Abstract
ObjectiveApparent mineralocorticoid excess (AME) is an autosomal recessive disorder caused by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme deficiency, traditionally assessed by measuring either the urinary cortisol metabolites ratio (tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone, THF+5αTHF/THE) or the urinary cortisol/cortisone (F/E) ratio. Exosomal mRNA is an emerging diagnostic tool due to its stability in body fluids and its biological regulatory function. It is unknown whether urinary exosomal HSD11B2 mRNA is related to steroid ratio or the HSD11B2 662 C>G genotype (corresponding to a 221 A>G substitution) in patients with AME and essential hypertension (EH).Aim of the StudyTo detect and quantify HSD11B2 mRNA from urinary exosomes in samples from family members affected by AME and EH, and to evaluate the relationship between exosomal HSD11B2 mRNA, steroid ratio, 662C>G genotype, and hypertension.MethodsIn this observational case–control study, urinary steroid ratios and biochemical parameters were measured. Urinary exosomes were extracted from urine and exosomal HSD11B2 mRNA was quantified by Droplet Digital PCR (ddPCR). B2M (β-2 microglobulin) gene was selected as the reference housekeeping gene.ResultsAmong family members affected by AME, exosomal urinary HSD11B2 mRNA expression was strictly related to genotypes. The two homozygous mutant probands showed the highest HSD11B2 mRNA levels (median 169, range 118–220 copies/µl) that progressively decreased in 221 AG heterozygous with hypertension (108, range 92–124 copies/µl), 221 AG heterozygous normotensives (23.35, range 8–38.7 copies/µl), and wild-type 221 AA subjects (5.5, range 4.5–14 copies/µl). Heterozygous hypertensive subjects had more HSD11B2 mRNA than heterozygous normotensive subjects. The F/E urinary ratio correlated with HSD11B2 mRNA copy number (p < 0.05); HSD11B2 mRNA strongly decreased while THF+5αTHF/THE increased in the two probands after therapy. In the AME family, HSD11B2 copy number correlated with both F/E and THF+5αTHF/THE ratios, whereas in EH patients, a high F/E ratio reflected a reduced HSD11B2 mRNA expression.ConclusionsHSD11B2 mRNA is detectable and quantifiable in urinary exosomes; its expression varies according to the 662 C>G genotype with the highest levels in homozygous mutant subjects. The HSD11B2 mRNA overexpression in AME could be due to a compensatory mechanism of the enzyme impairment. Exosomal mRNA is a useful tool to investigate HSD11B2 dysregulation in hypertension.
Highlights
Apparent mineralocorticoid excess (AME) is a rare autosomal recessive disorder caused by the 11b-hydroxysteroid dehydrogenase type 2 (11b-HSD2) enzyme deficiency that leads to an activation of the mineralocorticoid receptor (MR) not mediated by aldosterone [1]
To the best of our knowledge, this is the first report showing that HSD11B2 mRNA is detectable in urinary exosomes and correlates with the 11b-HSD2 activity estimated by urinary steroid ratios
The results highlight, that the urinary exosomal HSD11B2 mRNA expression varies according to the HSD11B2 662C>G genotype in an AME family, suggesting a potential use of it as a molecular marker of hypertensive disease, due to an impaired 11b-HSD2 enzyme function
Summary
Apparent mineralocorticoid excess (AME) is a rare autosomal recessive disorder caused by the 11b-hydroxysteroid dehydrogenase type 2 (11b-HSD2) enzyme deficiency that leads to an activation of the mineralocorticoid receptor (MR) not mediated by aldosterone [1]. The MR is characterized by similar affinity for both aldosterone and cortisol, but in spite of the higher plasma concentration of cortisol as compared to that of aldosterone (about 100-fold), specific MR activation by aldosterone is physiologically guaranteed because of the specific 11b-HSD2 enzymatic activity. An impaired 11b-HSD2 function leads to an accumulation of active steroid forms in the renal distal tubular cells, with subsequent MR activation and sodium retention with the development of a clinical syndrome characterized by sodium retention, hypokalemia, salt-dependent hypertension, low renin, and suppressed aldosterone concentrations [1, 4,5,6,7]. The activity of the 11b-HSD2 enzyme can be estimated either by measuring the urinary cortisol metabolites ratio (tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone, THF+5aTHF/THE) or by measurement of serum or urinary free cortisol/cortisone ratio (F/E). An increase in urinary (THF +5aTHF)/THE ratio or urinary F/E indicates a decreased 11b-HSD2 activity
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