1 Department of Physiology and Biophysics, 2 Division of Nephrology, Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California 90089-9142
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ABSTRACT |
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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 µl 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+-Pi 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.
kidney; sympathetic nervous system; phenol; sodium/hydrogen exchanger type 3
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INTRODUCTION |
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A RAT MODEL OF NEUROGENIC hypertension provoked by a renal injury was recently developed by Campese and colleagues (10, 43, 44). In this model, injection of 50 µl 10% phenol causes a rapid elevation of blood pressure, which is preceded by a rise in norepinephrine secretion from the posterior hypothalamus and an increase in renal sympathetic nervous system (SNS) activity. Renal denervation before the renal injury prevents the SNS activation and the subsequent rise in blood pressure (44). Thus renal injury activates renal afferent pathways, increases norepinephrine release from the posterior hypothalamus, activates renal efferent pathways, and raises blood pressure. Interestingly, the hypertension becomes established and persists long after the site of injury recedes to a microscopic scar and is reversed by removal of the injured kidney or renal denervation (43). The cellular and molecular bases for the hypertension are not understood. One plausible mechanism is that the renal efferent sympathetic nerve activity may stimulate Na+ and volume reabsorption and contribute to hypertension.
There is a dynamic relationship between blood pressure and renal Na+ reabsorption that is responsible for the blood pressure set point. A decrease in Na+ transport can be a homeostatic compensation to elevated blood pressure; an experimental increase in blood pressure acutely decreases proximal tubule Na+ reabsorption, which both increases NaCl at the macula densa, a transforming growth factor signal to normalize renal blood flow (RBF) and glomerular filtration rate (GFR), and causes a pressure natriuresis that reduces extracellular volume, which counteracts the hypertension (8, 13, 14). In contrast, inappropriately elevated Na+ transport, due to either excess production of an antinatriuretic substance (e.g., aldosterone) (40) or an activated Na+ transporter (Liddle's syndrome) (33), can generate and maintain hypertension. Both responses can occur together; if renal Na+ reabsorption is elevated, then blood pressure increases and induces a pressure-natriuresis variant known as "escape" in which Na+ reabsorption is depressed at sites along the nephron not primarily affected by the excess hormone or Na+ transport, a response that balances Na+ excretion to Na+ intake.
Our laboratory previously investigated the molecular mechanisms responsible for the decrease in proximal tubule Na+ reabsorption during an experimental 5-min increase in blood pressure and discovered that there is a rapid retraction of Na+/H+ exchangers [Na+/H+ exchanger type 3 (NHE3)] and Na+-Pi cotransporters (NaPi) from the apical brush border to intermicrovillar cleft and subapical endosomes, demonstrated by both subcellular fractionation and confocal microscopy, as well as a decrease in basolateral Na+-K+-ATPase activity (42, 47). Motivated by these findings, we aimed to test the hypothesis that acute phenol injury, via activation of sympathetic efferents, increases proximal tubule Na+ transport by recruiting Na+ transporters from subapical pools to the brush border, contributing to the genesis of the hypertension. There is support for this hypothesis from in vitro studies on adrenergic regulation of proximal Na+ transporters (3, 4, 34). The results of this study support the hypothesis that acute phenol injury provokes redistribution of proximal tubule NHE3 from subapical endosomal pools to apical brush border and that the response is mediated by sympathetic stimulation and may contribute to the generation and persistence of phenol injury-induced hypertension.
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METHODS |
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Animal preparation. 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 (1:1, vol/vol; Miles) and then placed on a thermostatically controlled warming table to maintain body temperature at 37°C. A polyethylene catheter was placed into the carotid artery to monitor blood pressure. The jugular vein was cannulated to infuse 4.0% BSA in 0.9% NaCl at 50 µl/min throughout the entire experimental period to maintain euvolemia. The left side ureter was cannulated to collect urine.
