Urinary proteomics: role of putative markers of Epithelial Sodium Channel (ENac) activation

La proteomica urinaria sta diventando un utile approccio metodologico per l\u2019identificazione di potenziali marcatori di malattia. Noi abbiamo studiato l\u2019ipertensione arteriosa ed, in particolare, l\u2019iperaldosteronismo primitivo (PA) analizzando urine ed esosomi urinari di soggetti sani e pazienti ipertesi. L\u2019iperaldosteronismo primitivo \ue8 la pi\uf9 comune causa di ipertensione secondaria causata da un eccessiva secrezione di aldosterone con riassorbimento di sodio e conseguente espansione volemica mediata dall\u2019attivazione del canale epiteliale del sodio (ENaC) a livello renale. Nonostante l\u2019alta prevalenza della malattia, la fisiologica regolazione dell\u2019ENaC non \ue8 pienamente chiara e inoltre l\u2019iter diagnostico per l\u2019iperaldosteronismo primitivo ancora oggi non presenta un\u2019ottimale accuratezza. Prostasina, una serin-proteasi GPI-ancorata, attiva l\u2019ENaC portando al riassorbimento di sodio a livello del tubulo distale; la prostasina \ue8 rilasciata nei fluidi extracellulari, comprese le urine. Dati precedenti hanno ipotizzato una diretta correlazione tra prostasina urinaria e l\u2019attivazione della cascata di segnale indotta dall\u2019aldosterone ma un\u2019associazione quantitativa non era mai stata dimostrata nei soggetti normotesi. Abbiamo analizzato le urine di soggetti sani con un metodo quantitativo (ELISA) e abbiamo riscontrato la presenza di prostasina in urine di soggetti di entrambi i sessi senza differenze significative; abbiamo descritto, inoltre, una correlazione diretta fra prostasina e natriuresi e tra prostasin e ARR ( rapporto tra aldosterone e renina, usato come test di screening per l\u2019iperaldosteronismo primitivo). Inoltre prostasina sembra essere modulata, come l\u2019ARR, dalla terapia estro-progestinica in giovani donne normotese. Prostasina presenta una variazione diurna simile all\u2019ARR e all\u2019ADH ( adiuretina o vasopressina). Nei pazienti ipertesi la concentrazione urinaria di prostasina \ue8 pi\uf9 elevata in pazienti affetti da PA rispetto a quelli affetti da ipertensione essenziale (EH). In conclusione la prostasina potrebbe essere un marcatore di attivazione dell\u2019ENaC in vivo.Urine proteomic is becoming an useful approach to discover potential biomarkers of disease. We investigated arterial hypertension and specifically primary aldosteronism (PA), analyzing urine and urinary exosomes in healthy subjects and hypertensive patients. Primary aldosteronism (PA) is the most common cause of secondary hypertension caused by an aldosterone excess with consistent sodium retention and volume expansion mediated by the renal epithelial sodium channel (ENaC) activation. Despite the high prevalence of the disease, the physiological regulation of ENaC activation is not yet fully elucidated, and the PA diagnostic work-up still lacks optimal accuracy. Prostasin, a GPI-anchored serine protease, activates ENaC leading to Na+ reabsorption; 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. We analyzed urine of healthy subjects with a quantitative method (ELISA assay) and we found prostasin in both the genders without differences; we described a direct correlation between prostasin and natriuresis and between prostasin and ARR (aldosterone to renin ratio, the screening test used for primary aldosteronism diagnosis). Moreover prostasin seemed to be modulated, like ARR, by estro-progestinic therapy in normotensive young women. Prostasin presented a diurnal variation pattern similarly to ARR and ADH. In hypertensive patients prostasin was higher in those affected by PA compared with those with essential hypertension (EH). In conclusion urinary prostasin seems to grossly reflect the extent of ENaC activation in vivo

Isolation of Urinary Exosomal miRNAs using various methods

Exosomes are involved in a wide spectrum of physiological mechanisms such as immune system modulation, paracrine functions and cell to cell communications. They can be detected in urine being released from every segment of the nephron. Urinary exosomes (UEs) constitutively contain RNA (microRNAs/mRNA/small RNAs) and harbour unique subset of proteins, reflecting their cellular source. With the aim to establish the best method both for urinary exosomes isolation and for the subsequent RNA profiling analysis, we compared three different UEs isolation methods and six RNA extraction techniques. Exosomal RNA yield, quality and size were assessed respectively by specific staining with fluorescent dyes (Ribogreen\uae, RNA specific quantification kit), spectrophotometric quantification (Nanodrop\uae ND \u2013 1000 spectrophotometer) and capillary electrophoresis (Agilent 2100 Bioanalyzer). All the samples were analysed for detection of selected miRNAs and mRNAs by Real Time PCR specific assays. Based on these results the most reliable and convenient method for UEs miRNAs extraction and analysis was selected. Advantages and drawbacks of each methodology were also discussed

Circadian exosomal expression of renal thiazide-sensitive NaCl cotransporter (NCC) and prostasin in healthy individuals

A circadian timing system is involved in the maintenance of fluid and electrolyte balance and blood pressure control. Aldosterone and vasopressin modulate ion transporters and channels crucial in sodium (Na) and water reabsorption such as the epithelium Na channel and the renal thiazide-sensitive NaCl cotransporter (NCC). We analyzed in urinary exosomes the intraday variations of NCC and prostasin expression and the association with electrolytes and water balance parameters

Increased urinary excretion of the epithelial Na channel activator prostasin in patients with primary aldosteronism

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

NT-proBNP, a useful tool in hypertensive patients undergoing a diagnostic evaluation for primary aldosteronism.

