Angiotensin II stimulates trafficking of NHE3, NaPi2, and associated proteins into the proximal tubule microvilli.

Angiotensin II (ANG II) stimulates proximal tubule (PT) sodium and water reabsorption. We showed that treating rats acutely with the angiotensin-converting enzyme inhibitor captopril decreases PT salt and water reabsorption and provokes rapid redistribution of the Na(+)/H(+) exchanger isoform 3 (NHE3), Na(+)/Pi cotransporter 2 (NaPi2), and associated proteins out of the microvilli. The aim of the present study was to determine whether acute ANG II infusion increases the abundance of PT NHE3, NaPi2, and associated proteins in the microvilli available for reabsorbing NaCl. Male Sprague-Dawley rats were infused with a dose of captopril (12 microg/min for 20 min) that increased PT flow rate approximately 20% with no change in blood pressure (BP) or glomerular filtration rate (GFR). When ANG II (20 ng x kg(-1) x min(-1) for 20 min) was added to the captopril infusate, PT volume flow rate returned to baseline without changing BP or GFR. After captopril, NHE3 was localized to the base of the microvilli and NaPi2 to subapical cytoplasmic vesicles; after 20 min ANG II, both NHE3 and NaPi2 redistributed into the microvilli, assayed by confocal microscopy and density gradient fractionation. Additional PT proteins that redistributed into low-density microvilli-enriched membranes in response to ANG II included myosin VI, DPPIV, NHERF-1, ezrin, megalin, vacuolar H(+)-ATPase, aminopeptidase N, and clathrin. In summary, in response to 20 min ANG II in the absence of a change in BP or GFR, multiple proteins traffic into the PT brush-border microvilli where they likely contribute to the rapid increase in PT salt and water reabsorption.

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.

Reducing blood pressure in SHR with enalapril provokes redistribution of NHE3, NaPi2, and NCC and decreases NaPi2 and ACE abundance.

To determine the effects of long-term angiotensin-converting enzyme inhibition (ACEI) and blood pressure (BP) lowering on renal sodium transporter abundance and distribution in spontaneously hypertensive rats (SHR), 9-wk SHR were treated with enalapril (30 mg.kg(-1).day(-1)) for 4 wk. BP decreased from 156 +/- 4 to 96 +/- 8 mmHg. Na(+)/H(+) exchanger isoform 3 (NHE3) and Na(+)-P(i) cotransporter type 2 (NaPi2) localized to the body of the microvilli (MV) in normotensive rat strains. In untreated SHR, NHE3 partially retracted from the body to base of the MV and NaPi2 retracted to subapical vesicles. After enalapril treatment of SHR, NHE3 fully retracted to the base of the MV and, by density gradient fractionation, NHE3, NaPi2, dipeptidyl peptidase IV, myosin VI, Na-Cl cotransporter, and cortical Na-K-Cl cotransporter redistributed from low-density (apical enriched) to high-density (endosome enriched) membranes. Enalapril decreased total abundance of myosin VI (to 0.51 +/- 0.18 of untreated), ACE (0.67 +/- 0.22), and cortical NaPi2 (0.83 +/- 0.10). Normalizing SHR BP with HRH (7.5 mg/day hydralazine, 0.15 mg/day reserpine, and 3 mg/day hydrochlorothiazide) did not change Na(+) transporter density distribution or abundance. We conclude that lowering BP to normal levels in SHR does not normalize Na(+) transporter distribution, rather, chronic ACEI treatment provokes retraction of Na(+) transporters and associated proteins from transport-relevant domains of apical membranes and/or reduces their abundance.

Phenol injury-induced hypertension stimulates proximal tubule Na+/H+ exchanger activity.

