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Am J Physiol Cell Physiol 274: C1090-C1100, 1998;
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Vol. 274, Issue 4, C1090-C1100, April 1998

Reversible effects of acute hypertension on proximal tubule sodium transporters

Yibin Zhang1, Clara E. Magyar1, John M. Norian1, Niels-H. Holstein-Rathlou2, Austin K. Mircheff1, and Alicia A. McDonough1

1 Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033; and 2 Department of Medical Physiology, The Panum Institute, DK-2200 Copenhagen N, Denmark

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Acute hypertension provokes a rapid decrease in proximal tubule sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of membranes containing apical membrane sodium/hydrogen exchangers (NHE3) [Y. Zhang, A. K. Mircheff, C. B. Hensley, C. E. Magyar, D. G. Warnock, R. Chambrey, K.-P. Yip, D. J. Marsh, N.-H. Holstein-Rathlou, and A. A. McDonough. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F1004-F1014, 1996]. To determine the reversibility and specificity of these responses, rats were subjected to 1) elevation of blood pressure (BP) of 50 mmHg for 5 min, 2) restoration of normotension after the first protocol, or 3) sham operation. Systolic hypertension increased urine output and endogenous lithium clearance three- to fivefold within 5 min, but these returned to basal levels only 15 min after BP was restored. Renal cortex lysate was fractionated on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase activity (but not subunit immunoreactivity) decreased one-third to one-half after BP was elevated and recovered after BP was normalized. After BP was elevated, 55% of the apical NHE3 immunoreactivity, smaller fractions of sodium-phosphate cotransporter immunoreactivity, and apical alkaline phosphatase and dipeptidyl-peptidase redistributed to membranes of higher density enriched in markers of the intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5), whereas density distributions of the apical cytoskeleton protein villin were unaltered. After 20 min of normalized BP, all the NHE3 and smaller fractions of the other apical membrane proteins returned to their original distributions. These findings suggest that the dynamic regulation of proximal tubule sodium transport by acute changes in BP may be mediated by rapid reversible regulation of sodium pump activity and relocation of apical sodium transporters.

sodium-potassium-adenosinetriphosphatase; NHE3; membrane trafficking

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

ACUTE INCREASES IN arterial pressure elicit rapid natriuretic and diuretic responses that occur in the absence of changes in renal blood flow (RBF) or glomerular filtration rate (GFR) (18). This autoregulation of RBF and GFR is mediated by an increase in volume flow to the macula densa, which provokes an increase in afferent arteriolar resistance. The increased volume flow at the macula densa during acute hypertension, in the face of a constant GFR, is due, at least in part, to a very rapid (within 1.5-2 min) inhibition of salt and water reabsorption in the proximal tubule (11, 12, 23); the mediating signals remain to be determined.

Active sodium reabsorption across the proximal tubule is mediated primarily by apical entry via sodium/hydrogen exchangers (NHE3) and extrusion via basolateral sodium pumps (Na-K-ATPase). The rapid decrease in sodium transporter activity in response to acute hypertension may be due to 1) decreased activity of transporters in the apical and/or basolateral plasma membranes, 2) trafficking of transporters from plasma membranes to endosomal stores, or 3) rapid degradation of transporters. There is evidence for all three types of transport regulation in the proximal tubule: 1) phosphorylation of sodium pumps has been reported to change ATPase and transport activity (3, 4), 2) there is evidence for trafficking of apical membrane proteins between the brush border and a large pool of subapical endosomes (33), as well as reversible wholesale internal retraction of microvilli with ATP depletion and repletion (16), and 3) apical membrane sodium-phosphate (Na-Pi) cotransporters are internalized and degraded following acute high-phosphate diet (26).

We recently reported that during a 5-min arterial hypertension Na-K-ATPase catalytic activity in the basolateral membranes decreased and the density of membranes containing NHE3 increased (47). In this study, we test the hypothesis that the responses are reversible when normal blood pressure is restored, and we examine the specificity of the NHE3 redistribution. The findings demonstrate that transport returns to control levels by 10-15 min after normalization of blood pressure and suggest that the dynamic regulation of proximal tubule sodium transport by fluctuations in blood pressure may be mediated by changes in sodium transporter characteristics at both the apical and basolateral membranes via 1) reversible inhibition of basolateral Na-K-ATPase activity and 2) relocation of a set of apical proteins, including NHE3 and Na-Pi but not villin, consistent with redistribution to intermicrovillar cleft region and/or internalization to endosomal pools.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Animal preparations. Experiments were performed on male Sprague-Dawley rats (300-350 g body wt) that had free access to food and water before the experiment. Rats were anesthetized intramuscularly with ketamine (Fort Dodge Laboratories) and xylazine (Miles; 1:1, vol/vol) and then placed on a thermostatically controlled warming table to maintain body temperature at 37°C. Polyethylene catheters were placed into the carotid artery for monitoring blood pressure, into the right jugular vein for infusion of 0.9% NaCl at 50 µl/min during the entire experimental period to maintain euvolemia, and into the ureter for urine collection.

Three groups of rats (n = 5 each) were compared: 1) control (sham operated), 2) hypertension (5 min of acute systolic hypertension), and 3) restored or restoration (normalizing blood pressure to control after 5 min of acute hypertension). To induce acute hypertension, the total peripheral resistance was increased as suggested by Roman and Cowley (38), without hormone infusion. Mean arterial pressure was increased 40-50 mmHg over basal levels for 5 min by constricting the superior mesenteric artery, celiac artery, and abdominal aorta below the renal artery with Schwantz vascular clamps (no. 18052-01, Fine Science Tool). Blood pressure was restored to basal levels by releasing clamps around arteries. Control sham-operated rats were processed in parallel, with arteries dissected but not constricted.

