1 Department of Physiology and Biophysics and 2 Division of Nephrology, Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California 90089-9142
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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+-Pi 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-µl 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
1- and
1-subunits, and epithelial
Na+ channel (ENaC)
- and
-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.
sodium transport; membrane traffic; sympathetic nervous system; phenol
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A MODEL OF NEUROGENIC HYPERTENSION provoked by intrarenal injection of phenol into the cortex of a pole of one kidney was recently developed by Ye and Campese (8, 33, 34). In this model, a 50-µl 10% phenol injection causes a rapid elevation of blood pressure, preceded by a rise in norepinephrine secretion from the posterior hypothalamus and an increase in renal sympathetic nervous system activity. Renal denervation before phenol injection prevents the sympathetic nervous system activation as well as the rise in blood pressure. These results are consistent with the interpretation that this phenol 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 the point when it is just a microscopic scar. The cellular and molecular bases for the hypertension are not clearly understood. One potential contributor is activation of sodium and volume reabsorption mediated by renal efferent sympathetic nerve activity.
A dynamic relationship between blood pressure and renal sodium
reabsorption is responsible, at least in part, for the blood pressure
set point (15). Increases in sodium transport can be responsible for the generation and maintenance of hypertension, whereas
decreases in sodium transport may be evidence of homeostatic compensation for elevated blood pressure. For example, an experimental increase in blood pressure acutely decreases proximal tubule sodium reabsorption, which both increases NaCl delivery at the macula densa, a
transglomerular feedback signal to normalize renal blood flow and
glomerualr filtration rate, and causes pressure-natriuresis that
reduces extracellular volume, which in turn counteracts the hypertension (6, 9, 10). In contrast, if renal sodium reabsorption is elevated due to excess production of an antinatriuretic (e.g., aldosterone) (30) or to a mutated epithelial sodium
channel (ENaC; Liddle's syndrome), then extracellular volume increases and blood pressure rises. The secondary hypertension depresses sodium
reabsorption at sites along the nephron, for example, the thiazide-sensitive Na+-Cl cotransporter (NCC)
(30), to match sodium excretion to sodium intake, a
pressure-natriuresis variant known as "escape." Although these
phenomena are well established as important for the maintenance of
extracellular volume and blood pressure, many questions remain regarding the molecular mechanisms responsible for regulation of sodium
transporters along the nephron in compensating for hypertension or in
generating and maintaining hypertension.
This laboratory has investigated the proximal tubule sodium transporter responses during experimental acute hypertension induced by increasing peripheral resistance as well as in the spontaneously hypertensive rat (SHR). In both models, there was a retraction of Na+/H+ exchangers (NHE3) and Na+-Pi cotransporters (NaPi) from the apical brush border to the intermicrovillar cleft and subapical endosomes, as demonstrated by both subcellular fractionation and confocal microscopy (21, 36, 37). In addition, there was a decrease in basolateral Na-K-ATPase activity with the onset of hypertension in both models (21, 37). Recently, we analyzed the acute response (30 min) to phenol injury in the rat cortex and discovered that NHE3 and NaPi redistributed from intracellular membranes to the apical microvilli, mediated by sympathetic nervous system activation, a response that could contribute to increased sodium/volume status (32). Motivated by these findings, we aimed to determine the chronic (5 wk) effects of phenol renal injury on proximal tubule sodium transporter distribution, namely, whether the proximal tubule sodium transporters would maintain a redistribution to the apical membranes or whether the hypertension would drive a retraction of proximal tubule sodium transporters from the apical microvilli as seen in the increased peripheral resistance and SHR models. It has been reported that the kidney can escape from certain sodium-retention disorders, such as hyperaldosterone states, by downregulating renal sodium transporters, such as the NaCl transporter of the distal tubule, to counteract the sodium retention (30). Therefore, in the present study we looked for evidence of escape during chronic phenol injury-induced hypertension by examining the total pool size of renal sodium transporters along the nephron. The results demonstrate that the redistribution of NHE3 and NaPi2 to apical microvilli and Na-K-ATPase activity to the plasma membranes persists for 5 wk after phenol injury and a decrease in cortical NHE3 abundance as evidence for a coincident escape mechanism in the same region of the nephron.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal preparation. Experiments were performed in male Sprague-Dawley (SD) rats (280-320 g body wt) that had free access to food and water. After anesthesia with an intramuscular injection of ketamine (Fort Dodge Laboratories) and xylazine (1:1, vol/vol, Miles), the left kidney was exposed via a dorsal incision, 50 µl of 10% phenol or saline were injected into the lower pole of the renal cortex, the incision was sutured closed, and the rats were returned to the vivarium, where they had free access to food and water. After 5 wk, rats were anesthetized as above and placed on a thermostatically controlled warming table to maintain body temperature at 37°C. A polyethylene catheter was placed into the carotid artery to record blood pressure. In one set, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (35 mg/kg).