Injection of 50 microl 10% phenol into rat renal cortex activates renal sympathetic nerve activity which provokes acute hypertension that persists for weeks. We have previously shown with membrane fractionation that phenol injury caused a redistribution of the main proximal tubule (PT) apical transporter NHE3 (Na+/H+ exchanger isoform 3) to low density membranes enriched in apical microvilli. The aim of this study was to determine whether phenol injury increases PT apical Na+/H+ exchanger (NHE) activity. NHE activity was measured in vivo as the initial rate of change in intracellular pH (dpH(i)/dt) during luminal Na+ removal in PT preloaded with the pH-sensitive fluorescence dye BCECF. Injection of 50 microl 10% phenol increased blood pressure from 113 +/- 5.2 to 130 +/- 4.6 mmHg without changing glomerular filtration rate or urine output. NHE activity increased 2.6-fold by 70 min after phenol injury. The increase of NHE activity was accompanied with an increase of tubular reabsorption. Total NHE activity/NHE3 protein in cortical brush-border membrane (BBM) vesicles, measured by acridine orange quench and immunoblot, respectively, was unchanged by phenol injury. In conclusion, acute phenol injury provokes coincident increases in PT apical NHE activity, redistribution of NHE3 into low density apical membranes, and hypertension. The increase in NHE activity may contribute to the lack of pressure-diuresis and the maintenance of chronic hypertension in this model.

Effects of ACE inhibition on proximal tubule sodium transport.

Angiotensin-converting enzyme (ACE) inhibitors such as captopril, which block ANG II formation, are commonly used for treatment of hypertension. There is substantial evidence that the proximal tubule (PT) is a primary target site for captopril but the molecular mechanisms for its action in PT are not well defined. The aim of this study was to determine the physiological and molecular changes in PT provoked by acute captopril treatment in the absence of changes in blood pressure or glomerular filtration rate (GFR). Captopril (infused at 12 microg/min for 20 min) did not change blood pressure or GFR but induced an immediate (<10 min) increase in PT flow measured with a nonobstructive optical method (to 117 +/- 14% of baseline) along with a rapid diuresis from 2.1 +/- 0.6 mg/min (baseline) to 3.7 +/- 0.9 mg/min (captopril). Captopril also provoked a significant retraction of PT Na(+)/H(+) exchanger isoform 3 (NHE3), NHE regulatory factor (NHERF)-1, myosin-VI, and Na(+)-P(i) cotransporter type 2 (NaPi2), but not ACE, out of apical microvillus-enriched membranes. Proteomic analysis with MALDI-TOF MS revealed an additional eight abundant membrane-associated proteins that redistributed out of the microvillus-enriched membrane during captopril treatment: megalin, myosin II-A, clathrin, aminopeptidase N, DPPIV, ezrin, moesin, and vacuolar H(+)-ATPase subunit beta(2). In summary, captopril can rapidly depress PT reabsorption in the absence of a change in GFR or BP and provokes the redistribution of a set of transporters and transporter-associated proteins that likely participate in the decrease in PT reabsorption and may also contribute to the blood pressure-lowering effect of ACE inhibitors.

Modest dietary K+ restriction provokes insulin resistance of cellular K+ uptake and phosphorylation of renal outer medulla K+ channel without fall in plasma K+ concentration.

Extracellular K(+) concentration ([K(+)]) is closely regulated by the concerted regulatory responses of kidney and muscle. In this study, we aimed to define the responses activated when dietary K(+) was moderately reduced from a control diet (1.0% K(+)) to a 0.33% K(+) diet for 15 days. Although body weight and baseline plasma [K(+)] (4.0 mM) were not reduced in the 0.33% K(+) group, regulatory responses to conserve plasma [K(+)] were evident in both muscle and kidney. Insulin-stimulated clearance of K(+) from the plasma was estimated in vivo in conscious rats with the use of tail venous and arterial cannulas. During infusion of insulin.(50 mU.kg(-1).min(-1)), plasma [K(+)] level fell to 3.2 +/- 0.1 mM in the 1.0% K(+) diet group and to only 3.47 +/- 0.07 mM in the 0.33% K(+) diet group (P < 0.01) with no reduction in urinary K(+) excretion, which is evidence of insulin resistance to cellular K(+) uptake. Insulin-stimulated cellular K(+) uptake was quantitated by measuring the K(+) infusion rate necessary to clamp plasma K(+) at baseline (in micromol.kg(-1).min(-1)) during 5 mU of insulin.kg(-1).min(-1) infusion: 9.7 +/- 1.5 in 1% K(+) diet was blunted to 5.2 +/- 1.7 in the 0.33% K(+) diet group (P < 0.001). Muscle [K(+)] and Na(+)-K(+)-ATPase activity and abundance were unchanged during the 0.33% K(+) diet. Renal excretion, which was measured overnight in metabolic cages, was reduced by 80%, from 117.6 +/- 10.5 micromol/h/animal (1% K(+) diet) to 24.2 +/- 1.7 micromol/h/animal (0.33% K(+) diet) (P < 0.001). There was no significant change in total abundance of key renal K(+) transporters, but 50% increases in both renal PTK cSrc abundance and ROMK phosphorylation in the 0.33% K(+) vs. 1% K(+) diet group, previously established to be associated with internalization of ROMK. These results indicate that plasma [K(+)] can be maintained during modest K(+) restriction due to a decrease in insulin-stimulated cellular K(+) uptake as well as renal K(+) conservation mediated by inactivation of ROMK, both without a detectable change in plasma [K(+)]. The error signals inciting and maintaining these responses remain to be identified.

