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Urinary extracellular vesicles (UEV) mainly derive from cells of the urogenital tract and their cargo (proteins, nucleic acids, lipids, etc.) reflects their cells of origin. Na chloride cotransporter (NCC) is expressed at the kidney level in the distal convoluted tubule, is involved in salt reabsorption, and is the target of the diuretic thiazides. NCC protein has been recognized and quantified in UEV in previous studies; however, UEV NCC mRNA has never been studied. This study aimed to identify and analyze NCC mRNA levels in primary aldosteronism (PA). The rationale for this investigation stems from previous observations regarding NCC (protein) as a possible biomarker for the diagnosis of PA. To evaluate modulations in the expression of NCC, we analyzed NCC mRNA levels in UEV in PA and essential hypertensive (EH) patients under different conditions, that is, before and after saline infusion, anti-aldosterone pharmacological treatment, and adrenal surgery. NCC mRNA was measured by RT-qPCR in all the samples and was regulated by volume expansion. Its response to mineralocorticoid receptor antagonist was correlated with renin, and it was increased in PA patients after adrenalectomy. NCC mRNA is evaluable in UEV and it can provide insights into the pathophysiology of distal convolute tubule in different clinical conditions including PA., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: Apparent 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 Study: To 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., Methods: In 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., Results: Among 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., Conclusions: HSD11B2 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 De Santis, Castagna, Danese, Udali, Martinelli, Morandini, Veneri, Bertolone, Olivieri, Friso and Pizzolo.)

Objectives: Prostasin is a glycosylphosphatidylinositol-anchored serine protease that is released in urine and is involved in epithelial Na channel activation. A direct association between urinary prostasin (u-prostasin) concentration and activation of the aldosterone-driven pathway has been suggested; however, in previous studies on primary aldosteronism, a semiquantitative evaluation, rather than a precise quantification, of prostasin was performed. We aim to investigate if u-prostasin concentrations are higher in patients with primary aldosteronism than in patients with essential hypertension and whether u-prostasin measurements could be a useful marker for diagnosing primary aldosteronism in hypertensive patients., Methods: A total of 62 primary aldosteronism and 56 essential hypertension patients were enrolled. Biochemical and hormonal parameters were measured by applying routine laboratory methods, and u-prostasin levels were assessed by ELISA., Results: Primary aldosteronism patients had higher u-prostasin levels than did essential hypertension patients. Prostasin levels were positively correlated with the aldosterone-to-renin ratio and inversely correlated with plasma K and urinary Na levels. In the highest concentration quartile, u-prostasin levels were associated with a several-fold higher probability of primary aldosteronism diagnosis in hypertensive patients. Receiver operating characteristic curve analysis showed that prostasin was specific but poorly sensitive as a diagnostic marker for primary aldosteronism., Conclusions: The study shows that an elevated u-prostasin concentration in humans is a specific marker for primary aldosteronism, which involves the classical model of epithelial Na channel activation. There was no statistically significant difference in prostasin concentrations among patients with different primary aldosteronism subtypes. Studies with a larger series of patients are necessary to clarify the clinical usefulness of the prostasin assay.

A correct diagnosis of primary aldosteronism (PA) requires adrenal venous sampling (AVS) for the classification of subtypes (bilateral hyperplasia, BAH, or adenoma, APA). Since such testing is not easily practicable, appropriate markers for the definition of subtypes are desirable. We hypothesized that an aldosterone excess was associated with abnormalities in urinary proteome, specific for PA subtypes. The project work was divided into 3 phases: (1) screening/identification by proteomic analysis and further characterization by RT-PCR and immunohistochemistry of the candidate protein; (2) clinical validation by quantitative ELISA assay of 57 (33 M, 24 F) PA patients and 50 normotensive controls (21 M, 29 F); (3) analysis of adrenal tissue of 8 individuals who had undergone adrenalectomy for APA or other adrenal tumors. The proteomic analysis showed a different expression of Serpin B3 Inhibitor-SCCA1 (SB3) in APA and BAH patients. Urine SB3 concentrations in normotensive controls, quantified by ELISA assay and normalized by urinary creatinine, resulted much lower in males (6.72 ng SB3 per mg creatinine, C.I. 4.43-10.19) than in females (20.56 ng SB3 per mg creatinine, C.I. 12.43-33.99, p < 0.00001). SB3 concentrations were not significantly different in males affected by different PA subtypes (BAH, n = 19 and APA, n = 14) compared with normotensive subjects (n = 21). In contrast, in PA females, SB3 was significantly higher in APA (n = 13) than in BAH patients (n = 11) or in normotensive controls (n = 29) (P < 0.01 and <0.05, respectively). Neither messenger RNA nor SB3 protein were identified in tissue obtained from adrenal tumors and from the surrounding normal gland. In conclusion urine SB3 concentrations are physiologically much lower in males than in females. Hypertensive women, affected by APA, present urinary SB3 concentrations significantly higher than women affected by BAH.