The experimental time course was as follows. Blood pressure was measured continuously. A baseline urine sample was collected over 20 min, and then 50 µl of 10% phenol or saline (sham control) was injected into the cortical lower pole of the left kidney (n = 7 in each group). Thirty minutes after injection, another urine sample was collected over 20 min, and then kidneys were removed for immediate analysis. On the same day, a phenol-injected animal was analyzed in parallel with a saline-injected animal and analyzed in a paired fashion. In two sets, the effects of denervation on the response to phenol or saline injection was assessed. The left renal nerve was isolated by dissection and removed, and blood pressure was allowed to stabilize for at least 30 min before phenol injection as described by Ye et al. (44). For the experiments comparing arterial constriction hypertension to phenol injury-induced hypertension, mean arterial pressure was increased 20-30 mmHg by constricting the superior mesenteric artery, celiac artery, and abdominal aorta below the renal artery by tying silk ligatures around the vessels, as reported previously (46).Urine collection and endogenous lithium clearance. Urine was collected from the left ureter catheter, and urinary volume 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 (47).
Homogenization and subcellular fractionation.
In preliminary fractionation experiments, the injected and the
contralateral kidneys were compared, and the results were
indistinguishable. The injected kidneys were chosen for analysis.
The procedure for subcellular fractionation of renal cortex membranes
has been described previously (46, 47). In brief, the
injected kidney from each phenol- or saline-injected animal was cooled
in situ by flushing with cold PBS and then excised. The renal cortex
was dissected (injured area, ~2-mm diameter, was cut off and
discarded), homogenized with a Tissuemizer (Tekmar Instruments) in
isolation buffer [5% sorbitol, 0.5 mM disodium EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 9 µg/ml aprotinin, and 5 mM
histidine-imidazole buffer (pH 7.5)], and centrifuged at 2,000 g for 10 min; the pellet was rehomogenized and centrifuged;
and the low-speed supernatants were pooled, loaded between two
hyperbolic sorbitol gradients, and centrifuged at 100,000 g
for 5 h. Twelve fractions were collected from the top, 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.
Immunoblot analysis and antibodies.
A constant volume of sample from each gradient fraction was denatured
in SDS-PAGE sample buffer for 30 min at 37°C, resolved on the same
7.5% SDS polyacrylamide gel according to Laemmli (20), and transferred to polyvinylidene difluoride membranes (Millipore Immobilon-P). Selected samples on each blot were run at one-half the
volume or protein to assure that the sample was in the linear range of
detection, and multiple exposures of autoradiograms were analyzed to
ensure that signals were within the linear range of the film. All
blots, except for NaPi2 analysis, were detected with the ECL enhanced
chemiluminescence kit (Amersham Pharmacia Biotech), and
autoradiographic signals were quantified with a Bio-Rad imaging
densitometer with Molecular Analyst software. For NHE3 detection, blots
were probed with polyclonal NHE3-C00 (42) at 1:1,000
dilution. Polyclonal antisera to dipeptidyl-peptidase IV (DPPIV) were
generously provided by M. Farquhar (University of California at San
Diego). For detection of Na-Pi cotransporter 2 (NaPi2),
blots were incubated with polyclonal anti-NaPi2 antibody generated by
Biber and Murer (University of Zürich, Zurich, Switzerland), then
with Alexa 680-labeled goat-anti-rabbit secondary antibody, and then
detected with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NB).
Each gradient and immunoblot was from a separate rat, i.e., no pooling
of samples. Results were quantitated by normalizing the density in each
fraction to the total sum density from all the fractions and expressed
as the percentage of total immunoreactivity within each sample. These
fraction-specific results were pooled into three "windows" to
simplify analyses. As previously reported (42, 46),
fractions 3-5 [window I (WI)] are enriched in apical
brush-border markers alkaline phosphatase, DPPIV, and NHE3;
fractions 6-8 (WII) contain most of the
intermicrovillar cleft marker megalin (5) as well as
apical markers; and fractions 9-11 (WIII) are enriched
in the endosomal marker rab 5a and the lysosomal marker
-hexosaminidase as well as megalin.