Redistribution of myosin VI from top to base of proximal tubule microvilli during acute hypertension.

During acute hypertension, Na(+)/H(+) exchangers (NHE3) retract from top to base of proximal tubule microvilli (MV) and Na(+) reabsorption decreases in proximal tubule. This study aimed to determine whether the actin-based motor myosin VI coordinately retracts with NHE3 in response to acute hypertension. BP was raised approximately 50 mmHg in rats for 20 to 30 min or sham treated, and kidneys were analyzed by subcellular fractionation or microscopy. During acute hypertension, myosin VI redistributed from low density apical MV-enriched membranes (from 23 +/- 2.4 to 11.4 +/- 2.2%) into higher density membranes (from 23.2 +/- 0.7 to 36.9 +/- 2.6%). By confocal microscopy, myosin VI was detected over the whole length of the MV in controls, then became completely focused at the base of MV during acute hypertension. For electron microscopic analysis using immunogold labeling, MV were divided into five zones from top (z1) to base (z5). In controls, myosin VI was evenly distributed through the five MV zones. In acute hypertension, myosin VI decreased in z1 (from 20.6 +/- 1.9 to 10.5 +/- 2.3%) and z2 (from 21.0 +/- 2.0 to 13.2 +/- 1.4%) and increased in z5 (from 21.1 +/- 3.3 to 38.6 +/- 3.0%). These results provide the first observation that acute hypertension causes myosin VI redistribution and support the idea that myosin VI may serve as the molecular motor for NHE3 retraction from top to base of MV during acute hypertension.

Differential traffic of proximal tubule Na+ transporters during hypertension or PTH: NHE3 to base of microvilli vs. NaPi2 to endosomes.

We previously reported that Na(+)/H(+) exchanger type 3 (NHE3) and NaPi2 are acutely retracted from the proximal tubule (PT) microvilli (MV) during acute hypertension [high blood pressure (BP)] or parathyroid hormone (PTH) treatment. By subcellular membrane fractionation, NHE3 and NaPi2 show indistinguishable redistribution patterns out of light-density into heavy-density membranes in response to either treatment consistent with a retraction from the apical MV to the intermicrovillar cleft region. This study aimed to examine the redistribution of PT NHE3 vs. NaPi2 by confocal and electron microscopy during high BP and during PTH treatment to determine whether their respective destinations overlap or are distinct. High-BP protocol: systolic BP was increased 50-60 mmHg by increasing peripheral resistance for 20 min; PTH protocol: rats were infused with 6.6 microg/kg iv of PTH followed by 0.1 microg.kg(-1).min(-1) infusion for 1 h. For light microscopy, rats were infused with 25 mg of horseradish peroxidase (HRP) 10 min before kidney fixation. Kidney slices were dual labeled with either NHE3 or NaPi2 and either clathrin-coated vesicle adaptor protein AP2 or endosome marker HRP. The results demonstrate retraction of NHE3 from the MV to the base of MV during either high-BP or PTH treatment: NHE3 staining did not retract below the AP2-stained domain or to HRP-labeled endosomes in either model. In comparison, NaPi2 was retracted from MV to below the AP2-stained region in both models, a little colocalizing with HRP staining. At the electron microscopic level with immunogold labeling, during high BP NHE3 was concentrated in a distinct domain in the base of the MV while NaPi2 moved to endosomes. The results demonstrate that there are divergent routes of retraction of PT NHE3 and NaPi2 from the MV during acute hypertension or PTH treatment: NHE3 is not internalized but remains at the base of the MV while NaPi2 is internalized.

