SUN-017 Impact of Steroid Precursors and Minor Metabolites Quantified by LC-MS-MS on Salt Wasting in 21-Hydroxylase Deficient Patients

Abstract

Congenital adrenal hyperplasia (CAH), associated with 21-hydroxylase deficiency (21OHD), is a severe autosomic recessive disease. It may lead to life-threatening adrenal insufficiency with cortisol and aldosterone secretion defect, requiring hormone replacement therapy with hydrocortisone and fludrocortisone, often difficult to handle especially in pediatric patients, with risks of stunting, obesity and hypertension. Plasma steroidomic profiling, obtained by LC-MS/MS technology, of 20 pediatric patients presenting with CAH, established a precise quantification of the entire steroidogenesis pathway, and confirmed previous results with substantial increase not only in 17-hydroxyprogesterone (17OHP) but also in 11-hydroxylated steroids, i.e 21-deoxycortisol (21DF) and for the first time 21-deoxycorticosterone (21DB) levels, up to 1 µM concentration in uncontrolled patients. To determine functional consequences upon mineralocorticoid signaling pathway of the accumulation of such steroids, previously shown for 17OHP, their biological activities were evaluated by transient transfection assays with Luc-reporter plasmid and in human renal cells stably expressing the human mineralocorticoid receptor (hMR) by analyzing expression of MR target genes such as aENaC, Gilz, Sgk1 or PER1. Here, we demonstrate that 21DF and 21DB both activate hMR in a partial agonist manner (ED50 (21DF) = 180 nM and ED50 (21DB) = 13 nM, vs ED50 (Aldosterone) = 0.1 nM), displaying de facto an intrinsic antagonist activity to a lesser extent than 17OHP, progesterone or spironolactone. Thereby, both 21DF and 21DB could repress the aldosterone-induced expression of target genes involved in sodium reabsorption. These findings have been further validated by tridimensional structural modeling. Docking within the hMR ligand-binding domain (LBD) showed that, in contrast to aldosterone, the lack of 21-hydroxyl group in 21DF and 21DB alter their anchoring to Asn770. Moreover, the 11β-hydroxyl group of both ligands forms an unfavorable contact with Ala773, likely explaining their weak agonistic and partial antagonistic characters. These original findings are of major importance and could explain why salt wasting remains so difficult to manage in some unbalanced CAH treated-patients, sometimes justifying the use of enhanced doses of fludrocortisone in those patients. These results also underscore the need to establish steroidomic profiles at the initial diagnostic phase of any adrenal enzymatic deficiency, but also during the patient follow-up for optimized therapy.