The Phosphorylated Sodium Chloride Cotransporter in Urinary Exosomes Is Superior to Prostasin as a Marker for Aldosteronism

Abstract

Abstract-Urinary exosomes are vesicles derived from renal tubular epithelial cells. Exosomes often contain several disease-associated proteins and are thus useful targets for identifying biomarkers of disease. Here, we hypothesized that the phosphorylated (active) form of the sodium chloride cotransporter (pNCC) or prostasin could serve as biomarkers for aldosteronism. We tested this in 2 animal models of aldosteronism (aldosterone infusion or low-sodium diet) and in patients with primary aldosteronism. Urinary exosomes were isolated from 24-hour urine or spot urine using ultracentrifugation. In rats, a normal or a high dose of aldosterone for 2, 3, or 8 days increased pNCC 3-fold in urinary exosomes (PϽ0.05 for all). A low-sodium diet also increased pNCC in urinary exosomes approximately 1.5-fold after 4 and after 8 days of treatment. The effects of these maneuvers on prostasin in urinary exosomes were less clear, showing a significant 1.5-fold increase only after 2 and 3 days of high-aldosterone infusion. In urinary exosomes of patients with primary aldosteronism, pNCC was 2.6-fold higher (PϽ0.05) while prostasin was 1.5-fold higher (Pϭ0.07) than in patients with essential hypertension. Urinary exosomal pNCC and, to a lesser extent, prostasin are promising markers for aldosteronism in experimental animals and patients. These markers may be used to assess the biological activity of aldosterone and, potentially, as clinical biomarkers for primary aldosteronism. 1 Exosomes are low-density membrane vesicles that originate from multivesicular bodies. Urinary exosomes have sparked interest as potential biomarkers for human disease. 1-3The presence of urinary exosomes and a reproducible method for their isolation were reported in 2004 by Pisitkun and colleagues. 4 Proteomic analysis of these exosomes showed that they contain many disease-related proteins 4,5 ; however, the question remained whether the presence of a given protein in urinary exosomes could provide information on physiological or disease processes in the kidney. Studies addressing this question analyzed urinary exosomes in patients with monogenetic diseases, resulting in inactivity or overactivity of renal sodium transport proteins. For example, in Bartter and Gitelman syndrome, in which the sodium potassium chloride cotransporter and the sodium chloride cotransporter (NCC) are genetically inactivated, these proteins were also found to be absent or reduced in urinary exosomes. 5,6 Conversely, Mayan et al found the abundance of NCC to be increased in patients with familial hyperkalemic hypertension, in which mutations in NCC-regulating kinases cause overactivity of this cotransporter.7 Thus, in these homogeneous groups, the expression of sodium-transport proteins in urinary exosomes correlated with what one would expect from their renal expression. The next step in assessing the potential of exosomes as urinary biomarkers is to analyze their performance in acquired disease. Therefore, in this study, we asked whether various forms of aldosteronism result...

The sodium chloride cotransporter SLC12A3: new roles in sodium, potassium, and blood pressure regulation

“…Urinary exosomes are vesicles derived from renal tubular epithelial cells that are thought to reflect the metabolic profile of these cells [93]. We recently showed that the abundance of phosphorylated NCC in urinary exosomes correlated with elevated aldosterone levels in animals and humans [117]. Although this review focused on NCC, sodium excretion by the kidney depends on many other sodium transporters, including NHE3, NKCC2, ENaC, and pendrin.…”

Differential protein profiling of renal cell carcinoma urinary exosomes

“…14 Accordingly, several proteomic studies on urinary exosomes have been performed to identify biomarkers predictive of urinary track diseases, both in experimental and clinical settings. 7,13,[15][16][17][18] For an exhaustive review see Moon et al 19 However no proteomic study of urinary exosomes has been yet accomplished in RCC. Therefore, we performed MS profiling and antibody-based validation and quantification of differential proteins in urinary exosomes from a RCC patient cohort in order to search for a potential tumor marker.…”

Role of the alternative splice variant of NCC in blood pressure control

“…They contain membrane and cytosolic proteins, DNA and RNA that are conserved and protected from degradation [65,66]. The cargo of uEVs reflects changes in the expression of different proteins present in the epithelial cells of the nephron, including the total and phosphorylated forms of NCC [20,67,68]. Indeed, it has been demonstrated that patients with Gitelman syndrome display a decreased NCC abundance in uEVs [67,69].…”

“…Indeed, it has been demonstrated that patients with Gitelman syndrome display a decreased NCC abundance in uEVs [67,69]. On the other hand, patients with Gordon syndrome show higher levels of NCC in the kidney and this increased abundance was also observed in the uEVs [68]. Moreover, a recent study on kidney transplant recipients treated with calcineurin inhibiters (CNIs) suggested the use of NCC abundance in uEVs as biomarker to predict hypertensive patient’s response to thiazide diuretics [70].…”