Towards an in vivo model to screen for antihypertensive drugs - understanding mineralocorticoid synthesis and action in zebrafish

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Hypertension is a major cardiovascular risk factor and the leading contributor to worldwide mortality. Recent evidence suggests that about 20% of hypertension is caused by overproduction of mineralocorticoids. Mineralocorticoid receptor (MR) blockers such eplenerone are used in clinical practice, but are suboptimal due to their limited effectiveness. Thus, the discovery of novel antihypertensive drugs is warranted. Zebrafish are a well-established for drug screening and our grouping has implemented zebrafish as a model to study steroid pathophysiology in vivo. The current dogma suggests MR-independent regulation of sodium homeostasis in zebrafish larvae by the glucocorticoid cortisol. However, the MR is expressed from early development paralleling the development of steroid biosynthesis. Our unpublished data suggests active mineralocorticoid synthesis including deoxycorticosterone (DOC) in zebrafish larvae supported by in silico evidence of 11-hydroxylase harbouring specific amino-acid residues essential for aldosterone biosynthesis. This projects aims to establish the physiology of mineralocorticoid synthesis and action in zebrafish by using several cutting edge approaches. This project will potentially result in a dogma shift within zebrafish physiology and establish a novel screening tool for antihypertensive drugs. We will establish the MR expression in zebrafish using whole mount in situ hybridisation (WISH) and immunohistochemistry. Whole organism effects of impaired mineralocorticoid synthesis and action will be dissected in a stepwise approach. By using CRIPSR/CAS9, a MR-null allele will be created to analyse the pathophysiological consequences of mineralocorticoid resistance in vivo. The data will be compared to wildtype larvae treated with the MR-antagonist eplenerone. To induce endocrine hypertension, wildtype larvae will be treated with DOC and aldosterone. These studies will be complemented by using a recently developed 21-hydroxylase-deficient line with impaired cortisol and mineralocorticoid synthesis representing a model of endocrine salt-loss. Furthermore, we will create a 17-hydroxylase (cyp17a2) null-allele line. This line will be cortisol deficient in combination with an overproduction of mineralocorticoid precursors modelling endocrine hypertension. Steroid metabolomes in mutants and pharmacological-treated wildtypes will be analysed by liquid-chromatography tandem mass spectrometry providing functional read-outs. Measuring the rate of in vivo sodium uptake using the 22Na isotope will provide novel in vivo insights of sodium homeostasis. After defining the best time point to detect significant changes during development, we will compare transciptomes of wildtype and MR-null alleles. We anticipate strong changes in MR-downstream genes including Sgk1, Need4-2, Fkbp5, Rasl12, Tns1 and Tsc22d3. These will be confirmed by quantitative real-time PCR. This will provide novel insights into MR-physiology and also establish genes showing the strongest response to mineralocorticoid deficiency and excess. To screen for anti-mineralocorticoid substances, we will use a standard library of 2000 compounds. WISH against genes showing the largest expression difference will be employed as readout for the screen. Hits detected in the first screen will be selected from the library and reanalyzed using the same method. Identified substances will be further analysed in vivo to study the mechanism altering mineralocorticoid synthesis and action. This project will provide fundamental novel insights into physiology relevant to research involving steroid hormones and has the potential to discover novel drugs for treatment of hypertension.