5, cause Dent’s disease, a kidney disease defined by excessive loss of proteins in the urine. ClC-5 resides on early endosomal membranes in proximal tubule epithelial cells, where it facilitates protein receptor-mediated endocytosis. Loss-of-function mutations in ClC-5 produce proximal tubule defects in protein reabsorption. This study characterized an epithelial cell phenotype for nonsense ClC-5 mutations, R648X and R704X. Both ClC-5 mutants displayed defective biosynthesis, mistrafficking and ER localization. This study showed that ClC-5 mutations, R718X and C221R, which are also misprocessed and ER retained, are targeted for proteasomal degradation as a means to be efficiently eliminated from the ER. In addition, we have shown that a missense mutation in ClC- 5, C221R, causes a global conformational change in the antiporter, which likely reflects protein misfolding, as evident by enhanced susceptibility to trypsin proteolysis. We have characterized ClC-5 disease-causing mutations in an epithelial cell model of the proximal tubule.

Mutations in the chloride-proton antiporter, ClC-5, cause Dent’s disease, a kidney disease defined by excessive loss of proteins in the urine. ClC-5 resides on early endosomal membranes in proximal tubule epithelial cells, where it facilitates protein receptor-mediated endocytosis. Loss-of-function mutations in ClC-5 produce proximal tubule defects in protein reabsorption. This study characterized an epithelial cell phenotype for nonsense ClC-5 mutations, R648X and R704X. Both ClC-5 mutants displayed defective biosynthesis, mistrafficking and ER localization. This study showed that ClC-5 mutations, R718X and C221R, which are also misprocessed and ER retained, are targeted for proteasomal degradation as a means to be efficiently eliminated from the ER. In addition, we have shown that a missense mutation in ClC- 5, C221R, causes a global conformational change in the antiporter, which likely reflects protein misfolding, as evident by enhanced susceptibility to trypsin proteolysis. We have characterized ClC-5 disease-causing mutations in an epithelial cell model of the proximal tubule.

Le tubule proximal participe à la diurèse en modifiant la composition de l'ultrafiltrat glomérulaire. Grâce à de nombreux transports transépithéliaux, il le glucose, les acides aminés et les protéines de bas poids moléculaires, ainsi que 80 % des ions HPO42- ou HCO3-, 60 % des ions Na+, Cl-, K+, Ca2+, 75 % de l’eau et 30 % des ions Mg2+ ultrafiltrés.Durant ma thèse, j'ai étudié les rôles physiologiques et physiopathologiques de deux protéines de transport exprimées dans le tubule proximal.Dans le cadre de ma première étude, j'ai évalué in vivo la fonction rénale de souris n'exprimant pas une protéine appelée Kir4.2, dont le rôle est inconnu. Nos résultats montrent que Kir4.2, associée à Kir5.1, forme un canal potassique basolatéral Kir4.2/Kir5.1 dans le tubule proximal. L'absence de Kir4.2 provoque chez la souris une acidose tubulaire proximale isolée, consécutive à une ammoniogénèse altérée. De fait, la perte de fonctionnalité de Kir4.2 pourrait être à l'origine d'acidoses tubulaires proximales isolées familiales idiopathiques.Dans le cadre de ma seconde étude, j'ai analysé in vitro la fonctionnalité d'un mutant pathogène de l'échangeur 2Cl-/H+ ClC-5 impliqué dans la maladie de Dent. Cette maladie, caractérisée par une protéinurie de bas poids moléculaire associées à divers troubles du tubule proximal, serait liée à un défaut d'acidification des endosomes précoces par ClC-5. Toutefois, le mutant de ClC-5 que nous avons étudié, converti en canal chlorure, acidifie autant les endosomes précoces que le ClC-5 sauvage. Surprenants, ces résultats suggèrent que la maladie de Dent puisse être causée par un défaut d'accumulation d'ions chlorure dans l'endosome. / The proximal tubule is involved in diuresis by modifying the content of the glomerular ultrafiltrate. Using a variety of transepithelial transports systems, it reabsorbs all ultrafiltrated glucose, amino-acids and low molecular weight proteins, as well as 80% of HPO42- and HCO3- ions, about 60% of Na+, Cl-, K+, and Ca2+ ions, 75% of water and 30% of Mg2+.During this thesis, I determined the physiological and physiopathological roles of two transport proteins present in proximal tubule. Firstly, I evaluated the renal function of mice invalidated for the Kir4.2 protein, whose role was undetermined. Our results show that Kir4.2, in association with Kir5.1, form a Kir4.2/Kir5.1 potassium channel at the basolateral membrane of proximal tubular cells. Furthermore, Kir4.2-null mice exhibit a reduced ammoniagenesis leading to an isolated proximal renal tubular acidosis. This study provides the gene encoding Kir4.2 as a candidate gene for the yet unexplained autosomal dominant isolated proximal renal tubular acidosis.Secondly, I evaluated in vitro the functional consequences of a pathogenic mutation of the 2Cl-/H+ exchanger ClC-5, involved in Dent’s disease. This disease, characterized by a low-molecular-weigth-proteinuria in the context of a general proximal tubule dysfunction, is currently thought to be due to an acidification defect of early endosomes linked to a loss of function of ClC-5. Surprisingly, our results show that ClC-5, converted into a chloride channel by this mutation, indeed acidifies the early endosomes as well as the ClC-5 wild-type. Thus, Dent’s disease may originate from a defect in the accumulation of chloride ions into the early endosomes.