Renal NHE3 and NaPi2 partition into distinct membrane domains.

Hypertension provokes differential trafficking of the renal proximal tubule Na(+)/H(+) exchanger 3 (NHE3) to the base of the apical microvilli and Na(+)-P(i) 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 +/- 2 to 142 +/- 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 +/- 9% of NHE3 localized to flotillin-enriched lipid raft domains and 69 +/- 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 +/- 10% detergent insoluble) to base (75 +/- 3%) of the apical microvilli, while NaPi2 partitioned into more insoluble domains as it moved from the microvilli (45 +/- 7% detergent insoluble) to endosomes (82 +/- 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.

Acute hypertension provokes acute trafficking of distal tubule Na-Cl cotransporter (NCC) to subapical cytoplasmic vesicles.

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.

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

The distal convoluted tubule (DCT) Na+-Cl(-) 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 microg/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.kg(-1).min(-1) along with 12 microg/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 +/- 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 +/- 6.9% (P < 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 +/- 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 II+captopril 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+ reabsorption, while ANG II provokes rapid trafficking of NCC from stores in subapical vesicles to the plasma membrane, which likely increases DCT Na+ reabsorption.