Homogenization and subcellular fractionation.
The procedure for subcellular fractionation of renal cortical membranes
has been described previously (38, 39). In brief, the
noninjected right kidney was cooled in situ by flushing with cold PBS
and then excised. The renal cortices and medullas were dissected,
homogenized 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)] with a Tissuemizer (Tekmar
Instruments), and centrifuged at 2,000 g for 10 min; the pellet was rehomogenized and centrifuged, and the low-speed
supernatants (So) were pooled. The cortical So
was loaded between two hyperbolic sorbitol gradients and centrifuged at
100,000 g for 5 h, and 12 fractions were then 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 assay. To simplify the
measurements, fractions were pooled into three windows based on
previous analyses (31, 37): window I
(WI; fractions 3-5) is enriched in apical
brush border and basolateral membrane markers, window II
(WII; fractions 6-8) is enriched in
intermicrovillar cleft and dense apical tubule markers, and
window III (WIII; fractions 9-11)
is enriched in endosomal markers.
Immunoblot analysis and antibodies.
To determine the distribution of proteins in the sorbitol gradient, a
constant volume of each window was assayed and expressed as the
percentage of the total in all three windows. To assess the total pool
size of transporters expressed along the nephron, a constant amount of
So protein (2,000-g supernatant of homogenate) was analyzed. In all assays, one-half the volume or one-half the protein was also assayed to verify linearity of the detection system.
Samples were denatured in SDS-PAGE sample buffer for 30 min at 37°C,
resolved on 7.5% SDS polyacrylmide gels according to Laemmli
(20), and transferred to polyvinylidene difluoride membranes (Millipore Immobilon-P). Polyclonal antisera to NHE3 [NHE3-C00; McDonough laboratory (31)] and to NaPi2 [J.
Biber and H. Murer, University of Zürich, Zurich, Switzerland]
were used at 1:2,000 dilution. Polyclonal antisera to dipeptidyl
peptidase IV (DPPIV; M. Farquhar, Univ. of California at San Diego)
were used at 1:1,000 dilution. A monoclonal antibody specific for
Na-K-ATPase -subunit (464.6) (M. Kashgarian, Yale Univ.) was used at
1:200 dilution. Polyclonal anti-Na-K-ATPase
-subunit (McDonough
laboratory) and a polyclonal antiserum to NaCl transporter (TSC
; D. Ellison, Oregon Health and Science Univ.) were used at 1:500 dilution. Monoclonal anti-Na-K-2Cl transporter antibody (T4; C. Lytle, Univ. of
California at Riverside) and polyclonal anti-NHERF1 antibody (R-1046;
E. Weinman, Univ. of Maryland School of Medicine) were used at 1:3,000
dilution. Polyclonal antisera to ENaC
- and
-subunits (Chemicon) were used at 1:1,000 dilution. Except for
- and
-ENaC, all blots were incubated with Alexa 680-labeled goat anti-rabbit (Molecular Probes, Eugene, OR) or goat anti-rabbit IRDye800 or goat
anti-mouse IRDye800 secondary antibody (both from LI-COR, Lincoln, NE),
detected with an Odyssey Infrared Imaging System (LI-COR), and
quantitated using the accompanying LI-COR software.