Urine collection and endogenous lithium clearance. Urine volume, collected from the ureter catheter, was determined gravimetrically. A blood sample was collected after the kidneys were removed. The concentrations of endogenous lithium in blood and urine samples were measured by flameless atomic absorption spectrophotometry (Perkin-Elmer 5100PC) as described previously (24).

Homogenization, differential sedimentation, and density gradient centrifugation. Kidneys were cooled in situ before excision by flushing the abdominal cavity with ice-cold PBS solution to block further membrane trafficking. After excision, the renal cortices were rapidly dissected in isolation buffer (5% sorbitol, 0.5 mM Na2EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 9 µg/ml aprotinin, and 5 mM histidine-imidazole buffer, pH 7.5). The procedure for subcellular fractionation of the renal cortex membranes has been described in detail previously (20, 21, 47). Briefly, cortex was homogenized in two rounds with a Tissuemiser (Tekmar Instrument) for 10 min at a thyristor setting of 45 and centrifuged at 2,000 g for 10 min. The two low-speed supernatants (So) were pooled, loaded at the interface between two hyperbolic sorbitol gradients (ranging between 35 and 70% sorbitol), and centrifuged in a swinging bucket rotor (100,000 g for 5 h). Twelve fractions were collected with a Buchler AutoDensi Flow apparatus from the top, and each fraction was diluted with isolation buffer, pelleted by centrifugation (250,000 g for 1.5 h), resuspended in 1 ml isolation buffer, and stored at -80°C, pending assays.

Phase partitioning. Methods for phase partitioning are described in detail elsewhere (30). Phase systems containing 5% dextran T-500 (Pharmacia, Piscataway, NJ), 3.5% polyethylene glycol (Carbowax 8000, Union Carbide, Danbury, CT), 5 mM sorbitol, 10 µM Na2EDTA, and 8.33 mM imidazole, pH adjusted to 7.3 with HCl, were prepared the day before use. Analyses were performed in an Albertsson thin-layer counter-current distribution apparatus. Samples were suspended in the upper phase and added to chambers 1 and 2. After 18 transfers, contents of adjacent chambers were pooled, producing 10 fractions. Thus membranes that partitioned into the stationary, dextran-rich phase remained near the origin, i.e., fraction 1, whereas membranes that partitioned into the mobile, polyethylene glycol-rich phase migrated toward fraction 10. Membranes were sedimented by centrifugation at 250,000 g for 75 min, resuspended, and analyzed in the same way as the density gradient fractions.

Na-K-ATPase and enzymatic marker measurements. Na-K-ATPase activity was measured by the potassium-dependent p-nitrophenylphosphatase (K-pNPPase) reaction (32), since our previous analysis demonstrated indistinguishable distribution patterns for K-pNPPase activity and ouabain-sensitive ATPase activity in kidney cortex. Standard assays were used for alkaline phosphatase (31) and protein (27).

Immunoblot analysis and antibodies. A constant volume of sample from each gradient fraction was prepared in SDS-PAGE sample buffer (final concentration: 2% SDS, 1% beta -mercaptoethanol, 0.25 mM Na2EDTA, and 2.5 mM H2PO4-HPO4 buffer, pH 7.0), which was denatured for 30 min at 37°C, resolved on 7.5% SDS polyacrylamide gels, and transferred to polyvinylidene difluoride membranes according to standard methods. The antibody incubation protocol has been detailed previously (1). The monoclonal antibody specifically against the rat Na-K-ATPase alpha 1-subunit (464.6), generously provided by M. Kashgarian (Yale), was used at 1:200 dilution, and a polyclonal anti-rat beta 1 fusion protein, generated in our lab, was used at 1:500 dilution. An NHE3 monoclonal cell (2B9) culture supernatant provided by D. Biemesderfer and P. Aronson (Yale) (7, 45) was used without dilution on blots and detected with an enhanced chemiluminescence kit (from Amersham). Monoclonal antibody to villin was obtained from Immunotech, used at 1:1,000, and detected with 125I-labeled protein A. Polyclonal antiserum to the Na-Pi cotransporter from F. Ghishan (University of Arizona) (14, 42) and polyclonal antisera to dipeptidyl-peptidase IV (DPPIV) and megalin [provided by M. Farquhar (University of California at San Diego)] and against Rab 5a and Rab 4 [obtained from Santa Cruz Biotechnology (Santa Cruz, CA)] were all used at 1:1,000 dilutions. These antibody-antigen complexes were detected with 125I-protein A (ICN). The resulting autoradiographic signals were quantified with a Bio-Rad imaging densitometer with molecular analyst software. Multiple exposures of autoradiograms were analyzed to ensure that signals were within the linear range of the film.