Indirect immunofluorescence. To compare the NHE3 distribution in two models of acute hypertension (arterial constriction vs. phenol injury), blood pressure was raised to the same level in each model (20-30 mmHg over baseline as described above), and then one kidney from each animal was analyzed by confocal microscopy. The kidney contralateral to the phenol or saline injection was analyzed in this series to focus on the effects of the similarly elevated blood pressure in the two models and to eliminate any potential effect of the phenol per se. Kidneys were fixed at 30-50 min in situ by placing the isolated kidney in a small plexiglass cup and bathing it in fixative [2% paraformaldehyde, 75 mM lysine, and 10 mM Na-periodate, pH 7.4 (PLP)] for 20 min. The kidneys were then removed, cut in half on a midsagittal plane, and postfixed in PLP for another 4-6 h. The fixed tissue was rinsed twice with PBS, cryoprotected by incubation overnight in 30% sucrose in PBS, embedded in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA), and frozen in liquid nitrogen. Cryosections (5 µM) were cut with a Microm Heidelgerg ultramicrotome, transferred to Fisher Superfrost Plus-charged glass slides, and air dried. For immunofluorescence labeling, the sections were rehydrated in PBS for 10 min, followed by 10-min washing with 50 mM NH4Cl in PBS and then with 1% SDS in PBS for 4 min for antigen retrieval (9). SDS was removed by two 5-min washes in PBS, and then sections were blocked with 1% BSA in PBS to reduce background. Double labeling was performed by incubating with polyclonal antiserum NHE3-C00 and monoclonal antibody against villin (Immunotech, Chicago, IL), both at a dilution of 1:100 in 1% BSA in PBS for 1.5 h at room temperature. After being washed for 5 min three times in PBS, the sections were incubated with a mixture of FITC-conjugated goat-anti-rabbit (Cappel Research Products, Durham, NC) and Alexa 568-conjugated goat-anti-mouse (Molecular Probes, Eugene, OR) secondary antibodies diluted 1:100 in 1% BSA in PBS for 1 h, washed three times with PBS, mounted in Prolong Antifade (Molecular Probes), and dried overnight at room temperature. Slides were viewed with a Nikon PCM quantitative measuring high-performance confocal system equipped with filters for both FITC and tetramethylrhodamine isothiocyanate fluorescence attached to a Nikon TE300 Quantum upright microscope. Images were acquired with Simple PCI C-Imaging hardware and Quantitative Measuring software and processed with Adobe PhotoDeluxe (Adobe Systems, Mountain View, CA).
Other assays. Na+-K+-ATPase activity was measured by the K+-dependent p-nitrophenyl phosphatase reaction (30). Standard assay was used for alkaline phosphatase activity (29). Protein concentrations were measured with a bicinchoninic acid assay kit (Pierce Technology, Iselin, NJ).
Quantitation and statistical analysis. Experiments were conducted and analyzed in a pairwise fashion, that is, one phenol injected and one saline injected in 1 day. Data are expressed as means ± SE. Two-way ANOVA was applied to determine whether there was a significant effect of treatment on the overall subcellular distribution pattern. If a significance was established (P < 0.05), the location of the difference in the pattern was assessed by two-tailed Student's t-test for paired samples, and the differences were regarded significant at P < 0.05.
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RESULTS |
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Physiological responses to acute phenol injury.
Ye et al. (43, 44) reported that acute renal injury by an
intrarenal injection of 50 µl 10% phenol caused an immediate and
permanent elevation in blood pressure. In this study, we verify that
the effect of phenol injury could be reproduced in our laboratory with
our animal preparation. Arterial blood pressure increased immediately
after 50 µl of 10% phenol injection, fluctuated somewhat over the
next 30 min, and then was maintained at 20-30 mmHg above baseline, an average increase from 110 ± 2.8 to 134 ± 2.4 mmHg (Fig. 1). These arterial
pressures are within the autoregulatory range for GFR and RBF
(13). Sham injection of 50 µl of saline caused a brief
transient fluctuation in blood pressure that returned to baseline
within 30 min (Fig. 1A, bottom).
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NHE3 redistribution in response to phenol injury.