- and
-ENaC
were detected with the enhanced chemiluminescence (ECL) kit (Amersham
Pharmacia Biotech), and 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.
Indirect immunofluorescence. Immunofluorescence analysis was conducted as described in detail previously (31). In brief, the kidney contralateral to the saline or phenol injection was fixed in situ (without perfusion of fixative to avoid changing renal perfusion pressure), cut in half on a midsagittal plane, and postfixed in periodate-lysine-paraformaldehyde, incubated overnight in 30% sucrose in PBS, embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA), and frozen in liquid nitrogen for 5-µm cryosectioning. Sections were incubated with 1% SDS in PBS for 4 min for antigen retrieval (7), SDS was removed by washing in PBS, and then sections were blocked with 1% bovine serum albumin in PBS. Double labeling was performed by incubating with polyclonal antiserum NHE3-C00 and a monoclonal antibody against villin (Immunotech, Chicago, IL) and then detected with a mixture of FITC-conjugated goat anti-rabbit (Cappel Research Products, Durham, NC) and Alexa 568-conjugated goat anti-mouse (Molecular Probes), as described previously (31). Slides were viewed with a Nikon PCM Quantitative Measuring High-Performance Confocal System equipped with filters for both FITC and TRITC 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).
Assays. Na+-K+-ATPase activity was measured by the potassium-dependent p-nitrophenyl phosphatase (K+-pNPPase) reaction (27), alkaline phosphatase activity was measured as described (25), and protein concentration was measured with the BCA assay kit (Pierce Technology, Iselin, NJ).
Quantitation and statistical analysis. Data are expressed as means ± SE. Differences were regarded significant at P < 0.05. In cellular fractionation assays, two-way ANOVA was applied to determine whether there was a significant effect of treatment on the overall pattern. If significance was established, the location of the difference in the pattern was assessed by two-tailed Student's t-test for paired samples. Differences in total cell sodium transporters were assessed by two-tailed Student's t-test for paired samples.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of intrarenal phenol injection on systolic arterial
pressure.
Ye et al. (33, 34) have reported that a limited renal
injury through an intrarenal injection of 50 µl of 10% phenol caused an immediate and permanent form of neurogenic hypertension. In this
study, we independently verified this finding in a different laboratory. Five weeks after 50 µl of 10% phenol or saline were adminstered in the lower pole of one renal cortex, arterial blood pressure was measured in ketamine- and xylazine-anesthetized rats (n = 6) via arterial cannulation, and systolic blood
pressure was significantly elevated in the phenol-injected rats
(129 ± 3 mmHg) compared with the saline-injected rats (115 ± 3 mmHg). In a subgroup of the ketamine/xylazine-anesthetized rats
(n = 3), blood pressure was measured by tail cuff
before injection and 5 wk thereafter in conscious rats: phenol
injection increased systolic pressure from 123 ± 1 to 157 ± 2 mmHg, whereas injection of 50 µl saline did not change blood
pressure. Because we and others have previously reported that blood
pressure measured in anesthetized SHRs is higher with pentobarbital
sodium compared with ketamine/xylazine (22, 37), we also
checked systolic arterial pressure in pentobarbital sodium-anesthetized
rats (n = 3) 5 wk after phenol injection (rats not used
in further experiments) and found that the measured blood pressure was
indeed higher than that in the ketamine/xylazine group (154 ± 8 vs. 113 ± 11 mmHg in phenol-injected vs. saline-injected rats).
This may be attributed to the observation that xylazine, a centrally
acting 2-adrenergic agonist, promotes urinary sodium
excretion by a renal nerve-dependent pathway (24). These
measurements confirm the previous report that a single phenol injection
provokes persistent hypertension (33) and that the
measured blood pressure is higher with pentobarbital sodium anesthesia
compared with ketamine/xylazine.
Immunoblot detection of NHE3 distribution in phenol injury-induced
chronic hypertension.