Quantitation and statistical analysis. Data are expressed as means ± SE. ANOVA was applied to determine whether there was a significant effect of treatment on the overall fractionation pattern of a given parameter. Treatment was one repeated factor, and fraction was another repeated factor. If the interaction between treatment and fraction was found to be significant (P < 0.05), it was concluded that the treatment had a significant effect. If so, the location of the difference in the pattern was assessed by two-tailed Student's t-test for paired samples, and differences were regarded to be significant at P < 0.05.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Physiological responses. Figure 1 summarizes the mean arterial blood pressure, urine output, and endogenous lithium clearance following constriction of the celiac artery, superior mesenteric artery, and the abdominal aorta and after the subsequent release of arterial constriction. Arterial blood pressure increased immediately by 50 mmHg above control level when arteries were constricted. This pressure is within the range in which GFR and RBF are autoregulated (11, 12) during similar protocols. During the 5 min of acute hypertension, urine output increased 4.8 ± 0.6-fold, and endogenous lithium clearance, an inverse measure of proximal tubule sodium reabsorption (43), increased 2.9 ± 0.3-fold. After release of constriction around the arteries, blood pressure returned immediately to the basal level of 100 mmHg. Recovery of sodium reabsorption, indicated by endogenous lithium clearance and urine output, lagged behind, returning to basal levels by 15 min after the return to normal blood pressure. The persistent elevation of urine output and endogenous lithium clearance after blood pressure was normalized indicates that either the physical stimulus of elevated blood pressure is not itself the signal that alone depresses sodium transport and that chemical mediators that persist after pressure restoration are likely involved or, alternatively, that reversing the modifications or redistribution in the transporters requires 15 min. On the basis of this time course, we chose a time point of 20 min after blood pressure restoration to analyze the restoration response.


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  		Fig. 1.   Physiological responses to constricting and then releasing clamps around celiac and superior mesenteric arteries and abdominal aorta as a function of time, where 0-5 min is acute hypertension duration. A: arterial pressure, recorded from carotid artery. B: urine output collected over 5-min intervals, expressed as urine weight in µg/5-min collection period. C: endogenous lithium clearance calculated for each 5-min collection period as urinary Li+ concentration · urine output · plasma Li+ concentration-1 (µl/min). Data are expressed as means ± SE, n = 5. * P < 0.05 vs. control period by paired Student's t-test.

Response of renal cortex Na-K-ATPase to acute hypertension and blood pressure restoration. The sodium pump drives active transepithelial sodium reabsorption and transports sodium ions from the cell into the extracellular fluid. We previously established that Na-K-ATPase activity decreases in response to acute hypertension (47). We aimed to determine if activity returned to control levels when normotension was restored. Figure 2 summarizes the subcellular distribution of Na-K-ATPase activity in renal cortex membrane fractions from control, acute hypertension, and restored protocols, measured under maximal reaction velocity conditions. The peak of Na-K-ATPase activity, the traditional marker for location of basolateral membranes, was between fractions 3 and 5. Acute hypertension did not change the density distribution pattern of Na-K-ATPase activity (Fig. 2A) but did decrease Na-K-ATPase activity by one-third in the basolateral peak region of the gradient, not in other regions with Na-K-ATPase activity (fractions 6-12) consistent with our previous findings (47). After blood pressure was restored for 20 min, Na-K-ATPase activity increased significantly in fractions 3-5 above the activity of samples taken during acute hypertension, although activity was not completely restored. When assayed in the cortex sample before fractionation, the So Na-K-ATPase activity decreased ~30% during 5 min of hypertension and returned to control levels after 20-min blood pressure restoration (Table 1).


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  		Fig. 2.   Distribution of Na-K-ATPase activity in membrane fractions of renal cortex from control, acute hypertension, and blood pressure-restored protocols following density gradient fractionation. Na-K-ATPase activity was assessed by measuring p-nitrophenylphosphatase (pNPPase) activity as described in EXPERIMENTAL PROCEDURES. Na-K-ATPase activity distribution, expressed as percentage of the total in the gradient, was unaltered by acute systolic hypertension. B: Na-K-ATPase-specific activity, corrected for total protein in the 12 fractions, was decreased by acute hypertension in the peak basolateral membrane fractions. Results are expressed as means ± SE, n = 5 in each group. * P < 0.05 vs. control and + P < 0.05 vs. acute hypertension, assessed by ANOVA and followed by paired t-test.

                              
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  		Table 1.   Apical membrane markers and Na-K-ATPase in So fraction

Regarding the specificity of the inhibition of enzyme activity, acute hypertension also significantly decreased activity of the apical membrane marker alkaline phosphatase by 40% but did not decrease the activity of the apical enzyme DPPIV (Table 1). Blood pressure restoration returned alkaline phosphatase enzyme activity to control levels. These results demonstrate that both the basolateral Na-K-ATPase and apical alkaline phosphatase activities are affected by alterations in blood pressure in a manner that persists through membrane isolation, supporting the hypothesis that during hypertension these membrane proteins (or their regulators) undergo reversible structural modification or translocation to lipid domains where their activity is decreased and that the decreases are not due to degradation.

We previously reported that there was a significant, albeit minor, redistribution of Na-K-ATPase subunits to heavier densities during 5 min of acute hypertension that would contribute to the decrease in ATPase activity in fractions 3-5. In this study, we again measured Na-K-ATPase alpha - and beta -subunit immunoreactivity after control, hypertension, and blood pressure-restored protocols (Fig. 3). Compared with the peak in Na-K-ATPase activity profile in Fig. 2, alpha  immunoreactivity is broadly distributed near 100 kDa between fractions 3 and 10, whereas the beta  immunoreactivity pattern near 50 kDa has a distinct peak between fractions 3 and 6, similar to ATPase activity. In this series of experiments, no significant decrease in immunoreactive alpha - or beta -subunits in fractions 3-5 occurred that would account for the corresponding decrease in enzymatic activity. The difference between these and the previous findings may reflect a subtle difference in methodology: in our previous study, animals were killed directly after 5 min of hypertension without chronic infusion, whereas in this study all of the animals were infused, as described in EXPERIMENTAL PROCEDURES, to maintain euvolemia during the more lengthy protocols.