Na+/H+ exchanger is the major route for apical
Na+ entry across the proximal tubule, and NHE3 is
responsible for virtually all the Na+/H+
exchange activity in this region (2, 6). Our previous
studies using confocal microscopy established that acute hypertension due to arterial constriction is associated with a rapid redistribution of NHE3 immunoreactivity out of the brush-border microvilli to intermicrovillar cleft and endosomal membranes (42); the
same conclusion was reached when analyzed by subcellular fractionation, NHE3 redistributed from lower-density membranes enriched in markers of
apical microvilli to higher-density membranes enriched in markers of
intermicrovillar cleft, dense apical tubules, and endosomes (42,
47). These same techniques were applied to the kidneys after
acute phenol injection to test the hypothesis that NHE3 would
redistribute into the brush border in response to the increased sympathetic efferent activity; the alternative hypothesis was that the
NHE3 would retract out of the brush border in response to the
hypertension per se, identical to the response to arterial constriction. Representative immunoblots of NHE3 in 12 gradient fractions from saline- and phenol-injected kidneys are shown in Fig.
2A. Samples were pooled into
three windows to simplify analyses, as described in
METHODS. After phenol injection, a significant fraction of
NHE3 (expressed as percentage of total in the gradient) shifts out of
WIII into WI (Fig. 2B): NHE3 in WI increased from 13.07 ± 1.97 to 27.15 ± 4.08% of total, NHE3 in WII remained
unchanged (63.7 ± 3.53 to 61.45 ± 2.15%), and NHE3 in WIII
decreased from 22.79 ± 4.75 to 10.79 ± 2.01% after phenol
injury (P < 0.05 vs. saline, assessed by ANOVA and
followed by paired Student's t-test). These results support
the hypothesis that phenol injury provokes a redistribution of NHE3
from intracellular and intermicrovillar membrane pools to the apical
microvilli, a response that could favor increased salt and water
transport and the generation and persistence of hypertension after
phenol injury.
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NHE3 distribution in denervation vs. denervation followed by phenol injection. Renal injury activates renal afferent pathways, increases norepinephrine release from the posterior hypothalamus, activates renal efferent pathways, and raises blood pressure. To test the hypothesis that the activation of renal efferent pathways provokes the apical redistribution of NHE3, we performed two sets of experiments in which the left kidney was denervated before phenol injection. As previously shown by Ye et al. (44), we found that denervation of the left renal nerves before the phenol injection into the left kidney prevented the increase in blood pressure (not shown). As shown in Fig. 2, denervation per se did not affect the distribution pattern of NHE3 compared with the saline-injected kidney; however, denervation before phenol injection did prevent the redistribution of NHE3 to WI (Fig. 2C). This provides direct evidence that the NHE3 redistribution to apical brush border in phenol injury (Fig. 2, A and B) is associated with the activated renal efferent pathways and not to the local effects of the injected phenol.
NHE3 distribution in phenol injury vs. arterial
constriction-induced hypertension.
The change in the density gradient distribution pattern of NHE3 after
phenol injection-induced hypertension is reciprocal to that seen with
acute hypertension due to arterial constriction, providing evidence for
bidirectional regulation of NHE3 between apical microvilli and
intermicrovillar/subapical membranes. In our previous study of the
arterial constriction model of acute hypertension, blood pressure was
raised 50-70 mmHg (42, 47), whereas after phenol
injection blood pressure increased 20-30 mmHg, which allows for
the possibility that the distinct responses were a function of the
different levels of hypertension. NHE3 redistribution responses were
reexamined by immunocytochemistry after blood pressure was increased by
20-30 mmHg in both the phenol-induced and the
arterial-constriction models of hypertension. Thirty minutes after
saline or phenol injection or arterial constriction, kidneys were fixed
in situ for another 20 min, as described in METHODS. Double
labeling was performed on cryosections harvested from each of the three
groups. NHE3 was imaged with polyclonal NHE3-C00 with FITC-conjugated
anti-rabbit secondary, and villin, the actin bundling protein localized
to the microvilli, was imaged with monoclonal anti-villin with Alexa
568-conjugated anti-mouse secondary antibody. We previously
demonstrated that the subcellular distribution of villin was unaltered
during acute hypertension, so it provides a consistent background
marker for the microvilli as the NHE3 redistributes (47).
