Vasopressin regulates the renal Na+-Cl- cotransporter.

THE RENAL THIAZIDE-SENSITIVE Na -Cl cotransporter (NCC) is the major salt reabsorption pathway in the distal convoluted tubule (DCT), a region of the nephron that is located beyond the macula densa. Because of this location, salt reabsorption in DCT is not subjected to the tubular-glomerular balance mechanism, affecting the final concentration of salt in urine and, thus, arterial blood pressure. Additionally, salt reabsorption in DCT is essential to define the amount of salt delivery to the collecting duct, which is required for the Na /K exchange between the epithelial Na channel (ENaC) and the apical K channels ROMK and BK to promote K secretion regulation. These roles of NCC in blood pressure and K secretion regulation have been demonstrated extensively. On one hand, inactivating mutations of the SLC12A3 gene encoding NCC produce Gitelman’s disease that features arterial hypotension and hypokalemia. On the other hand, dysregulation of NCC by mutations in the with-no-lysine serine/threonine kinases WNK1 and WNK4 is the major mechanism producing arterial hypertension and hyperkalemia in pseudohypoaldosteronism type II (10). Thus NCC activity is critical to define arterial blood pressure levels and K secretion; therefore, a lot of attention has been given in the last years to regulatory mechanisms of this cotransporter. It is widely accepted that NCC is regulated by both aldosterone and ANG II. Aldosterone modulates NCC activity by increasing its expression (9). This is an effect that is likely to be more important for DCT2, rather than DCT1, since the early DCT lacks expression of 11 -hydroxysteroid dehydrogenase type 2 (11HSD2). ANG II, in contrast, is known to modulate NCC activity by promoting trafficking of NCC from intracellular vesicles to the plasma membrane (18), an effect that is expected to occur in both DCT1 and DCT2. It has been demonstrated in studies in vitro (13, 15, 16) and in vivo (1) that activity of NCC, mainly because of vesicular trafficking of the cotransporter to the plasma membrane, is associated with phosphorylation of the NH2-terminal domain threonines-53 and -58 and serine-71 in rat (55, 60, and 73 in human NCC). The same conserved sites become phosphorylated during activation of the Na -K -2Cl cotransporters (NKCC1 and NKCC2) that are closely related to NCC (degree of identity 50%) (2, 3, 7, 14, 16). One of these studies demonstrated that acute vasopressin administration increased the phosphorylation of NKCC2 NH2-terminal domain threonines and its trafficking toward apical membrane (7). Activation/ phosphorylation of all three cotransporters by different stimuli has been linked to signaling through at least two families of threonine/serine kinases (WNKs and the STE20 related kinases SPAK and OSR1). Evidences suggest that WNKs lie upstream of SPAK/OSR1 and that NKCC1/2 or NCC are actually phosphorylated in the conserved NH2-terminal threonines by SPAK/OSR1 (4, 6, 8, 14–17, 19, 20). One potential regulatory hormone for NCC is vasopressin. This nanopeptide hormone produced in hypothalamus and secreted in the posterior pituitary gland in response to increased plasma osmolarity or decreased blood pressure is very well known for its capacity to activate urinary concentration mechanisms and also to promote an increase in arterial pressure. Like many other hormones affecting blood pressure (ANG II, aldosterone, atrial natriuretic peptide, catecholamines, etc.), vasopressin does it by modulating both vascular smooth muscle contraction and urinary salt/water reabsorption. These effects are traduced in the smooth muscle and renal tubular cells through the G coupled receptors V1 and V2, respectively. In the kidney, vasopressin is known to modulate the activity of NKCC2, ENaC, and aquaporin 2. Its effects in DCT, however, have not been clearly demonstrated. Studies from Elalouf et al. (5) using micropuncture in homozygous diabetes insipidus (DI) Brattleboro rats strongly suggested that vasopressin increases salt reabsorption in DCT. Mutig et al. (11) demonstrated by in situ hybridization and immunohistochemistry of human, mouse, and rat kidney the presence of the V2 mRNA and protein in DCT1. The study of vasopressin on the NCC, however, has been limited by the absence of reliable cultured cell lines from DCT. In the American Journal of Physiology-Renal Physiology, the study of Mutig et al. (12) for the first time provides direct evidence of NCC regulation by vasopressin, taking advantage of the molecular tools that have been developed to study NCC regulation/phosphorylation. Short-term stimulation of NCC by vasopressin in physiological or pharmacological doses was assessed in DI Brattleboro rats 30 min after intraperitoneal injection of desmopressin (dDAVP). Using confocal microscopy, immunogold staining of NCC in electron microscopy analysis, and Western blot of vesicle-enriched fraction, the authors observed that dDAVP administration was associated with significant trafficking of NCC toward DCT apical plasma membrane. With the aid of phosphospecific antibodies recognizing phosphorylation of NCC threonine-53 or serine-71, it was demonstrated that dDAVP administration resulted in increased phosphorylation of these residues. Next, the authors observed that dDAVP was able to induce NCC serine-71 phosphorylation in renal tubule suspensions, suggesting that the vasopressin effect on DCT can be direct and not necessarily related to activation of other hormonal pathways. Finally, consistent with the previous observation that V2 receptors are more abundant in DCT1 than in DCT2 (11), it was observed that dDAVP-induced phosphorylation of NCC serine-71 was confined to DCT1, since 11HSD2-positive DCT2 cells showed no major change in pS71 NCC after dDAVP administration. Thus, although aldosterone seems to be a regulator of NCC in DCT2, vasopressin probably affects NCC activity only in Address for reprint requests and other correspondence: G. Gamba, Vasco de Quiroga No. 15, Tlalpan 14000, Mexico City, Mexico (e-mail: gamba @biomedicas.unam.mx or [email protected]). Am J Physiol Renal Physiol 298: F500–F501, 2010; doi:10.1152/ajprenal.00723.2009. Editorial Focus