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  		Fig. 3.   Relative immunoreactive pools of Na-K-ATPase alpha 1- and beta 1-subunits in membrane fractions of renal cortex from control, acute hypertension, and blood pressure-restored protocols fractionated on sorbitol density gradients. A constant volume of each fraction was resolved by SDS-PAGE, blotted, probed with alpha 1- and beta 1-specific antibodies, and quantitated by scanning densitometry. Subunit relative immunoreactivity is expressed as the percentage of total signal in all 12 fractions. Left: summary of 5 independent experiments. Results are expressed as means ± SE, n = 5 in each group. Right: autoradiograms of a typical experimental set, with alpha  detected at 97.4 kDa and beta  at ~50 kDa.

The density gradient results do not rule out the possibility that during hypertension the inactivated sodium pumps were transported out of the basolateral membranes to internal membranes that share the same density distribution as basolateral membranes. Indeed, there are previous reports that sodium pump activity can be decreased by internalization (reviewed in Refs. 2, 4, and 10). To test this possibility, we made use of a fractionation strategy that separates membranes on the basis of their partitioning in an aqueous, dextran-polyethylene glycol two-phase system.

Figure 4 summarizes the distributions of Na-K-ATPase catalytic activity and alpha - and beta -subunit immunoreactivities after phase-partitioning analysis of density gradient fraction 4 (in which Na-K-ATPase was decreased 35% without detectable changes in alpha  or beta  immunoreactivity). In the baseline blood pressure control sample, all three markers exhibited peaks, with maxima in partitioning fraction 8, evidently marking the basolateral membranes. The alpha - and beta -subunits both exhibited minor peaks with maxima in fraction 2, but these were without a peak in catalytic activity. Hypertension was associated with leftward shifts in the major peaks of all three markers, from maxima in fraction 8 to maxima in fraction 7, and a >50% decrease in Na-K-ATPase activity localized to fraction 8. A similar leftward shift and decrease in catalytic activity was observed in a second experiment, performed with a different phase system pH. The most economical interpretation of this result is that a change in the basolateral membrane physical properties that determine phase-partitioning behavior accompanies the modification that decreases Na-K-ATPase catalytic activity.


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  		Fig. 4.   Phase-partitioning analysis of Na-K-ATPase in fraction 4 from paired control and acute hypertension samples in a polyethylene glycol-dextran two-phase system as described in EXPERIMENTAL PROCEDURES. Top: Na-K-ATPase activity is corrected for total protein applied to the two-phase system. Na-K-ATPase subunit immunoreactivity is expressed as the percentage of the total alpha 1- or beta 1-subunit immunoreactivity detected in all 10 samples. Bottom: autoradiograms scanned for quantitation. Experiment shown is representative of the 2 separate independent assays conducted.

Response of renal cortex apical membrane proteins to acute hypertension and blood pressure restoration. The NHE is a major transporter for sodium entry across the proximal tubule apical membrane, and NHE3 is responsible for virtually all the NHE activity in this region (1, 6, 45). We previously reported that an acute hypertension provoked a rapid redistribution of apical membrane NHE3 immunoreactivity to higher-density membranes. In this study, we aimed both to determine whether this response was reversible and to characterize the specificity of the response. NHE3, detected at 80 kDa by immunoblot (7), distributes to a major peak at fractions 4-5 after the control protocol, containing 75% of the total NHE3, defined as the apical membrane population (Fig. 5). After 5 min of acute hypertension, the immunoreactive pool of NHE3 in fractions 4-5 is reduced to 20% of total due to redistribution to a peak centered around fraction 6, with a small shoulder appearing at fractions 8-10. After 20 min of blood pressure restoration, the apical membrane NHE3 immunoreactivity returns to its starting level and distribution in fractions 4-5. This result demonstrates reversible redistribution of NHE3 associated with the changes in sodium transport provoked by blood pressure fluctuations.


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  		Fig. 5.   Top: Distribution of Na/H exchanger (NHE3) in membrane fractions of renal cortex from control, acute hypertension, and blood pressure-restored protocols. A constant volume of each fraction was resolved by SDS-PAGE, blotted, and probed with NHE3-specific monoclonal antibody. Antibody antigen complexes were detected by enhanced chemiluminescence. NHE3 immunoreactivity in each fraction is expressed as the percentage of total signal in all 12 fractions. Results are expressed as means ± SE, n = 5 in each group. * P < 0.05 vs. control, and + P < 0.05 vs. acute hypertension, assessed by ANOVA and followed by paired Student's t-test. Bottom: representative immunoblots from a typical experiment with NHE3 detected at 80 kDa.