In saline-injected rats, the staining of NHE3 is restricted to the
brush border, as evidenced by colocalization with staining for villin
(Fig. 3, top). When blood
pressure is increased 20-30 mmHg by arterial constriction
hypertension (Fig. 3, bottom), NHE3 moves out of the apical
brush-border microvilli, leaving the tops of the villi stained red with
anti-villin, NHE3 is detected in the intermicrovillar cleft region
where it coincides with villin (Fig. 3, yellow, arrow), and NHE3
appears in subapical vesicles where it does not overlay villin (Fig. 3,
green, arrowhead). This response is indistinguishable from that
observed when blood pressure is increased 50-60 mmHg by arterial
constriction. After phenol injection associated with a 20-30 mmHg
hypertension, NHE3 remained colocalized with villin staining (Fig. 3,
middle). This technique was not sensitive enough to detect
the low levels of subapical NHE3 at baseline blood pressure, thus no
redistribution was evident, but the finding provides strong visual
confirmation that NHE3 was not internalized during phenol
injury-induced hypertension. This comparison demonstrates that although
blood pressure was increased 20-30 mmHg over baseline in both
animal models, NHE3 is not internalized from apical membranes during
phenol injury-induced hypertension as it is during arterial
constriction hypertension.
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Effect of acute phenol injury on the distributions of other apical
membrane proteins.
To determine the specificity of the redistribution of NHE3 during acute
phenol injury, the distribution of NaPi2 was investigated. NaPi2 is
mainly expressed in the proximal tubule apical brush border. NaPi2 has
been shown to move from apical membranes to intracellular membranes in
response to acute hypertension due to arterial constriction and during
a high-Pi diet (24, 47). Figure
4A shows representative
immunoblots of NaPi2 and summarized data expressed as percentage of
total in the three windows from saline- vs. phenol-injected rats. In
phenol-injected rats, NaPi2 increased in WI from 9.5 ± 1.62 to
18.7 ± 1.45% of total, NaPi2 decreased in WII from 68.72 ± 1.95 to 59.76 ± 2.21% of total, and there was no change in WIII
(P < 0.05 vs. saline, assessed by ANOVA and followed
by paired Student's t-test). The results indicate that
NaPi2 may, similarly to NHE3, move from intermicrovillar cleft region
and/or dense apical tubules (WII) to apical membranes (WI) during
phenol injury-induced hypertension.
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Effect of acute phenol injury on basolateral membrane Na+-K+-ATPase. We previously determined that renal cortical Na+-K+-ATPase was inhibited in response to arterial constriction hypertension (26), so we investigated whether Na+-K+-ATPase activity was altered during phenol injury-induced hypertension. The subcellular distribution of Na+-K+-ATPase activity in renal cortex (Fig. 5B) indicates a peak of Na+-K+-ATPase activity in WI that did not change in activity or distribution pattern after acute phenol injury.
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DISCUSSION |
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Abundant evidence has accumulated supporting a role of increased
SNS activity in renal injury-induced hypertension. Campese and
colleagues (10, 43, 44) developed a rat model of renal injury-induced hypertension caused by an injection of 50 µl 10% phenol to the lower pole of one kidney, which leads to an immediate elevation of norepinephrine secretion from posterior hypothalamus and a
rise in blood pressure. They also measured an increase in plasma
norepinephrine level and renal sympathetic activity recorded directly
from renal nerves after phenol injection. The injury per se is not
sufficient to provoke the hypertension, because renal denervation
before phenol injection prevents the increase in blood pressure and
norepinephrine secretion from posterior hypothalamus (44).