Acute hypotension induced by aortic clamp vs. PTH provokes distinct proximal tubule Na+ transporter redistribution patterns.

Renal parathyroid hormone (PTH) action is often studied at high doses (100 microg PTH/kg) that lower mean arterial pressure significantly, albeit transiently, complicating interpretation of studies. Little is known about the effect of acute hypotension on proximal tubule Na(+) transporters. This study aimed to determine the effects of acute hypotension, induced by aortic clamp or by high-dose PTH (100 microg PTH/kg), on renal hemodynamics and proximal tubule Na/H exchanger isoform 3 (NHE3) and type IIa Na-P(i) cotransporter protein (NaPi2) distribution. Subcellular distribution was analyzed in renal cortical membranes fractionated on sorbitol density gradients. Aortic clamp-induced acute hypotension (from 100 +/- 3 to 78 +/- 2 mmHg) provoked a 62% decrease in urine output and a significant decrease in volume flow from the proximal tubule detected as a 66% decrease in endogenous lithium clearance. There was, however, no significant change in glomerular filtration rate (GFR) or subcellular distribution of NHE3 and NaPi2. In contrast, high-dose PTH rapidly (<2 min) decreased arterial blood pressure to 51 +/- 3 mmHg, decreased urine output, and shifted NHE3 and NaPi2 out of the low-density membranes enriched in apical markers. PTH at much lower doses (<1.4 microg.kg(-1).h(-1)) did not change blood pressure and was diuretic. In conclusion, acute hypotension per se increases proximal tubule Na(+) reabsorption without changing NHE3 or NaPi2 subcellular distribution, indicating that trafficking of transporters to the surface is not the likely mechanism; in comparison, hypotension secondary to high-dose PTH blocks the primary diuretic effect of PTH but does not inhibit the PTH-stimulated redistribution of NHE3 and NaPi2 to the base of the microvilli.

Mechanisms of pressure natriuresis: how blood pressure regulates renal sodium transport.

An acute increase in blood pressure provokes a rapid decrease in proximal tubule salt and water reabsorption that is central to tubuloglomerular feedback regulation of renal blood flow and glomerular filtration rate and contributes to pressure natriuresis. The molecular mechanisms responsible for this critical homeostatic adjustment were studied. When blood pressure is acutely elevated, apical proximal tubule NHE3 are rapidly redistributed out of the microvilli to intermicrovillar clefts and then endosomal pools, and Na,K-ATPase activity is suppressed. Depressing apical Na(+) entry without hypertension is not sufficient to decrease Na,K-ATPase activity, and depressing Na,K-ATPase activity alone is not sufficient to decrease proximal tubule Na(+) and water reabsorption; thus, it appears that coordinated decreases in both NHE3 surface distribution and Na,K-ATPase activity may be important for the response to acute hypertension. Clamping plasma angiotensin II levels blunts the retraction of NHE3 from the cell surface to endosomal pools. The increased volume flow of salt and water to the loop of Henle stimulates Na,K-ATPase activity in this region and provides evidence for a downstream shift in sodium transport during acute hypertension. These same responses in the proximal tubule and loop develop and persist in the spontaneously hypertensive rat. These studies demonstrate that sodium transporters along the nephron are very dynamic, responding quickly to normal fluctuations of blood pressure, and are key to generating the macula densa tubuloglomerular feedback signal and for accommodating increased volume flow through the loop of Henle.

Chronic renal injury-induced hypertension alters renal NHE3 distribution and abundance.

