ANG II provokes acute trafficking of distal tubule Na⁺-Cl⁻cotransporter to apical membrane

Abstract

May 16, 2007; doi:10.1152/ajprenal.00064.2007.-The distal convoluted tubule (DCT) Na ϩ -Cl Ϫ cotransporter (NCC), the target of thiazide diuretics, is responsible for the reabsorption of 5-10% of filtered NaCl. The aim of this study was to test the hypothesis that acute infusion of the angiotensin-converting enzyme (ACE) inhibitor captopril (at 12 g/min) for 20 min provokes trafficking of NCC from apical plasma membranes (APM) to subapical cytoplasmic vesicles (SCV), which is reversed by acute ANG II infusion (ANG II at 20 ng ⅐ kg Ϫ1 ⅐ min Ϫ1 along with 12 g/min captopril) for 20 min in male Sprague-Dawley rats (250 -350 g). By immuno-electron microscopy using an anti-NCC (D. Ellison) 71.5 Ϯ SD 4.9% of the NCC gold labeling was associated with the APM in control, sham operated, and infused rats, while captopril infusion reduced NCC in APM to 54.9 Ϯ 6.9% (P Ͻ 0.001) and markedly increased immunogold labeling of SCV. Subsequent infusion of ANG II with captopril restored NCC immunogold labeling of APM to 72.4 Ϯ 4.2%, that is, 20% of the total NCC trafficked between APM and SCV. Likewise, on density gradients of cortex, captopril provoked redistribution of 27.3% of total NCC from low-density APM-enriched membranes to higher-density membranes and ANG IIϩcaptopril restored 20.3% of the NCC to APM-enriched fractions. Redistribution occurred independent of a change in NCC total abundance. In conclusion, this study demonstrates that ACE inhibition provokes acute trafficking of NCC out of the plasma membrane, which likely decreases DCT Na ϩ reabsorption, while ANG II provokes rapid trafficking of NCC from stores in subapical vesicles to the plasma membrane, which likely increases DCT Na ϩ reabsorption. sodium transport; thiazide receptor; immunoelectron microscopy THE NA ϩ -CL Ϫ COTRANSPORTER (NCC) is expressed in the apical membrane of the distal convoluted tubule (DCT) and is the target of the thiazide diuretics, which are frequently used in the treatment of hypertension and edema (1, 29). The importance of NCC in the regulation of blood pressure and salt balance is demonstrated in the genetic disorder Gitelman's syndrome in which loss of function mutations in NCC results in salt wasting, hypokalemia, and hypotension (32). Studies in mice with NCC knocked out show that on low-sodium diets, blood pressure is significantly reduced from control (31).There are multiple mechanisms by which NCC could be regulated to control Na ϩ transport in the DCT. Many studies have shown that NCC abundance is regulated by stimuli, such as dietary salt, aldosterone escape, and mineralocorticoid receptor blockade (22,26,33,36), less is known about trafficking of NCC to and from the apical membrane as a way of regulating NCC. The potential importance of trafficking in the regulation of NCC has been demonstrated in vitro in Gitelman's syndrome where oocyte studies indicate that NCC is not processed properly in the endoplasmic reticulum, resulting in deficient trafficking of NCC to the plasma membrane (8,14). A previous study from this labora...

Activation of the renal Na + :Cl − cotransporter by angiotensin II is a WNK4-dependent process

“…This hypothesis was supported by observations in Xenopus oocytes in which WNK4 was required for AngII to increase the activity of NCC in a SPAK-dependent fashion and in mpkDCT cells in which AngII induced SPAK and NCC phosphorylation (19). Additionally, AngII increased the surface expression and phosphorylation of NCC by an aldosterone-independent mechanism in the distal tubule of rats (20,21,27). Supporting this proposal, we observed that renal sodium excretion and blood pressure were normal in WNK4 −/− mice, but the activity of AngII was increased, constituting the expected physiological response when a component of a negative feedback system is lost.…”

Phosphorylation Decreases Ubiquitylation of the Thiazide-sensitive Cotransporter NCC and Subsequent Clathrin-mediated Endocytosis

“…The role of phosphorylation has focused predominantly on the direct activation of the cotransporter, with increased phosphorylation resulting in greater NaCl transport activity (16,17). However, under certain conditions, NCC abundance has been shown to be increased or decreased in the apical plasma membrane of DCT cells in vivo (31,32), which presumably also modulates the total NaCl transport capacity of this nephron segment. However, very little is known about whether phosphorylation plays a role in modulating this plasma membrane targeting of NCC and whether both forward trafficking (exocytosis), retrograde transport (endocytosis), or both mechanisms are involved in maintaining the surface pool of NCC.…”

Hypertrophy in the Distal Convoluted Tubule of an 11β-Hydroxysteroid Dehydrogenase Type 2 Knockout Model

“…Dysfunction of the NaCl cotransporter (NCC) in the DCT perturbs BP not only in the extreme phenotypes of patients with Gitelman's 1 and Gordon's 2 syndromes but also in healthy individuals, 3 and the inhibition of NCC by thiazide diuretics has a potent antihypertensive effect. 4 In the short term, NCC activity is regulated at the molecular level; neurohormonal inputs converge on intracellular signaling networks (WNK-SPAK/OSR1 5 and SGK1/Nedd4-2 pathways 6 ) that shuttle NCC to and from the plasma membrane 7 and induce posttranslational protein modifications that modify transport function (phosphorylation 8 and ubiquitylation 6 ). Sustained physiologic perturbations promote structural remodeling of DCTepithelium.…”