Recent results from the same laboratory indicate that afferent impulses
triggered by the phenol injury activate ANG II formation in brain
nuclei, which inhibits IL-1 and nNOS, leading to activation of
central and peripheral SNS activity. Injection of the ANG II
AT1 receptor antagonist losartan (into the lateral
ventricle or intravenously) inhibited the effect of phenol on blood
pressure and increased IL-1
and nNOS mRNA in three regions of the
brain (45). In addition, the
-adrenergic receptor
blocker phentholamine normalized blood pressure (45a), all
supporting a role of
-adrenergic receptor activation in the genesis
of hypertension in this model. The effect of phenol injury on
blood pressure is long lasting; 5 wk after phenol injection, the site
of injection is reduced to a microscopic scar while hypertension persists (43). The phenol-injected kidney is a necessary
component of the chronic hypertension because its removal at 4 wk
normalizes blood pressure, likely because of elimination of the renal
afferent impulses (10). Taken together, these studies
suggest that renal and central SNS activation are responsible for
phenol injury-induced hypertension. Our present study extends these
observations to the level of molecular mechanisms regulating proximal
tubule Na+ transporters in this neurogenic hypertensive
model. Acute phenol injection provoked a rapid redistribution of NHE3
and NaPi2 to the apical microvilli, a response that may contribute to
the generation or maintenance of hypertension in this model.
Formal analysis of the molecular mechanisms of SNS activation of
Na+ transport in vivo have not been previously conducted or
reported; however, there are a number of studies on the in vitro
effects of norepinephrine on Na+/H+ exchangers.
The proximal tubule contains numerous -adrenergic receptor binding
sites (19, 37, 38) and conditions that increase receptor
number or postreceptor components responsible for
1- and
-adrenergic-mediated Na+ reabsorption in proximal tubule
can contribute to Na+ retention and elevated blood pressure
(19). Liu and colleagues (22, 23) found that
proximal nephron Na+/H+ exchange transport
activity is increased by activation of
1A- and
1B-adrenergic receptor subtypes facilitated by the MAPK
signaling pathway. The findings of the present study provide the first
direct in vivo evidence that SNS stimulation activates apical
Na+/H+ exchange activity by increasing NHE3
transporters at the apical surface, a response that can be prevented by
renal denervation. The increase in apical NHE3 may be accomplished by
decreasing endocytosis or increasing exocytosis from intracellular
stores, or both. Because the subapical endosomal pool of NHE3 is
difficult to detect at baseline blood pressures by confocal microscopy, it is quite plausible that the apical NHE3 and NaPi2 accumulate due to
depressed endocytosis.
In vivo studies have provided mixed results regarding adrenergic
regulation of basolateral Na+-K+- ATPase in
isolated and cultured renal proximal tubules. Norepinephrine was found
to increase solute and fluid reabsorption and
Na+-K+-ATPase activity (1, 3) in
isolated proximal tubules, an effect that may be driven by increased
apical Na+ entry (34). In another system,
adrenergic stimulation drives exocytic insertion of Na+
pumps into the plasma membrane of cultured lung cells (4). However, Holtback et al. (18) concluded that
norepinephrine has no net effect on proximal tubule
Na+-K+-ATPase activity because of combined
activation of - and
-adrenergic receptors in the proximal tubule.
In the present study, there was no effect of phenol injection-induced
hypertension on Na+-K+-ATPase activity in
isolated membranes resolved on sorbitol gradients. There was a slight
tendency to redistribute Na+-K+-ATPase from WII
to the WI basolateral membrane peak, which may reflect insertion of
Na+-K+-ATPase from intracellular vesicles to
the plasma membranes, similar to the effect of adrenergic agents in
cultured lung cells (4), but establishing this will
require an improved fractionation strategy or pharmacological
manipulation of
- vs.
-adrenergic receptor levels. In contrast,
our laboratory previously determined that an acute increase in blood
pressure by artery constriction inhibits proximal tubule
Na+-K+-ATPase activity measured in isolated
membranes (25, 47). It is possible that the opposing
forces of SNS stimulation to increase activity and hypertension to
decrease activity may counteract each other, an issue that could be
tested by SNS stimulation in a setting of servocontrolled blood
pressure. Can Na+ reabsorption increase without a change in
Na+-K+-ATPase activity?