Renal cortical phenol injection provokes acute sympathetic nervous system-dependent hypertension and a shift of proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3) and Na(+)-P(i) cotransporter type 2 (NaPi2) to apical microvilli. This study aimed to determine whether proximal tubule (PT) Na(+) transporter redistribution persists chronically and whether the pool sizes of renal Na(+) transporters are altered. At 5 wk after a 50-microl 10% phenol injection, blood pressure is elevated: 154 +/- 8 vs. 113 +/- 11 mmHg after saline injection. Cortical membranes were fractionated into three "windows" enriched in apical brush border (WI), mixed apical and intermicrovillar cleft (WII), and intracellular membranes (WIII). NHE3 relative distribution in these windows, assessed by immunoblots and expressed as %total, remained shifted to apical from intracellular membranes (WI: 25.3 +/- 3 in phenol vs.12.7 +/- 3% in saline and WIII: 9.1 +/- 1.3 in phenol vs. 18.9 +/- 3% in saline). NaPi2 and dipeptidyl-peptidase IV also remained shifted to WI, and alkaline phosphatase activity increased 100.9 +/- 29.7 (WI) and 51.4 +/- 17.5% (WII) in phenol-injected membranes. Na(+) transporter total abundance [NHE3, NaPi2, thiazide-sensitive Na-Cl cotransporter, bumetanide-sensitive Na-K-2Cl cotransporter, Na-K-ATPase alpha(1)- and beta(1)-subunits, and epithelial Na(+) channel (ENaC) alpha- and beta-subunits] was profiled by immunoblotting. Only cortical NHE3 abundance was altered, decreasing to 0.56 +/- 0.06. The results demonstrate that phenol injury provokes a persistant shift of PT NHE3 and NaPi2 to the apical microvilli, along with a 44% decrease in total NHE3, evidence for an escape mechanism that would counteract the redistribution of a larger fraction of NHE3 to the apical surface by normalizing the total amount of NHE3 in apical membranes.

Responses of proximal tubule sodium transporters to acute injury-induced hypertension.

Renal injury-induced by phenol injection activates renal sympathetic afferent pathways, increases norepinephrine release from the posterior hypothalamus, activates renal efferent pathways, and provokes a rapid and persistent hypertension. This study aimed to determine whether phenol injury provoked a redistribution of proximal Na(+) transporters from internal stores to the apical cell surface mediated by sympathetic activation, a response that could contribute to generation or maintenance of hypertension. Anesthetized rats were cannulated for arterial blood pressure tracing and saline infusion and then 50 microl 10% phenol or saline was injected into one renal cortex (n = 7 each). Fifty minutes after injection, kidneys were removed and renal cortex membranes from injected kidneys were fractionated on sorbitol gradients and pooled into three windows (WI-WIII) that contained enriched apical brush border (WI); mixed apical, intermicrovillar cleft and dense apical tubules (WII); and intracellular membranes (WIII). Na(+) transporter distributions were determined by immunoblot and expressed as percentage of total in gradient. Acute phenol injury increased blood pressure 20-30 mmHg and led to redistribution of Na(+)/H(+) exchanger type 3 (NHE3) out of WIII (from 22.79 +/- 4.75 to 10.79 +/- 2.01% of total) to WI (13.07 +/- 1.97 to 27.15 +/- 4.08%), Na(+)-P(i) cotransporter 2 out of WII (68.72 +/- 1.95 to 59.76 +/- 2.21%) into WI (9.5 +/- 1.62 to 18.7 +/- 1.45%), and a similar realignment of dipeptidyl-peptidase IV immunoreactivity and alkaline phosphatase activity to WI. Renal denervation before phenol injection prevented the NHE3 redistribution. By confocal microscopy, NHE3 localized to the brush border after phenol injection. The results indicate that phenol injury provokes redistribution of Na(+) transporters from intermicrovillar cleft/intracellular membrane pools to apical membranes associated with sympathetic nervous system activation, which may contribute to phenol injury-induced hypertension.

Angiotensin II clamp prevents the second step in renal apical NHE3 internalization during acute hypertension.

