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...

Hypertension provokes differential trafficking of the renal proximal tubule Na ϩ /H ϩ exchanger 3 (NHE3) to the base of the apical microvilli and Na ϩ -Pi cotransporter 2 (NaPi2) to endosomes. The resultant diuresis and natriuresis are key to blood pressure control. We tested the hypothesis that this differential trafficking of NHE3 vs. NaPi2 was associated with partitioning to distinct membrane domains. In anesthetized rats, arterial pressure was increased (104 Ϯ 2 to 142 Ϯ 4 mmHg, 15 min) by arterial constriction and urine output increased 23-fold. Renal membranes were fractionated by cold 1% Triton X-100 extraction then centrifugation through OptiPrep flotation gradients. In controls, 84 Ϯ 9% of NHE3 localized to flotillin-enriched lipid raft domains and 69 Ϯ 5% of NaPi2 localized to transferrin receptor-enriched nonrafts. MyosinVI and dipeptidyl peptidase IV, associated with NHE3 regulation, coenriched in lipid rafts with NHE3, while NHE regulatory factor-1 coenriched in nonrafts with NaPi2. Partitioning was not altered by hypertension. Detergent insoluble membranes were pelleted after detergent extraction. NHE3 detergent insolubility decreased as it redistributed from body (80 Ϯ 10% detergent insoluble) to base (75 Ϯ 3%) of the apical microvilli, while NaPi2 partitioned into more insoluble domains as it moved from the microvilli (45 Ϯ 7% detergent insoluble) to endosomes (82 Ϯ 1%). In conclusion, NHE3 and NaPi2, while both localized to apical microvilli, are segregated into domains: NHE3 to lipid rafts and NaPi2 to nonrafts. These domain properties may play a role in the distinct trafficking patterns observed during elevated pressures: NHE3 remains in rafts and settles to the base of the microvilli while NaPi2 is freely endocytosed. subcellular fractionation; hypertension; lipid raft; kidney; proximal tubule ALTHOUGH THE CELLULAR and molecular mechanisms responsible for determining the blood pressure set point are not completely understood, it is clear that the kidney plays a central role (9,16). An acute or chronic increase in blood pressure triggers a compensatory increase in fluid and salt excretion that normalizes blood pressure, a phenomenon known as pressure diuresis and natriuresis (16,17). About two-thirds of sodium reabsorption along the nephron occurs in the proximal tubule where the apical membrane facing the lumen is highly differentiated into tall and densely packed microvilli, providing a large surface area for reabsorption. An acute increase in blood pressure causes a significant decrease in Na ϩ and water reabsorption in the proximal tubule, resulting in a 50% increase in volume flow out of this region (7, 8). The Na ϩ /H ϩ exchanger isoform 3 (NHE3) is the main transporter mediating sodium reabsorption in the proximal tubule (27,39). The sodium phosphate cotransporter 2 (NaPi2), another major sodium transporter in this region, is essential for the reabsorption of filtered phosphate (32). We have reported that during acute high blood pressure the decrease in proximal tubule salt and volume r...

When blood pressure (BP) is elevated above baseline, a pressure natriuresis-diuresis response ensues, critical to volume and BP homeostasis. Distal convoluted tubule Na(+)-Cl(-) cotransporter (NCC) is regulated by trafficking between the apical plasma membrane (APM) and subapical cytoplasmic vesicles (SCV). We aimed to determine whether NCC trafficking contributes to pressure diuresis by decreasing APM NCC or compensates for increased volume flow to the DCT by increasing APM NCC. BP was raised 50 mmHg (high BP) in rats by arterial constriction for 5 or 20-30 min, provoking a 10-fold diuresis at both times. Kidneys were excised, and NCC subcellular distribution was analyzed by 1) sorbitol density gradient fractionation and immunoblotting and 2) immunoelectron microscopy (immuno-EM). NCC distribution did not change after 5-min high BP. After 20-30 min of high BP, 20% of NCC redistributed from low-density, APM-enriched fractions to higher density, endosome-enriched fractions, and, by quantitative immuno-EM, pool size of APM NCC decreased 14% and SCV pool size increased. Because of the time lag of the response, we tested the hypothesis that internalization of NCC was secondary to the decrease in ANG II that accompanies high BP. Clamping ANG II at a nonpressor level by coinfusion of captopril (12 microg/min) and ANG II (20 ng.kg(-1).min(-1)) during 30-min high BP reduced diuresis to eightfold and prevented redistribution of NCC from APM- to SCV-enriched fractions. We conclude that DCT NCC may participate in pressure natriuresis-diuresis by retraction out of apical plasma membranes and that the retraction is, at least in part, driven by the fall in ANG II that accompanies acute hypertension.