NHE is the major route for apical sodium entry across the proximal
tubule, and NHE3 is responsible for virtually all the
Na+/H+ exchange activity in this region
(1, 4). We recently discovered that the acute hypertension
established 30 min after intrarenal injection of 50 µl of 10% phenol
is associated with redistribution of NHE3 immunoreactivity from
intermediate-density membranes enriched in markers of intermicrovillar
cleft and endosomal pools to lower density membranes enriched in apical
brush-border microvillar markers, a response that may contribute to the
generation of phenol injury-induced hypertension and a response blocked
by prior renal denervation (32). Our previous studies in a
model of chronic hypertension found that as chronic hypertension
developed with age in the SHR, NHE3 redistributed in the opposite
direction: from lower density membranes enriched in markers of apical
microvilli to higher density membranes enriched in markers of the
intermicrovillar cleft and endosomes, a response also verified by
confocal microscopy (36), which provides evidence for a
homeostatic compensation to the developing hypertension
(21). These disparate findings stimulated us to examine
whether NHE3 would remain shifted to the apical membranes, as evidenced
in the acute response to phenol injury, or would retract to the
intermicrovillar membranes, as evidenced in the chronic hypertensive
SHR. NHE3 distribution was studied in the contralateral kidney 5 wk
after phenol or saline injection. Figure
1A shows representative
immunoblots of NHE3 in the renal cortical membranes fractionated into
the three defined windows (WI is enriched in apical
brush-border markers alkaline phosphatase, DPPIV, and NHE3;
WII contains most of the intermicrovillar cleft marker
megalin as well as the apical markers; and WIII is enriched
in megalin as well as the endosomal marker rab 5a and the lysosomal
marker -hexosaminidase) (31, 37). Because a constant
volume, rather than protein, of each window was analyzed, the total
immunoreactivity in the saline-vs.-phenol samples is not expected to be
identical. The differences in total NHE3 are analyzed subsequently. The
results indicate that the phenol injection-induced shift of NHE3 out of
WIII into WI seen at 30 min persists for 5 wk
(Fig. 1B): WI NHE3, expressed as %total NHE3 in
the gradient, contains 25.3 ± 3% in the phenol group and
12.7 ± 2.7% in the saline group; WII NHE3 is
unchanged, 65.6 ± 2.3% in the phenol group and 68.4 ± 1.9% in the saline group; WIII NHE3 contains 9.1 ± 1.4% in the phenol group and 18.9 ± 3.4% in the saline group. These results indicate that there is a persistent signal for net traffic of sodium transporters to the surface in the chronic
hypertensive phenol-injected group compared with saline-injected
controls. The pattern is distinct from the internalization of NHE3 that was observed in the chronic hypertension of SHRs or Goldblatt two
kidney, one-clip (2K1C) hypertension (21, 36).
|
Indirect immunocytochemistry of NHE3 in phenol injury-induced
chronic hypertension.
NHE3 distribution in phenol-induced hypertension was also visualized by
immunocytochemistry. Five weeks after phenol or saline injection,
kidneys were fixed in situ for 20 min as described in
EXPERIMENTAL PROCEDURES. Cryosections harvested from both
groups were double labeled: NHE3 was detected with polyclonal NHE3-C00- and FITC-conjugated goat anti-rabbit secondary antibody; villin, the
actin-bundling protein found in the microvilli, was detected with
monoclonal anti-villin together with Alexa 568-conjugated goat
anti-mouse secondary antibody. In saline-injected rats, the staining of
NHE3 is restricted to the brush border, as evidenced by colocalization
with staining of villin (Fig. 2,
top). In phenol injury-induced
hypertension, there was no discernable change in NHE3 distribution
pattern, as it still colocalized with villin staining (Fig. 2,
bottom). While not quantitated directly, there is evidence
for lower levels of NHE3 in the phenol injury group, a point confirmed
by immunoblots and discussed in Fig. 7. This technique provides visual
confirmation that NHE3 is not distributed out of the villi and is
internalized to endosomes during phenol injury-induced chronic
hypertension as reported in the chronic hypertension in both SHRs and
Goldblatt 2K1C (36) and in acute hypertension by
increasing peripheral resistance (31, 36).
|
Distribution of other apical membrane proteins in phenol
injury-induced hypertension.
