Detection of Urinary Exosomal HSD11B2 mRNA Expression: A Useful Novel Tool for the Diagnostic Approach of Dysfunctional 11β-HSD2-Related Hypertension

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.

Urinary extracellular vesicles carry valuable hints through mRNA for the understanding of endocrine hypertension

“…Heterozygous hypertensive subjects had higher concentrations of HSD11B2 mRNA transcripts than heterozygous normotensive subjects, suggesting a modulation of exosomal HSD11B2 mRNA associated with both mutation carrier status and hypertensive status. Interestingly, the F/E urinary ratio also correlated with the HSD11B2 mRNA copy number, thus validating the use of the F/E ratio as a possible useful surrogate marker of 11β-HSD2 enzyme activity ( 8 ).…”

“…The results for HSD11B2 mRNA transcripts, on the other hand, were surprising, showing an opposite trend than expected based on genotypes. In fact, highest HSD11B2 gene expression (as measured by mRNA transcripts) was observed in 662C>G homozygous, with a decreasing trend for heterozygous and then for wild types ( 8 ).…”

Apparent mineralocorticoid excess: comprehensive overview of molecular genetics

“…In addition, it is reported that the download expression of miRNA (miR‑192‑5p and miR‑204‑5p) in urinary exosomes also plays a potential role regulating the phenotype [ 68 ]. Santis et al identified that the expression level of exosomal urinary HSD11B2 mRNA was closely associated with the hypertension phenotype [ 69 ].…”