To determine the specificity of the redistribution phenomenon with acute hypertension, the distribution of the renal Na-Pi cotransporter, a type II Pi transporter, was investigated. Like NHE3, the Na-Pi cotransporter is expressed in the proximal tubule apical brush border. The Na-Pi cotransporter has been shown to move from apical membranes to internal membranes in response to acute high-phosphate diet (26). Figure 6A is an autoradiogram illustrating the immunoblot detection of the Na-Pi cotransporter as a series of bands between 75 and 90 kDa and smaller bands at 37 kDa, as previously reported (14). The autoradiogram in Fig. 6A also establishes that the density distribution of the Na-Pi cotransporter coincides with that of NHE3, with a single major peak in fractions 4-6 and baseline expression restricted to fractions 4-7. Figure 6B shows the results of two independent experimental sets of control, hypertension, and restoration samples, specifically fractions 4-7 between 75 and 90 kDa, that were analyzed by immunoblot. In both sets, there was a pronounced decrease in the immunoreactivity in fraction 4, increases in signals in fractions 6-7, and a shift of the peak from fraction 5 to 6, analogous to the redistribution of NHE3 with acute hypertension. With blood pressure restored, the distribution shifted back toward the lower densities (although not completely restored as observed for NHE3 in the same experimental sets).


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  		Fig. 6.   Distribution of Na-Pi cotransporter immunoreactivity in membrane fractions of renal cortex from control, acute hypertension, and blood pressure-restored protocols resolved by density gradient fractionation. A constant volume of each fraction was resolved by SDS-PAGE and blotted and probed with Na-Pi cotransporter-specific polyclonal antibody. Antibody-antigen complexes were detected with 125I-labeled protein A. A: entire blot of a fractionation of control, sham-operated renal cortex, which illustrates immunodetection of a series of bands between 75 and 90 kDa and a smaller molecular mass bands at 37 kDa, as previously reported for this antiserum (14). Essentially, all of the Na-Pi is restricted to fractions 4-7 in the 12 fraction set. Brush-border membranes (bbm) are included as a positive control in the last lane. B: series of immunoblots of fractions 4-7 from control, acute hypertension, and blood pressure-restored cortices from 2 independent experiments; upper series of bands between 75 and 90 kDa, not the bands at 37 kDa, are shown.

The distribution response of two classical apical membrane markers that are not sodium transporters was investigated as well. Alkaline phosphatase activity and DPPIV immunoreactivity (detected by immunoblot at 105 kDa) both have distribution patterns with a main peak centered at fractions 5 coincident with the peaks of NHE3 and the Na-Pi cotransporter, indicative of apical plasma membranes (6). However, both are broader than the sodium transporter peaks, suggesting expression in multiple membrane populations. A dotted line is provided in Fig. 7 as a reference to the location of the apical sodium transporter peaks. During hypertension, the alkaline phosphatase activity and DPPIV immunoreactivity in fractions 1-5 decreased 50% compared with control. During hypertension, the position of the peak shifted to fraction 6 and the fraction of DPPIV in the higher-density shoulder increased significantly, as also seen for the NHE3 pattern. Twenty minutes after blood pressure restoration, both alkaline phosphatase activity and DPPIV immunoreactivity in fractions 1-5 increased back to 70% of control levels (Fig. 7). These data provide evidence that several apical brush-border proteins are found in higher-density membranes after an acute increase in blood pressure.


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  		Fig. 7.   Distribution of apical membrane proteins in membrane fractions of renal cortex from control, acute hypertension, and blood pressure-restored protocols. Top left: alkaline phosphatase activity, corrected for total protein recovered from all 12 fractions. Top right: dipeptidyl-peptidase IV (DPPIV) immunoreactivity detected at 105 kDa by immunoblot (bottom) of a constant volume of each fraction with specific antibodies, expressed as percentage of total signal density from all 12 fractions. A dotted line at fraction 5 is provided as a reference, indicating the separation between the apical plasma membranes and higher-density intracellular membranes. Data are expressed as means ± SE; n = 5. * P < 0.05 vs. control and + P < 0.05 vs. acute hypertension, assessed by ANOVA and followed by paired t-test.

Redistribution of a set of apical membrane proteins in response to acute hypertension could result from 1) modification in the array of proteins associated with microvilli that changes its density equilibrium without leaving the apical plasma membrane, 2) wholesale internal retraction of microvilli, as recently reported to occur during ATP depletion (16), 3) concerted trafficking of several apical proteins to a new membrane population with a different density, or 4) redistribution of NHE3 within the apical membrane from the microvillar domain to the intermicrovillar cleft membrane. To address this question, we examined the distribution patterns of apical proteins relative to that of 1) villin, a 95-kDa microvillar cytoskeleton bundling (and severing) protein associated with F-actin and restricted to the microvillar cytoskeleton, 2) megalin (gp330, 330 kDa), a scavenger receptor for filtered proteins found mainly in the intermicrovillar domain, coated pits, endocytotic vacuoles, and lysosomes of the early proximal tubule, with patchy distribution in the brush-border domain in later proximal tubules (5, 13), and 3) Rab 5a and Rab 4 (both detected at ~25 kDa), monomeric GTPases associated with endocytotic and exocytotic vesicles, respectively (8, 25, 40, 44).

Villin was broadly distributed between fractions 4 and 12 (Fig. 8), in contrast to the sharper peak of NHE3 and Na-Pi cotransporter, but overlapping with that of alkaline phosphatase and DPPIV. The overall distribution of villin and the percentage of total villin in the sodium transporter peak (fractions 4-6) are unchanged by acute hypertension. That is, sodium transporters redistribute out of fractions 4 and 5 to higher densities without an accompanying shift in villin from these fractions, indicating that the shift is not likely to result from a change in the density of microvillar membranes associated with villin and containing NHE3 and the Na-Pi cotransporter.