When blood pressure (BP) is elevated above baseline, a pressure natriuresis response ensues which is critical to volume and blood pressure homeostasis. The response involves a decrease in salt and volume reabsorption from proximal tubule and a load dependent increase in salt reabsorption in loop of Henle. The role of distal nephron is less clear. Distal convoluted tubule Na+‐Cl− cotransporter (NCC) is regulated by trafficking between apical plasma membrane (APM) and sub‐apical cytoplasmic vesicles (SCV). We aimed to determine whether NCC trafficking contributes to pressure natriuresis (increase APM NCC) or compensates for increased volume flow to the DCT (decrease APM NCC). BP was raised 50 mmHg (hiBP) for 30 min in rats by arterial constriction. Kidneys were excised and NCC subcellular distribution analyzed on sorbitol density gradients by immunoblot or kidneys were perfusion fixed and NCC analyzed by immuno‐electron microscopy. After 30 min hiBP urine output increased 11‐fold. By density gradient, 20% of the total NCC redistributed from low density APM to higher density fractions (p<0.05). By quantitative immuno‐EM pool size of APM NCC decreased 13% with a corresponding increase in the SCV pool (p<0.05). In summary, the results provide evidence for an acute decrease in APM NCC during hypertension that likely contributes to pressure diuresis‐natriuresis. HL085388, DK34316, Danish NRF.

Previous studies of renal responses to dietary potassium (K) restriction compared control chow containing 0.74%NaCl with and without 2% KCl.Aimre‐examine renal responses to K restriction with 2% NaCl chow, the % found in the average American diet. Sprague Dawley rats (n=4‐6) were fed one of these diets 6 d: control chow CKCNa= 2%KCl‐0.74%NaCl, low K chow LKCNa = 0.05%KCl‐0.74%NaCl, high Na chow CKHNa= 2%KCl‐2%NaCl, low K high Na chow LKHNa= 0.05%KCl‐2%NaCl. Rats were placed in metabolic cages overnight, anesthetized for BP, sacrificed, and kidneys removed. Plasma [K] fell from 3.8 to 2.4 mM and [Na] remained at 140 mM in both LKCNa and LKHNa. Compared to LKCNa, LKHNa gained twice as much weight (6 vs 3 g/d), had lower BP (93 vs 101 mmHg) and higher urine osmolality (500 vs 325 mOsm). In renal homogenates of LKCNa vs CKCNa, as previously reported, pool size of NHE3, HK‐ATPase(g) and HK‐ATPase(c) and NaK‐ATPase(b) were increased and pool size of ROMK, ENaC(b) and AQP2 were decreased. In LKHNa vs CKHNa pool size of ROMK and ENaC(b) were reduced but NHE3, HK‐ATPases, NaK‐ATPase, AQP2 were unchanged.Conclusiondietary K restriction and/or hypokalemia provoke BP to fall and decrease ROMK and ENaC abundance which are key to conserving K. The additional physiologic and transporter changes seen with lower NaCl diet may be secondary to drastically lowering total electrolytes and/or chloride. DK34316 and UKRO fellowship to LEY.

Chronic AngII infusion raises blood pressure (BP) in mice which depends on renal AngII receptors (AT1R). AngII is anti‐natriuretic while high BP is natriuretic.Aim 1To determine how AngII infusion affects Na+ transporter abundance and phosphorylation (by immunoblot of homogenates). AngII infusion (1 μg/kg/min x 14d) increases systolic BP ~30 mmHg. Versus sham infused (=1.0), AngII decreased total NHE3 to 0.7±0.1, NHE3‐P (at Ser552) to 0.8±0.1 without altering NHE3 ‐P/total ratio. NaPi2 and myosin VI were unchanged. Distal convoluted tubule (DCT) total NCC increased to 2.2±0.3, NCC‐P (at Thr60) to 5.4 ± 1.6 and NCC‐P/total ratio to 2.2±0.5 fold.Aim 2To determine the role of proximal tubule (PT) AT1R in regulation of Na+ transporters in mice lacking PT AT1R (KO). BP decreased 10mmHg in KO vs. WT and baseline expression of NHE3, NaPi2, and NCC were unchanged in KO vs. WT. AngII infusion increased BP 20mmHg less than in WT. AngII infusion in KO decreased: total NHE3 to 0.55±0.1, NHE3‐P to 0.66±0.1 (no change in ‐P/total NHE3 ratio), myosin VI to 0.56±0.1, NaPi2 to 0.64±0.1. NCC total and NCC‐P were unchanged in the AngII infused KO.Conclusions1) increased NCC contributes to AngII induced high BP while decreased PT NHE3 compensates for high BP in WT, 2) AngII dependent increase in NCC depends on PT AT1R, and 3) the removal of PT AT1R facilitates pressure natriuresis by further decreasing PT transporters during AngII infusion.