Intitulé du sujet: Understanding the genetic susceptibility for primary aldosteronism

Sujet

Codirection:

Nombre de mois: 48 mois

Ecole Doctorale: ED 562 - Bio Sorbonne Paris Cité

Unité de recherche et équipe:

UMR_S 970 / Centre de recherche cardiovasculaire PARCC

Equipe ZENNARO - Génétique des pathologies associés à l'aldostérone - vers une médecine de précision intégrée

Coordonnées de l’équipe:

INSERM UMR-S-970

56 rue Leblanc

75015 Paris

France

Secteur: Sciences de la vie / Life Sciences

Langue attendue: Anglais

Niveau de langue attendu: B2

Description

Description du sujet:

Introduction and State-of-the-Art         

High blood pressure (arterial hypertension) is a major cardiovascular risk factor affecting around 25% of the population (1). Primary aldosteronism (PA) is the most common form of secondary hypertension, with a prevalence of 5% -10% in hypertensive patients and as high as 20% in patients with treatment resistant hypertension (2). PA results from the autonomous production of aldosterone by the adrenal cortex, which is caused in the majority of cases by an aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia (2). Despite guidelines for the screening, diagnosis and treatment of PA, the identification and therapy of PA often fails due to the complexity of investigative procedures. Beyond categorically overt PA, recent evidence suggests a pathophysiologic continuum of renin-independent aldosterone production, ranging from mild to severe, which affects a substantial proportion of patients with primary hypertension (3). As such, understanding mechanisms driving inappropriate aldosterone production, in primary hypertension and overt PA, is a critical gap in knowledge and unmet medical need.

Our laboratory has made major contributions to the knowledge of genetic and pathogenic mechanisms of PA (4). Different germline and somatic mutations have been identified in familial forms of PA and in APA. A majority of those mutations affect genes coding for ion channels, transporters and pumps, which lead to autonomous aldosterone biosynthesis by increasing intracellular Ca²⁺ signaling, the main trigger for aldosterone production. However, it is still unclear whether those mutations also lead to increased cell proliferation and nodule formation. In particular, we have recently shown that increased calcium signaling in adrenocortical cells does not lead to increased cell proliferation, but rather to a decrease in cell proliferation and an increase of apoptosis (5). Recently, a genome-wide association study (GWAS) performed by our laboratory has identified different susceptibility genes for PA (6). Two candidate genes located within the two main loci, CASZ1 on chromosome 1 and RXFP2 on chromosome 13, were found to be expressed in human and mouse adrenal cortex and in APA. Those genes have been investigated for their functional relevance by performing phenotypic and mechanistic studies in differentiated cell lines from the human adrenal cortex. Overexpression of CASZ1 and RXFP2 in adrenocortical H295R-S2 cells significantly suppressed mineralocorticoid output under basal and stimulated conditions, without affecting cortisol biosynthesis. In cells expressing CASZ1, this was paralleled by decreased expression of aldosterone synthase. RXFP2 overexpression did not affect aldosterone synthase expression, but rather increased 21-deoxycortisol levels, suggesting a shift in the steroidogenic cascade. Remarkably, loci identified in our GWAS have been associated with hypertension related traits and are in part shared between unilateral and bilateral forms of PA, providing a genetic basis for accumulating evidence of a continuum between these conditions. These results represent a paradigm shift in our understanding of PA, suggesting a genetically determined continuum between dysregulated aldosterone production and PA in hypertension.

Research hypothesis and objectives

Our previous work on susceptibility genes for PA suggests a mechanism whereby increased expression of RXFP2 in stem/progenitor cells in carriers of risk alleles may lead to modifications of adrenal cell lineage and lineage conversion in the adrenal cortex (6). We make the hypothesis that genetically determined reduction in basal or Ang II-stimulated aldosterone production would lead to PA through lifelong increased stimulation of the adrenal cortex to ensure appropriate aldosterone levels. This, in turn, would generate a drive for replenishment of adrenocortical cells from progenitors, which ultimately leads to adrenal cortex hyperplasia, creating a favorable environment for the occurrence of somatic mutations.

The aim of this project is to investigate how this newly discovered susceptibility gene affects aldosterone production and cell proliferation in the adrenal cortex, eventually leading to PA development. The specific objectives are: 1. To evaluate the role of RXFP2 in the development of PA by using genetically modified mouse models, and 2. To decipher the molecular mechanisms underlying the involvement of RXFP2 in the function of the adrenal cortex.

Methodology              
To achieve our objectives, we will use a range of cutting-edge techniques including genetically modified mouse models, single cell and spatial transcriptomics, 3D and multispectral imaging, and intra-vital bi-photon microscopy.

1. Animal models.

Our results show that RXFP2 is expressed in a set of sub-capsular stem/progenitor cells in the adrenal cortex. We will generate tissue-specific transgenic mice in adrenocortical stem cells by crossing Shh-Cre mice (Jackson Laboratory) with mice carrying a FLEX switch (FLipEXcision switch) Rxfp2 allele (collaboration Phenomin iCS, France), mimicking the effect of risk alleles on Rxfp2 expression. The adrenal phenotype of these mice will be explored in terms of: i) adrenal morphology (histological and immunohistochemical analyses, bi-photon microscopy) and function (circulating steroid hormone profiling and renin levels, steroidogenic gene expression); and ii) response to different dynamic challenges (high and low salt diets, dexamethasone suppression) known to modify aldosterone production and adrenocortical structure and function. Mice will also be crossed with Cyp11b2-Cre::a7-5HT3 mice generated in our laboratory, to model a two-hit system, in which modification of adrenocortical function due to inherited risk alleles combined with a genetically induced model of hyperaldosteronism mimicking somatic mutations in PA driver genes can fully recapitulate the development of APA.