In our previous study, we demonstrated that 30 min after acute phenol
injection the percentage of proximal tubule NaPi2 in WI
apical microvilli increased from 9.5 ± 1.6 to 18.7 ± 1.5%
and NaPi2 in WII decreased by a similar percentage. DPPIV,
an NHE3-associated protein in microvilli (14), increased
in WI from 19.2 ± 1.6 to 28.6 ± 2.4% of the
total 30 min after phenol injection. Figure 3A summarizes the NaPi2
distribution 5 wk after phenol injection during the chronic
hypertension phase. There was a significant difference in the NaPi2
distribution in the phenol- compared with saline-injected rats,
indicating a shift out of WII into WI: NaPi2 in
WI, expressed as %total in the gradient, was 22 ± 2.3 (phenol) vs. 11.2 ± 2.1% (saline); WII NaPi2 was
60.7 ± 1.8 (phenol) vs. 70.7 ± 2.4% (saline); and there
was no change in WIII. The results indicate that NaPi2 may
redistribute, as in the acute phase of hypertension (30 min after
phenol injection), from the intermicrovillar cleft region and/or dense
apical tubules (WII) to apical membranes (WI)
during phenol injury-induced hypertension. The redistribution of the
classic apical membrane protein DPPIV also persisted, similar to that
of DPPIV at 30 min after phenol injection: a slight but significant
increase in WI to 24.4 ± 11% of total from 18.1 ± 8.1% 5 wk after phenol injury (Fig. 3B), supporting a
functional link between NHE3 and DPPIV.
|
|
Basolateral membrane Na-K-ATPase activity in phenol injury-induced
hypertension.
Our previous investigations suggest that renal cortical Na-K-ATPase
activity falls as hypertension develops in both acute hypertension from
arterial constriction (22, 37) and chronic hypertension,
as in the developing SHR (21). However, 30 min after
phenol injection we did not observe any change in Na-K-ATPase activity
or distribution (32). During the chronic
hypertension phase 5 wk after phenol injection (Fig.
5), Na-K-ATPase activity was
significantly shifted to the basolateral membranes found in WI, perhaps mechanistically similar to the report that the
sympathetic -agonist isoproterenol increases surface expression of
Na-K-ATPase in cultured lung cells (2).
|
Profiling of sodium transporter abundance in renal cortex and
medulla.
To determine whether the total pool size of sodium transporters located
along the nephron was altered 5 wk after phenol injection during
chronic hypertension, So samples (homogenates subjected to
a 2,000-g spin to remove poorly homogenized bits) of
cortical and medullary membranes from saline- and phenol-injected rats were assayed and immunoreactivity was quantified. Figure
6 demonstrates the linearity of the
infrared imaging system (LI-COR). The relative distribution of these
transporters in the cortex vs. medulla was also verified: NHE3, the
thiazide-sensitive NCC, and NaPi2 were enriched in the cortex, the
bumetanide-sensitive Na+-K+-2Cl
cotransporter was expressed in the medulla, and Na-K-ATPase
1- and
-subunits were found in both the cortex and
medulla, with more in the latter. Figure
7 summarizes the immunoblots of these transporters and proteins in saline- vs. phenol-injected rats. To
ensure linearity, each sample was run twice, with one-half the protein
loaded in the second lane. The densitometric quantitation is shown in
Table 1. In the cortex, there was a
remarkable decrease in NHE3 protein in phenol-induced hypertensive rats
to 58.1 ± 7.4% of that measured in the saline-injected controls.