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  		Fig. 8.   Top: distribution of villin immunoreactivity in membrane fractions of renal cortex from control and acute hypertension protocols. A constant volume of each fraction was resolved by SDS-PAGE and blotted and probed with villin-specific monoclonal antibody. Antibody-antigen complexes were detected with 125I-protein A. Villin relative abundance in each fraction is expressed as the percentage of total signal in all 12 fractions. Results are expressed as means ± SE; n = 5 in each group. Distributions were not significantly different, assessed by ANOVA. Bottom: representative immunoblots from a typical experiment with villin at 95 kDa.

Distribution of megalin (gp330) is also distinct from that of NHE3 and other apical proteins and overlaps with the distribution of villin (Fig. 9). As expected from the complex pattern of subcellular expression previously described and discussed above (5, 13), there are multiple peaks of megalin in the gradient. Statistical analysis (by two-way repeated ANOVA) did not detect a significant difference in the overall distribution pattern of megalin immunoreactivity among the three treatment groups, but there was a tendency for fractional expression to be lower in fractions 4 and 5 and greater in fractions 8-10 with acute hypertension, reminiscent of the NHE3 redistribution with hypertension. This could reflect redistribution of the megalin expressed in the microvillar domain in the later proximal tubule. The major peak of megalin, which likely marks the peak in intermicrovillar membranes, is at fraction 6 in all three protocol groups. This location is coincident with the peaks of redistributed apical proteins, suggesting that these proteins move from the microvilli to the intermicrovillar cleft region when blood pressure is elevated. This is a plausible path, since it has been demonstrated for internalization of another microvillar protein, the insulin receptor (33) using electron microscopy. Rab 4 is detected in fractions 8-10 and Rab 5a is detected in fractions 10 and 11 (Fig. 9), a pattern unaffected by blood pressure perturbations (not shown). Thus classical endosomal membranes are localized to fractions 8-11, coincident with the high-density shoulder of apical proteins increased with hypertension and decreased with pressure restoration.


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  		Fig. 9.   Distribution of intermicrovillar cleft and endosomal protein markers in membrane fractions of renal cortex from control, acute hypertension, and blood pressure-restored protocols. Top: distribution of megalin (a marker for the intermicrovillar cleft, detected by immunoblot of a constant volume of each fraction probed with specific antibodies), expressed as percentage of total signal density from all 12 fractions, was unaltered by increasing or restoring blood pressure. Data are expressed as means ± SE, n = 5. Distributions were not significantly different, assessed by ANOVA. Middle: representative immunoblots of megalin at 330 kDa from a typical experiment. Bottom: distribution patterns of the endosomal markers Rab 4 and Rab 5a, detected by immunoblot at 25 kDa of a constant volume of each fraction probed with specific antibodies.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We previously demonstrated cellular mechanisms that could, at least in part, account for the decrease in proximal tubule sodium transport during acute hypertension: sodium pump activity decreases and apical NHE3 shifts to membranes with higher density. This current study demonstrates that these responses are reversed after blood pressure is returned to basal levels. Specifically, 20 min after blood pressure restoration, Na-K-ATPase activity is restored to control levels and NHE3 immunoreactivity shifts back from a higher- to a lower-density position in the gradient, which is typical of apical microvilli markers. The study also demonstrates that the response to acute hypertension is not restricted to Na-K-ATPase and NHE3: alkaline phosphatase activity is reversibly inhibited and the distributions of apical alkaline phosphatase and DPPIV are reversibly shifted.

Two assays demonstrate that the inactivation of Na-K-ATPase enzymatic activity in hypertension is reversible: in density gradient fractions containing basolateral membranes (fractions 3-5), mean activity decreases to 67% of control with hypertension and increases back to 83% of control with restoration, whereas, in the starting So before fractionation, activity decreases to 72% with hypertension and fully increases back to control levels with restoration. It should be noted that the ATPase activities, although reported in the same units (µmol Pi · mg protein-1 · h-1), are calculated slightly differently in the two assays. In the assays of So Na-K-ATPase, specific activity is expressed as the Pi liberation divided by the amount of protein in that So sample, whereas, in the density gradient fractions, Pi liberated in each fraction is divided by the total protein recovered in all 12 fractions to normalize for variation in protein content between sample sets. Within the peak basolateral membranes (fraction 3) pNPPase activity is enriched about fivefold (to 7 µmol Pi · mg protein-1 · h-1) compared with activity in So.

There is a burgeoning literature on mechanisms responsible for short-term regulation of Na-K-ATPase activity (reviewed in Refs. 2 and 4). Pathways linked to both generation of protein kinase C (PKC) and/or cAMP-dependent protein kinase A (3, 4) are postulated to regulate Na-K-ATPase activity by changing the alpha  catalytic subunit phosphorylation status. However, phosphorylation has been associated with both decreased activity (3, 29, 39) and increased activity (9, 28, 36) and no change in activity (15). There is also evidence that PKC causes a withdrawal of sodium pumps from the basolateral membranes independent of their PKC phosphorylation site [demonstrated by mutating at Thr-15 and Ser-16 to Ala (S16A/T15A)] (2). Proximal tubule Na-K-ATPase activity is also inhibited (whether directly or indirectly is not known) by activation of phospholipase A2, which stimulates production of arachidonate metabolites of cytochrome P-450 such as 20-hydroxyeicosatetraenoic acid (34, 35, 37), and sodium pump transport activity is inhibited by apical ATP mediated by purinergic receptors (22). Although the precise signaling mechanisms for the response to altered blood pressure remains to be elucidated, our results indicate that the inhibition of the sodium pump activity is due to structural modification of the pump itself or an associated regulator, rather than solely mediated by trafficking of active pumps to a new location; the data demonstrate significant changes in total ATPase activity that persist through membrane fractionation and phase-partitioning analysis. However, the minor change in the partitioning properties of the Na-K-ATPase alpha - and beta -subunits may reflect either a modification of the basolateral membranes containing the inhibited pumps or a transfer of inhibited pumps to membranes with different partitioning properties.