2. Mechanistic analysis.

To get mechanistic insight on phenotypic alterations, we will perform single nuclei RNA sequencing on the adrenal cortex of wild-type and Shh-Cre::Rxfp2fl/fl mice. In parallel, spatial transcriptome analysis of the mouse adrenal cortex of Shh-Cre::Rxfp2fl/fl and control mice will be performed. Bioinformatic analyses will be used to identify targets and pathways modified in the adrenal cortex.  Experiments will be performed in both sexes independently, to address the well know sexual dimorphism of the adrenal cortex. Investigations will be followed up by multispectral imaging of candidate proteins (Lunaphore techologies). To explore a potential effect of Rxfp2 overexpression on adrenal cortex cell lineage, Shh-Cre::Rxfp2fl/fl mice will be crossed with Rosa26-mTmG reporter mice, and trans-differentiation of adrenocortical cells will be followed at day 1, and 2, 5 and 8 weeks of age, under basal conditions and in response to dynamic challenges (high and low salt diets, dexamethasone suppression). All experimental procedures are routinely used in the laboratory; animal studies will be conducted following EC guidelines after approval by the local ethics committee. RNAseq experiments will be performed in collaboration with omics platforms of Université Paris Cité, as already described (6); bioinformatic analyses will be performed in collaboration with the bioinformatics platform of the PARCC.

Expected results, originality and feasibility       

Knowledge acquired through this project should allow understanding the role of RXFP2 in the adrenal cortex and how frequent polymorphisms of the gene may lead to the development of PA. Identifying genes and signaling pathways of the RXFP2 regulatory network that lead to increased susceptibility to develop PA can open new perspectives for the screening and treatment of patients with PA.

Compétences requises:

We are looking for a highly enthusiastic and motived candidate, interested in the field of basic and translational research. Knowledge in aldosterone related disorders and experience in molecular and cellular biology will be an advantage. Experience with animals would be a plus but is not a prerequisite. Applicant must be fluent in English. He/she must be strongly inclined to learn, very resourceful, and must have a creative approach to problem-solving. The candidate will develop his/her ability to work independently and will have to show initiative.

Références bibliographiques:

1. GBD 2019 Risk Factors Collaborators. Global burden of 87 risk factors in 204 countries and territories, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet. 2020 Oct 17;396(10258):1223-1249. doi: 10.1016/S0140-6736(20)30752-2. PMID: 33069327; PMCID: PMC7566194.
2. Zennaro MC, Boulkroun S, Fernandes-Rosa FL. Pathogenesis and treatment of primary aldosteronism. Nat Rev Endocrinol. 2020 Oct;16(10):578-589. doi: 10.1038/s41574-020-0382-4. Epub 2020 Jul 28. PMID: 32724183.
3. Brown JM, Siddiqui M, Calhoun DA, Carey RM, Hopkins PN, Williams GH, Vaidya A. The Unrecognized Prevalence of Primary Aldosteronism: A Cross-sectional Study. Ann Intern Med. 2020 Jul 7;173(1):10-20. doi: 10.7326/M20-0065. Epub 2020 May 26. PMID: 32449886; PMCID: PMC7459427.
4. Fernandes-Rosa FL, Boulkroun S, Fedlaoui B, Hureaux M, Travers-Allard S, Drossart T, Favier J, Zennaro MC. New advances in endocrine hypertension: from genes to biomarkers. Kidney Int. 2023 Mar;103(3):485-500. doi: 10.1016/j.kint.2022.12.021. Epub 2023 Jan 13. PMID: 36646167.
5. Fedlaoui B, Cosentino T, Al Sayed ZR, Giscos-Douriez I, Faedda N, Fayad M, Hulot JS, Magnus C, Sternson SM, Travers-Allard S, Baron S, Fernandes-Rosa FL, Zennaro MC, Boulkroun S. Modulation of calcium signaling on demand to decipher the molecular mechanisms of primary aldosteronism. bioRxiv 2024.04.29.591306. doi: https://doi.org/10.1101/2024.04.29.591306
6. Le Floch E, Cosentino T, Larsen CK, Beuschlein F, Reincke M, Amar L, Rossi GP, De Sousa K, Baron S, Chantalat S, Saintpierre B, Lenzini L, Frouin A, Giscos-Douriez I, Ferey M, Abdellatif AB, Meatchi T, Empana JP, Jouven X, Gieger C, Waldenberger M, Peters A, Cusi D, Salvi E, Meneton P, Touvier M, Deschasaux M, Druesne-Pecollo N, Boulkroun S, Fernandes-Rosa FL, Deleuze JF, Jeunemaitre X, Zennaro MC. Identification of risk loci for primary aldosteronism in genome-wide association studies. Nat Commun. 2022 Sep 3;13(1):5198. doi: 10.1038/s41467-022-32896-8. PMID: 36057693; PMCID: PMC9440917.