Acute hypertension inhibits proximal tubule (PT) sodium reabsorption. The resultant increase in NaCl delivery to the macula densa suppresses renin release. We tested whether the sustained pressure-induced inhibition of PT sodium reabsorption requires a renin-mediated decrease in ANG II levels. Plasma ANG II concentration of anesthesized Sprague-Dawley rats was clamped by simultaneous infusion of the ANG I-converting enzyme inhibitor captopril (12 microg/min) and ANG II (20 ng. kg(-1). min(-1)). Blood pressure was increased 50 mmHg for 20 min by arterial constriction +/- ANG II clamp or by sham operation. This acute hypertension increased urine output and endogenous Li(+) clearance, and these responses were blunted 40-50% in ANG II clamped rats. Acute hypertension provoked a rapid redistribution of Na(+)/H(+) exchanger isoform 3 (NHE3) out of apical brush-border membranes (21 +/- 4% decrease of total NHE3 abundance) to endosomal/lysosomal membranes (16 +/- 6% increase of total). In ANG II-clamped rats, acute hypertension also provoked disappearance of NHE3 from the apical membranes (27 +/- 2% decrease of total), but NHE3 was shifted to membranes enriched in intermicrovillar cleft and dense apical tubules (step 1) rather than endosomal/lysosomal membranes (step 2). This difference was independently confirmed by confocal analysis. In contrast, the pressure-induced redistribution of Na(+)-P(i) cotransporter type 2 was not altered by ANG II clamp. We conclude that the responses to acute hypertension, including diuresis and redistribution of PT NHE3 into intracellular membranes, require a responsive renin-angiotensin system and that the responses may be induced by the sustained increase in NaCl delivery to the macula densa during acute hypertension.

Diuretic response to acute hypertension is blunted during angiotensin II clamp.

Acute hypertension inhibits proximal tubule (PT) fluid reabsorption. The resultant increase in end proximal flow rate provides the error signal to mediate tubuloglomerular feedback autoregulation of renal blood flow and glomerular filtration rate and suppresses renal renin secretion. To test whether the suppression of the renin-angiotensin system during acute hypertension affects the magnitude of the inhibition of PT fluid and sodium reabsorption, plasma ANG II levels were clamped by infusion of the angiotensin-converting enzyme (ACE) inhibitor captopril (12 microg/min) and ANG II after pretreatment with the bradykinin B(2) receptor blocker HOE-140 (100 microg/kg bolus). Because ACE also degrades bradykinin, HOE-140 was included to block effect of accumulating vasodilatory bradykinins during captopril infusion. HOE-140 increased the sensitivity of arterial blood pressure to ANG II: after captopril infusion without HOE-140, 20 ng x kg(-1) x min(-1) ANG II had no pressor effect, whereas with HOE-140, 20 ng x kg(-1) x min(-1) ANG II increased blood pressure from 104 +/- 4 to 140 +/- 6 mmHg. ANG II infused at 2 ng x kg(-1) x min(-1) had no pressor effect after captopril and HOE-140 infusion ("ANG II clamp"). When blood pressure was acutely increased 50-60 mmHg by arterial constriction without ANG II clamp, urine output and endogenous lithium clearance increased 4.0- and 6.7-fold, respectively. With ANG II clamp, the effects of acute hypertension were reduced 50%: urine output and endogenous lithium clearance increased two- and threefold, respectively. We conclude that HOE-140, an inhibitor of the B(2) receptor, potentiates the sensitivity of arterial pressure to ANG II and that clamping systemic ANG II levels during acute hypertension blunts the magnitude of the pressure diuretic response.

Acute hypertension provokes internalization of proximal tubule NHE3 without inhibition of transport activity.

Acute hypertension rapidly decreases proximal tubule (PT) Na(+) reabsorption, facilitated by a redistribution of PT Na(+)/H(+) exchangers (NHE3) out of the apical brush border, increasing NaCl at the macula densa, the signal for autoregulation of renal blood flow and GFR. This study aimed to determine whether NHE3 activity per transporter decreases during acute hypertension and the time dependence of the response. Blood pressure was elevated by 50-60 mmHg in male Sprague-Dawley rats for 5 or 30 min by constricting arteries. Renal cortical membranes were fractionated by density gradient centrifugation. NHE3 transport activity was assayed as the rate of appearance of acridine orange (AO) from AO-loaded vesicles in response to an inwardly directed Na(+) gradient. After 5-min hypertension, 20% of total NHE3 protein, assayed by immunoblot, redistributed from low-density apical membranes to middensity membranes enriched in intermicrovillar cleft markers; by 30 min, a similar percentage shifted to heavier density membranes containing markers of endosomes. NHE3 activity shifted to higher density membranes along with NHE3 protein, that is, no change in activity/transporter during acute hypertension. Confocal analysis of NHE3 distribution also verified removal from apical microvilli and appearance in subapical vesicles. We conclude that the decrease in renal PT Na(+) transport during acute hypertension is mediated by removal of transport-competent NHE3 from the apical brush border to subapical and internal reserves.