The findings of this study add to a growing body of evidence for rapid regulation of renal proximal tubule solute transport by trafficking of the transporters between surface and internal membrane domains (2, 10, 20, 26) In isolated proximal tubules, Hensley et al. (20) provided evidence, using similar subcellular fractionation strategy, for redistribution of NHE activity from apical to internal membranes mediated by parathyroid hormone (PTH) stimulation, although the trafficking route and isoform were not identified. Proximal tubule Na-Pi cotransporter, present in both subapical and apical pools, is rapidly recruited to the apical brush border by acute dietary Pi restriction mediated by microtubule-dependent translocation of presynthesized Na-Pi cotransporters, and surface expression is rapidly downregulated with acute high-Pi diet independent of microtubules (26). By high-resolution immunocytochemistry, Biemesderfer and colleagues (7) found NHE3 in subapical vesicles in the proximal tubule consistent with possible regulation by membrane recycling. In the present study, we demonstrate that the decrease in proximal tubule sodium transport provoked by acute hypertension is associated with a redistribution of both NHEs and Na-Pi cotransporters to membranes of higher density. Immunofluorescence studies have demonstrated that both the NHE3 and the renal Na-Pi cotransporter are highly enriched in the apical brush border under control conditions (7, 26). The observation that there is no detectable shift of the brush-border cytoskeletal protein villin to higher densities with hypertension argues against the interpretation that the density of the apical membranes has increased. We postulate that the apical sodium transporters have redistributed out of the brush border to the subapical vesicles containing NHE3 demonstrated by Biemesderfer and colleagues (7).

The proximal tubule has such a rich array of vesicles under the apical membrane, also referred to as dense apical tubules, that it is difficult to interpret trafficking events when studied by light microscopy. However, Yip (46) has provided preliminary evidence that NHE3 is actually internalized in response to acute hypertension. Using electron microscopic techniques, Nielsen (33) studied early events in trafficking of labeled insulin receptors of the apical membrane and provided evidence that it involves two steps: first, lateral migration of membrane receptors from microvilli to the intermicrovillar cleft region and, subsequently, internalization into endocytotic vacuoles and dense apical tubules. Our results with subcellular fractionation suggest a similar route for redistribution of apical proteins in the response to acute hypertension. Within 5 min of acute hypertension, the apical proteins move to a region of the gradient in which there is the greatest percentage of megalin (gp330), a marker for the intermicrovillar cleft and coated pits (13), and to regions containing endosomal Rab markers. However, it should be noted that Biemesderfer and co-workers (7) did not observe NHE3 in coated pits in unstimulated rat kidney.

The fact that several apical proteins moved to higher densities during acute hypertension suggested the possibility that the response involved internal retraction of the brush-border microvilli, analogous to the response seen in cultured cells during ATP depletion (16) with PTH treatment (17) and that the restoration response involved the insertion of preformed microvilli seen during recovery from ATP depletion in cultured renal cells (16) or with epidermal growth factor treatment of enterocytes (19). Although this has not been ruled out, the results do not support this mechanism, since no accompanying shift in the microvilli marker villin was detected with acute hypertension, and there was, likewise, no detectable change in villin distribution in the density gradient fractions in which there was >50% decrease in NHE3 and Na-Pi with hypertension.

During membrane recycling, endocytosed proteins can be either returned to the plasma membrane or routed to lysosomes for degradation. For example, there is evidence that Na-Pi is colocalized with lysosomes during the transition from low- to high-Pi diet (26). The demonstration that the redistribution and inactivation of transporters and apical markers are reversible and occur without a change in starting pool size in the So fraction argues that changes in the degradation rate do not correlate to rapid decreases in sodium transport provoked by acute hypertension.

The onset of proximal tubule responses to acute hypertension (11, 12, 47) and the onset of pressure natriuresis (41) are almost instantaneous with the increase in blood pressure. In this study, we observed that the reversal of the natriuretic responses is gradual even though the restoration of blood pressure to basal levels is nearly instantaneous. The time courses of return in lithium clearance and urine output were indistinguishable, suggesting that the two parameters are linked to similar signaling mechanisms. The sustained elevation in lithium clearance, an inverse indicator of sodium handling in the proximal tubule (43) in the absence of the physical stimulus of elevated pressure, suggests mediation by chemical regulators. For example, acute hypertension may stimulate the release of a mediator (or may inhibit a tonically released regulator) from cells that sense the elevated blood pressure, and, when blood pressure is rapidly restored and the stimulus is removed, the return of the mediator to basal levels around the responding cells will be a function of the rate of removal from (or addition to) the regional pool. The rate of recovery of proximal tubule sodium reabsorption and urine output will also be a function of the rate of reversibility of sodium transporter trafficking and inhibition. Another explanation for the lag in response is that the reversal of the sodium pump modifications or rerouting of apical proteins requires 20 min.