Na+-K+-ATPase activity in vivo in the tubule is
very high to start with (28) and may indeed be activated
by increased Na+ availability or adrenergic stimulation
while not detected enzymatically in a Vmax assay
in broken membranes in vitro.
Regarding the phenol injury itself, the hypertension occurs in the face of minimal renal injury (1- to 2-mm wide) (43). In this study, before cell fractionation, a small area surrounding the injection site was removed, and areas beyond this injection area appeared normal when examined by electron microscopy (43). Activation of the SNS reflex does not appear to be the result of an unspecific renal injury as lesions of the same dimensions caused by burning, administration of alkali (NaOH), acid (HCl), or methanol do not raise blood pressure (not shown). In addition, phenol injections to other sites, including the spleen and peritoneum, change blood pressure only transiently over <10 min (44).
We have previously reported that an acute increase in arterial blood pressure brought about by arterial constriction provokes a rapid decrease in proximal tubule Na+ reabsorption, the key to increasing NaCl at the macula densa, which activates tubuloglomerular feedback to autoregulate RBF and GFR (13, 14, 47). The accompanying natriuresis and diuresis are a compensatory response to restore elevated blood pressure toward normal levels (16). Using CLi as a measure of volume flow out of the proximal tubule, we have consistently observed that arterial constriction hypertension causes a three- to fourfold increase in CLi and urine output (42, 47). The role of proximal tubule Na+ reabsorption, estimated by CLi, in the generation or maintenance of hypertension has been assessed by different investigators with differing results (12). This is likely because elevated Na+ and volume reabsorption may be causal to some varieties of hypertension, whereas decreased Na+ and volume reabsorption may be compensatory in other varieties of hypertension. An increase in proximal reabsorption has been demonstrated in unanesthetized spontaneously hypertensive rats (7) and in hypertensive patients (11, 27, 35). A decrease in proximal Na+ transport in hypertensive patients (32, 41) or no change (17, 36) has been reported as well. In the present study, hypertension caused by acute phenol injury did not cause a significant change in either urinary output or CLi, which could be explained by the combined effect of SNS activation to increase Na+ and volume reabsorption and elevated blood pressure, which would evoke a compensatory decrease in Na+ and volume reabsorption. Whether there is a significant increase in proximal tubule volume reabsorption associated with the Na+ transporter redistribution during phenol-induced hypertension is an important question that remains to be answered. In any case, the hypertension evoked by the acute phenol injection does not lead to the homeostatic compensation known as "pressure natriuresis" or to the three- to fourfold increase in CLi observed during arterial constriction hypertension, evidence for a significant resetting of the renal function curve (16).
The distinct responses to hypertension provoked by phenol injection vs. arterial constriction persist at the molecular level. Our laboratory previously studied the molecular mechanisms responsible for the decrease in proximal tubule Na+ reabsorption during arterial constriction hypertension and discovered there is an accompanying retraction of transport competent NHE3 as well as NaPi2 from apical brush border to intermicrovillar cleft and subapical membrane pools and inhibition of basolateral Na+-K+-ATPase (25, 42, 47). In contrast, during acute phenol injury-induced hypertension, NHE3 and NaPi2 redistribute in the opposite direction from subapical endosomes to the apical brush border. The two hypertension-dependent patterns appear remarkably distinct when analyzed by confocal microscopy. Although blood pressure was raised to the same extent (~20-30 mmHg) in both models, proximal tubule NHE3 was internalized in the arterial constriction hypertension, presumably a compensatory response, but not in the phenol injection-induced hypertension. By confocal analysis, there is no obvious shift of NHE3 from subapical stores to the microvilli. There are a variety of interpretations that could be resolved by electron microscopy analysis; NHE3 may redistribute laterally from stores in the intermicrovillar cleft to the microvilli (decreased traffic to the cleft or increased traffic to the microvilli) or redistribution from the endosomal pools (decreased internalization or increased exocytosis) is below the level of detection by immunofluorescence, because the concentration of NHE3 is too low at baseline. Perhaps SNS activation without hypertension would lead to a more obvious redistribution of NHE3 to the brush border.