This fall in cortical NHE3 was region specific, as there was no
significant change in NHE3 total abundance in the medulla. There was
also a tendency toward a decrease in total renal cortical Na-K-ATPase activity (Fig. 5C), which parallels the decrease in apical
NHE3 abundance, but this did not reach statistical significance and was
not paralleled by a change in total Na-K-ATPase subunit pool sizes
(Table 1). There were no other significant differences in
immunoreactivity of the other sodium transporters or in the NHE3-associated proteins DPPIV and NHE regulatory factor (NHERF) in
phenol-induced hypertension compared with saline-injected rats (Fig. 7,
Table 1).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ye and Campese (8, 33, 34) have characterized many
features of this phenol injury model that support a role for central and renal sympathetic nervous system (SNS) activation in the genesis and maintenance of hypertension. In brief, the injection of 50 µl
10% phenol into the cortex of one kidney leads to an immediate elevation of norepinephrine secretion from the posterior hypothalamus, a rise in blood pressure, and an increase in plasma norepinephrine level. Renal denervation before phenol injection prevents the increase
in both blood pressure and norepinephrine secretion from posterior
hypothalamus. Sympathetic activity recorded directly from renal nerves
increases after phenol injection, and the -adrenergic-receptor blocker phentholamine normalizes blood pressure (35).
Five weeks after phenol injection, the site of injection is reduced to
a microscopic scar, yet hypertension and elevated norepinephrine secretion from posterior hypothalamus persist; ablation of the injured
kidney at 4 wk normalizes blood pressure, perhaps due to elimination of
the renal afferent impulses (8). These findings all
support a role for central and renal SNS activation and
-adrenergic-receptor activation in the genesis and maintenance of
hypertension induced by phenol injury. These results complement the
evidence supporting a role of SNS activation in the pathogenesis of
hypertension induced by renal diseases, including chronic renal failure
(CRF) (5, 11, 34).
Our laboratory recently investigated the acute effects of phenol injury on renal sodium transport (32). Thirty minutes after phenol injection, NHE3 and NaPi2 were redistributed from the intermicrovillar cleft and intracellular membranes to the apical microvilli and apical alkaline phosphatase activity doubled. Additionally, the responses were prevented by prior denervation. These findings provide the first in vivo evidence that SNS stimulation activates proximal tubule apical sodium entry by recruiting NHE3 and NaPi2 transporters to the apical surface, responses that may contribute to the generation and maintenance of elevated blood pressure by hampering the pressure-natriuresis response. The results of this study demonstrate that a single injection of phenol into one kidney can cause permanent hypertension associated with a persistent redistribution of renal cortical NHE3, NaPi2, and Na-K-ATPase to the plasma membrane and an increase in alkaline phosphatase activity, rather than a return to the basal pre-phenol injection pattern or a change in the pattern observed in other models of chronic hypertension (SHR, 2K1C), where NHE3 is retracted out of the microvilli (see below). In addition, this study demonstrates a significant decrease in NHE3 pool size in the renal cortex 5 wk after phenol injury, an escape phenomenon that would counter the effect of redistributing a larger fraction of sodium transporters to the apical cell surface.
Comparing the acute and chronic proximal tubule responses to hypertension of distinct origins illustrates that an alteration in renal sodium transport may be causal with regard to some varieties of hypertension and compensatory in other varieties. The proximal tubule sodium transporter responses in the phenol injury model of chronic hypertension are quite distinct from that seen in chronic hypertension in the SHR. In young prehypertensive SHRs, NHE3 and Na-K-ATPase activity in renal cortex are higher vs. in age-matched Wistar-Kyoto (WKY) (16, 26) or SD rats (21), suggesting that elevated sodium transport may contribute to the development of hypertension. These differences in activity disappear in adult SHRs with established hypertension vs. WKY or SD rats (13, 21). Biochemical (21) and confocal immunofluorescence (36) studies reveal that NHE3 is localized to the apical brush border in young prehypertensive SHRs and then redistributes to the intermicrovillar cleft and subapical membranes as hypertension becomes established in adult SHRs, mimicking the redistribution