In conclusion, this study demonstrates rapid, reversible redistribution and inactivation of apical and basolateral sodium transporters in the proximal tubule in response to acute hypertension and blood pressure restoration. This complex coordinated set of cellular mechanisms can potentially account for the altered proximal tubule sodium reabsorption in response to blood pressure fluctuations.

    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants DK-34316 to A. A. McDonough and HL-45623 to N.-H. Holstein-Rathlou.

    FOOTNOTES

Y. Zhang was supported by a fellowship award from the American Heart Association, Greater Los Angeles Affiliate.

Portions of this work were presented at the 1995 and 1996 Annual Meetings of the American Society of Nephrology.

Address for reprint requests: A. A. McDonough, Dept. of Physiology and Biophysics, Univ. of Southern California School of Medicine, 1333 San Pablo St., Los Angeles, CA 90033.

Received 8 September 1997; accepted in final form 19 December 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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Diuretic response to acute hypertension is blunted during angiotensin II clamp
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Na+/H+ exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells
Am J Physiol Cell Physiol, September 1, 2002; 283(3): C927 - C940.
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J. Klisic, M. C. Hu, V. Nief, L. Reyes, D. Fuster, O. W. Moe, and P. M. Ambuhl
Insulin activates Na+/H+ exchanger 3: biphasic response and glucocorticoid dependence
Am J Physiol Renal Physiol, September 1, 2002; 283(3): F532 - F539.
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C. E. MAGYAR, Y. ZHANG, N.-H. HOLSTEIN-RATHLOU, and A. A. MCDONOUGH
Downstream Shift in Sodium Pump Activity along the Nephron during Acute Hypertension
J. Am. Soc. Nephrol., November 1, 2001; 12(11): 2231 - 2240.
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X. X. Li, J. Xu, S. Zheng, F. E. Albrecht, J. E. Robillard, G. M. Eisner, and P. A. Jose
D1 dopamine receptor regulation of NHE3 during development in spontaneously hypertensive rats
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2001; 280(6): R1650 - R1656.
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D. W. Good, J. F. Di Mari, and B. A. Watts III
Hyposmolality stimulates Na+/H+ exchange and HCO3- absorption in thick ascending limb via PI 3-kinase
Am J Physiol Cell Physiol, November 1, 2000; 279(5): C1443 - C1454.
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K.-P. Yip, A. J. Wagner, and D. J. Marsh
Detection of apical Na+/H+ exchanger activity inhibition in proximal tubules induced by acute hypertension
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2000; 279(4): R1412 - R1418.
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C. E. Magyar, Y. Zhang, N.-H. Holstein-Rathlou, and A. A. McDonough
Proximal tubule Na transporter responses are the same during acute and chronic hypertension
Am J Physiol Renal Physiol, August 1, 2000; 279(2): F358 - F369.
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X. X. Li, F. E. Albrecht, J. E. Robillard, G. M. Eisner, and P. A. Jose
Gbeta regulation of Na/H exchanger-3 activity in rat renal proximal tubules during development
Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2000; 278(4): R931 - R936.
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F. E. Albrecht, J. Xu, O. W. Moe, U. Hopfer, W. F. Simonds, J. Orlowski, and P. A. Jose
Regulation of NHE3 activity by G protein subunits in renal brush-border membranes
Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2000; 278(4): R1064 - R1073.
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O. W. MOE
Acute Regulation of Proximal Tubule Apical Membrane Na/H Exchanger NHE-3: Role of Phosphorylation, Protein Trafficking, and RegulatoryFactors
J. Am. Soc. Nephrol., November 1, 1999; 10(11): 2412 - 2425.
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Y. Zhang, J. M. Norian, C. E. Magyar, N.-H. Holstein-Rathlou, A. K. Mircheff, and A. A. McDonough
In vivo PTH provokes apical NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition
Am J Physiol Renal Physiol, May 1, 1999; 276(5): F711 - F719.
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L. Fan, M. R. Wiederkehr, R. Collazo, H. Wang, L. A. Crowder, and O. W. Moe
Dual Mechanisms of Regulation of Na/H Exchanger NHE-3 by Parathyroid Hormone in Rat Kidney
J. Biol. Chem., April 16, 1999; 274(16): 11289 - 11295.
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R. Collazo, L. Fan, M. C. Hu, H. Zhao, M. R. Wiederkehr, and O. W. Moe
Acute Regulation of Na+/H+ Exchanger NHE3 by Parathyroid Hormone via NHE3 Phosphorylation and Dynamin-dependent Endocytosis
J. Biol. Chem., October 6, 2000; 275(41): 31601 - 31608.
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M. C. Hu, L. Fan, L. A. Crowder, Z. Karim-Jimenez, H. Murer, and O. W. Moe
Dopamine Acutely Stimulates Na+/H+ Exchanger (NHE3) Endocytosis via Clathrin-coated Vesicles. DEPENDENCE ON PROTEIN KINASE A-MEDIATED NHE3 PHOSPHORYLATION
J. Biol. Chem., July 13, 2001; 276(29): 26906 - 26915.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Renal Physiol.Home page
L. Yang, P. K. K. Leong, J. O. Chen, N. Patel, S. F. Hamm-Alvarez, and A. A. McDonough
Acute hypertension provokes internalization of proximal tubule NHE3 without inhibition of transport activity
Am J Physiol Renal Physiol, April 1, 2002; 282(4): F730 - F740.
[Abstract] [Full Text] [PDF]

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