Girardi et al. (15) have studied proteins that associate with proximal tubule NHE3 by coimmunoprecipitation and discovered that DPPIV associates with NHE3 predominantly in the microvillar fraction in which NHE3 is active, as opposed to the intermicrovillar cleft region, suggesting that the association may affect NHE3 surface expression and/or activity. During phenol injection-induced hypertension, we found that DPPIV was recruited to the apical membranes in WI along with NHE3, which is consistent with the findings of Girardi et al. (15) that this would indicate increased association and NHE3 activity in the microvilli. In contrast, during arterial-constriction hypertension, DPPIV is internalized along with NHE3 (47), which is also consistent with a functional link between NHE3 and DPPIV.
The pattern of redistribution of NaPi2 to WI apical-enriched membranes during phenol injury-induced hypertension was distinct from that of NHE3 redistribution to WI; NHE3 was redistributed from WIII (intermicrovillar cleft, dense apical tubules, and endosomes) and NaPi2 was redistributed from WII (apical, intermicrovillar cleft, and dense apical tubule). This finding suggests that NaPi2 is translocated from intermicrovillar cleft or dense apical tubules, not intracellular endosomes, to the apical surface. This interpretation is consistent with Murer et al. (31), demonstrating that when NaPi2 is translocated from apical brush border to endosomes during parathyroid hormone treatment or high dietary Pi, it is directly routed to lysosomes for degradation, and recovery of transport activity requires de novo synthesis of NaPi2 rather than redistribution from an endosomal pool. A relevant in vivo study demonstrated that a rapid adaptive increase in renal proximal tubule apical NaPi2 abundance in response to acute administration of a low-Pi diet is independent of de novo protein synthesis mediated by microtubule-dependent translocation of presynthesized NaPi2 protein to the apical brush border (21, 24), suggesting the existence of an intracellular NaPi2 pool of limited size that could be involved in the fine adjustment of renal Pi reabsorption. Whether the NaPi2 that moved to WI is nascent NaPi2 en route to the apical membrane or from a recruitable pool was not determined.
The activity of the apical brush-border marker alkaline phosphatase is also differentially regulated by hypertension induced by phenol injury vs. arterial constriction. After phenol injection, alkaline phosphatase activity shifts to the apical membrane-enriched WI from WII and WIII. During arterial constriction, total alkaline phosphatase activity is decreased and the peak shifts out of lower density apical membranes (fractions 3-5, WI) into higher density membranes (fraction 6, WII) (47). These results suggest alkaline phosphatase may also be involved in the regulation of NHE3 and/or NaPi2 traffic through its association or dissociation with these apical transporters.
In summary, in this neurogenic hypertensive model, acute phenol injection provokes a rapid redistribution of NHE3 and NaPi2 to the apical microvilli. Renal denervation prevented the NHE3 redistribution, suggesting that SNS activation of proximal tubule Na+ transport in vivo is mediated by recruiting transporters to the apical brush border. Renal denervation also prevented the development of hypertension, suggesting that the proximal tubule response may play a role in the generation or maintenance of the phenol injury-induced hypertension. Finally, the results provide evidence for bidirectional regulation of NHE3 and NaPi2 during hypertension; transporters may be internalized consistently with a compensatory response, as observed in arterial constriction hypertension, or may be recruited to the microvilli in a fashion that would contribute to hypertension, as observed in neurogenic phenol injury-induced hypertension.
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ACKNOWLEDGEMENTS |
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We are grateful to Michaela Mac Veigh for assistance with confocal microscopy.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34316, fellowship support from the AHA Western States Affiliate (L. E. Yang and P. K. K. Leong), and National Institutes of Health Core Center Grant DK-48522.
Address for reprint requests and other correspondence: A. A. McDonough, Dept. of Physiology and Biophysics, Univ. of Southern California Keck School of Medicine, 1333 San Pablo St., Los Angeles, CA 90089-9142.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 11, 2002;10.1152/ajprenal.00134.2002
Received 10 April 2002; accepted in final form 10 September 2002.
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