Primary aldosteronism (PA) is the most frequent form of secondary hypertension, but diagnostic tools for this disease still lack optimal accuracy. The heart is one important target tissue for damage due to excess aldosterone, and the role of natriuretic peptides is well recognized in diagnosing heart failure. We hypothesized that measuring the NT-proBNP could improve the diagnostic evaluation of PA. We enrolled 132 hypertensive patients, who underwent aldosterone to renin ratio (ARR) screening, and 81 underwent an intravenous saline loading test (ivSLT) because of a high ARR. The NT-proBNP level positively correlated with the ARR and inversely correlated with the renin level. The NT-proBNP level was higher in patients with a high ARR than in those with a low ARR and higher in patients with a positive ivSLT than in those with a negative ivSLT. After logistic regression analysis, an NT-proBNP value above the median and male gender were predictors of a positive ivSLT. The proportion of patients with a positive ivSLT ranged from only 23 % in females with a low NT-proBNP to 93 % in males with a high NT-proBNP. NT-proBNP and gender are predictors of a positive PA confirmatory test. These findings highlight the possibility of using NT-proBNP to identify which patients with a high ARR should receive a complete PA diagnostic evaluation

Effects Of Female Sex Hormones And Contraceptive Pill On The Diagnostic Work-Up For Primary Aldosteronism

OBJECTIVES: Due to the widespread use of the aldosterone to renin ratio (ARR),primary aldosteronism is currently recognized as a frequent cause of secondaryhypertension. After a positive screening, primary aldosteronism diagnosis needsconfirmation by an inhibitory test such as intravenous saline load (ivSLT). Theaim of the present study was to investigate the role of female hormones inprimary aldosteronism diagnosis, by evaluating possible differences by sex on ARRscreening, on the rate of ivSLT response and analyzing the influence of free and oral contraceptive-induced menstrual cycle on ARR.METHODS: We examined ARR in 103 healthy normotensive volunteers, 81 hypertensive patients who underwent ivSLT, 33 healthy women during free menstrual cycle andafter oral contraceptive therapy.RESULTS: A significantly higher proportion of normotensive women than men had an elevated ARR (13.6 versus 2.3%, P < 0.05). In 44 out of 81 hypertensive patients,diagnosis of primary aldosteronism was confirmed by ivSLT. Patients with positiveand negative ivSLT differed only for sex distribution: 85.2% of men had theprimary aldosteronism diagnosis confirmed, compared with 38.9% of women. Inhealthy women, renin and aldosterone concentrations increased from the follicularto luteal phase of menstrual period, with unchanged ARR. By contrast, reninnearly halved, aldosterone slightly decreased and ARR doubled after oralcontraceptive therapy.CONCLUSION: 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 ARRcontributing to the diagnostic inaccuracy in women

Effects of female sex hormones and contraceptive pill on the diagnostic work-up for primary aldosteronism.

OBJECTIVES: Due to the widespread use of the aldosterone to renin ratio (ARR), primary aldosteronism is currently recognized as a frequent cause of secondary hypertension. After a positive screening, primary aldosteronism diagnosis needs confirmation by an inhibitory test such as intravenous saline load (ivSLT). The aim of the present study was to investigate the role of female hormones in primary aldosteronism diagnosis, by evaluating possible differences by sex on ARR screening, on the rate of ivSLT response and analyzing the influence of free and oral contraceptive-induced menstrual cycle on ARR. METHODS: We examined ARR in 103 healthy normotensive volunteers, 81 hypertensive patients who underwent ivSLT, 33 healthy women during free menstrual cycle and after oral contraceptive therapy. RESULTS: A significantly higher proportion of normotensive women than men had an elevated ARR (13.6 versus 2.3%, P < 0.05). In 44 out of 81 hypertensive patients, diagnosis of primary aldosteronism was confirmed by ivSLT. Patients with positive and negative ivSLT differed only for sex distribution: 85.2% of men had the primary aldosteronism diagnosis confirmed, compared with 38.9% of women. In healthy women, renin and aldosterone concentrations increased from the follicular to luteal phase of menstrual period, with unchanged ARR. By contrast, renin nearly halved, aldosterone slightly decreased and ARR doubled after oral contraceptive therapy. CONCLUSION: 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

Urinary prostasin in normotensive individuals: correlation with the aldosterone to renin ratio and urinary sodium.

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\u2009nM) 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\u2009nmol 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\ub11.27 to 4.85\ub15.28\u2009nM), 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