Project New-BP-KIT (Characterization of a New pathway of Blood Pressure regulation through Kidney Ion...) | ANR - Agence Nationale de la Recherche ANR funded project | ANR - Agence Nationale de la Recherche

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ANR funded project

Blanc International II - SVSE 1 - Physiologie, physiopathologie, santé publique (Blanc Inter II - SVSE 1) 2012
Projet New-BP-KIT

Characterization of a New pathway of Blood Pressure regulation through Kidney Ion Transport

Arterial hypertension is a complex trait caused by environmental and genetic factors. This complexity has limited the identification of susceptibility genes for this major cardiovascular risk factor to controversial loci and candidate genes. Conversely, search for genes implicated in rare Mendelian forms of the disease has been particularly successful. However, most of the identified genes encoded products already known or suspected to play a role in blood pressure regulation. The story we initiated more than 10 years ago is of another nature since it ended up with the identification of completely unsuspected genes.
Familial hyperkalemic hypertension (FHHt) syndrome, also known as Gordon syndrome or pseudohypoaldosteronism type 2, is a rare inherited form of low-renin hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis. The clinical and biological phenotypes suggest that this syndrome is due to an altered ionic transport in the renal distal tubule. Eleven years ago, linkage analysis led to the identification of two genes mutated in this syndrome, WNK1 and WNK4, whose involvement in blood pressure regulation was completely unsuspected. In the following years, numerous studies provided crucial informations about the mechanisms by which these serine-threonine kinases regulate ion transport in the distal nephron. However, the events leading to the development of FHHt are still unclear. We therefore undertook further genetic analysis of two informative FHHt families in order to identify new causative genes. Indeed, FHHt displays genetic heterogeneity, with at least four genes involved, and mutations in WNK1 and WNK4 are only found in 4 patients of our cohort, which includes 55 index cases. Linkage analyses combined with whole exome sequencing allowed us to identify KHL3 as a new "FHHt gene", mutated in 35% of our cohort. This gene encodes a homolog of the Drosophila melanogaster actin-binding and ring-canal organiser Kelch. Like WNKs eleven years ago, none of the available data could have predicted its contribution to the regulation of ion transport of the distal nephron. We showed that KLHL3 is ubiquitously expressed, with a stronger expression in the cerebellum. In the kidney, KLHL3 is detected in all nephron segments, with a higher expression in the Distal Convoluted Tubule. This nephron segment also expresses the thiazide-sensitive Na+-Cl- co-transporter NCC, which plays a key role in the pathogenesis of FHHt. We showed that KLHL3 inhibits NCC membrane localisation. This could be the consequence of KLHL3-mediated ubiquitination and proteosomal degradation of the co-transporter, as several KLHL proteins have been to shown to recruit substrates for the RING-type Cullin 3-based ubiquitin-ligase complexes. This hypothesis is supported by the recent discovery of mutations in the gene encoding Cullin 3 (Cul3) in FHHt patients of a North-American cohort. We found similar mutations in 7 patients of our cohort. However, another well-characterised partner of kelch domains is actin and we showed that a pool of endogenous KLHL3 is associated with the actin skeleton in HEK293 cells. We therefore cannot rule out that KLHL3-mediated inhibition of NCC membrane insertion is not actin-dependent, as insertion in or retrieval from the plasma membrane of ion transporters and channels are known to be microtubule-dependent.
The aims of the project developed in the present proposal are are to elucidate the mechanisms of regulation of ion transport in the distal nephron by KLHL3/Cul3 and understand how their mutations lead to the development of FHHt. We will characterize the molecular mechanisms underlying KLHL3-mediated inhibition of NCC, using protein-protein, ubiquitination and videomicroscopy studies in HEK293 cells as well as electrophysiology in Xenopus laevis oocytes. We will also use genetically engineered mice to define the roles played by KLHL3 in physiological and pathological ion transport in the distal nephron.

Partners

CNRS Institut Curie, CNRS UMR144

PARCC Paris Cardiovascular Research Center

Universidad Panamericana Molecular Physiology Unit - Escuela de Medicina

ANR grant: 288 165 euros
Beginning and duration: juin 2013 - 36 mois

 

ANR Programme: Blanc International II - SVSE 1 - Physiologie, physiopathologie, santé publique (Blanc Inter II - SVSE 1) 2012

Project ID: ANR-12-ISV1-0001

Project coordinator:
Madame Juliette Hadchouel (Paris Cardiovascular Research Center)
juliette.hadchouel@nullinserm.fr

 

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.