ARR screening fails to predict positive ivSLT in most (60.2%) hypertensive women as compared with 14.8% of hypertensive men. ARR is more often increased in normotensive women than men. Oral contraceptive may affect ARR contributing to the diagnostic inaccuracy in women.

Abstract-Prostasin is a serine peptidase hypothesized to regulate epithelial sodium channel (ENaC) activity in animals or on in vitro cultured cells. We investigated whether urinary prostasin may be a candidate marker of ENaC activation in humans. We studied 10 healthy volunteers and 8 hypertensive patients with raised aldosterone-to-renin ratio before and after spironolactone or saline/Florinef suppression test, respectively. Four healthy subjects were also studied before and after saline. Urinary prostasin was evaluated by SDS-PAGE, 2D maps, and Western blotting. Every sample of normotensive individuals was compared with the corresponding sample of urine collected after spironolactone or saline; every sample of hypertensive patients was compared with the corresponding sample of urine collected after saline or Florinef. Prostasin was detectable in all subjects regardless of gender, dietary sodium intake, and spironolactone treatment. Spironolactone (100 mg) increased urinary Na ϩ /K ϩ ratio and decreased urinary prostasin in normotensives in whom the renin/aldosterone axis was activated by a low Na ϩ intake, but it was ineffective in individuals with high Na ϩ intake. Saline infusion also reduced prostasin in normotensive subjects. In contrast, prostasin paradoxically increased in urine of patients affected by primary aldosteronism after volume expansion. By 2D immunoblotting, several protein isoforms were observed, some of them being overexpressed after inhibition tests in patients with primary aldosteronism. In addition to a "basal" aliquot of prostasin, constitutively released in human urine regardless of sodium balance and aldosterone activation, there exists a second "aldosterone-responsive" aliquot modulated by Na ϩ intake and potentially suitable as candidate marker of ENaC activation.

In urinary exosomes, NCC and prostasin had a diurnal pattern parallel to ADH and aquaporin 2, confirming that, in healthy subjects, both prostasin and NCC relate to water balance. These results provide suggestions for a possible chronotherapeutic approach in patients treated with thiazides, diuretic drugs acting as specific inhibitors of NCC-mediated Na reabsorption.

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.

Prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease, activates the epithelial sodium (Na) channel (ENaC), and prostasin is released in extracellular fluids, including urine. Previous data have suggested a direct association between urinary prostasin and the activation of an aldosterone-driven pathway, but a quantitative association has never been demonstrated in normotensive subjects. Similarly, physiological relationships with natriuresis or possible gender- or female hormone-related changes in urinary prostasin concentrations have never been investigated. We measured urinary prostasin by enzyme-linked immunosorbent assay in 43 healthy normotensive subjects of similar age presenting different urinary Na levels and in 15 women during the menstrual cycle and after oral estro-progestinic contraceptive (OC) therapy. Exosomal urinary prostasin was also estimated by western blotting of samples from six healthy subjects twice during the morning. Urinary prostasin presented a wide range of values (from 0.5 to 18.9 nM) without gender differences. It was positively correlated with the aldosterone to renin ratio (ARR) but not with circulating aldosterone or renin individually. Urinary prostasin was directly correlated with U-Na levels (up to 200 nmol Na), whereas it decreased for higher Na concentrations. In women, no significant changes of prostasin concentration were observed during menstrual phases. After OC therapy, prostasin increased (from 2.37±1.27 to 4.85±5.28 nM), although the increase was not statistically different (P=0.07). Prostasin was detectable in urinary exosomes and displayed a pattern similar to urinary prostasin in relation to urinary Na. In conclusion, urinary prostasin correlates with the ARR, and it is physiologically modulated by natriuresis in normotensive individuals.