in SD rats challenged by acute hypertension (37, 38). NHE3 is similarly retracted in the Goldblatt 2K1C model (36). The retraction of NHE3 as hypertension develops (acutely or chronically) is likely a homeostatic compensation to normalize salt and water balance as well as a key mechanism to stimulate transglomerular feedback. The proximal tubule responses in the phenol-induced hypertension model are the opposite to that seen in the SHR. Specifically, the NHE3 distribution patterns are nearly indistinguishable between the acute phase (30 min) of phenol injury and chronic phase of hypertension (5 wk), demonstrating a persistent shift of NHE3 to apical microvilli from internal membranes: in the acute phase, the NHE3 percentage in WI is 27.2 ± 4.1 (phenol) vs. 13.1 ± 2% (saline), that in WII is unchanged, and in WIII is 10.8 ± 2 (phenol) vs. 22.8 ± 4.8% (saline); after 5 wk, the percentage in WI is 25.3 ± 3 (phenol) vs. 12.7 ± 2.7% (saline), that in WII is unchanged, and in WIII is to 9.1 ± 1.4 (phenol) vs. 18.9 ± 3.4% (saline). NaPi2 and DPPIV distribution patterns, as well as alkaline phosphatase activation, are also similar in acute and chronic phases of phenol-induced hypertension. The fact that the percentages of NHE3, NaPi2, and DPPIV and the activity of alkaline phosphatase in the microvilli are persistently increased goes along with the observed persistence of SNS stimulation. In summary, the responses of proximal tubule transporters to the chronic hypertension in the phenol injury model are the opposite to those seen in the chronic hypertension of the SHR and Goldblatt 2K1C models. We speculate that the signals driving the internalization of NHE3 and NaPi2 during hypertension per se are overridden by opposing signals, most likely the activated SNS, which drive transporters to the apical membrane, associated with a significant blunting of the pressure-induced diuresis and natriuresis.
There have been many reports of the effects of norepinephrine on
proximal tubule sodium transport and transporters in isolated proximal
tubules. In brief, norepinephrine stimulates sodium reabsorption and
ouabain-sensitive rubidium uptake and decreases intracellular sodium,
evidence for an increase in plasma membrane Na-K-ATPase number or
activity (12). Studies of the signaling mechanisms in
isolated proximal tubule have shown that -agonist oxymetazoline stimulation of Na-K-ATPase transport activity was prevented by either
1- or
2-receptor antagonism
(17), and studies in cultured proximal tubule cells
demonstrate a
2-adrenoreceptor-mediated increase in
Na-K-ATPase transport activity secondary to increased apical sodium
entry (29). In this study, we observed a redistribution of
Na-K-ATPase activity to WI, where the peak basolateral
membrane Na-K-ATPase resides (37), and did not measure
stimulation of total Na-K-ATPase Vmax activity
in a membrane preparation after phenol injury-induced hypertension.
These findings are in agreement with the norepinephrine studies in cell
and tubules, where transport activity in the membrane was assessed. The
findings are also in agreement with studies in cultured lung cells,
where adrenergic agents have been shown to stimulate Na-K-ATPase via
insertion of Na-K-ATPase from intracellular vesicles to the plasma
membranes (2). We have previously shown that hypertension
per se decreases Na-K-ATPase in the renal cortex (22, 23,
37), so it is likely that the Na-K-ATPase activity and
distribution after phenol injury may be the product of the combined
multiple stimuli of SNS stimulation and hypertension.
Hypertension and other renal pathologies can alter the total abundance
of sodium transporters along the nephron in a pathology-dependent fashion. For example, in certain disorders of extracellular volume expansion, kidneys can escape from sodium retention by downregulating one or more of renal sodium transporters, thereby depressing sodium reabsorption to match sodium excretion to sodium intake. The molecular mechanisms responsible for regulation of sodium transporters along the
nephron have been investigated in a number of models (30). For example, when the aldosterone level is inappropriately elevated, e.g., primary aldosteronism, the reabsorptive activity of ENaCs is
increased while the renal abundance of NCC is profoundly and selectively decreased. This response appears to be the chief molecular mechanism by which the kidney overcomes the sodium-retentive effect of
aldosterone. Similarly, long-term pressure-natriuresis has been
reported to be associated with inhibition of distal tubule sodium
transporters (23). In contrast, increased Na-K-ATPase, NCC, and ENaC protein pool sizes are increased in the obese Zucker rat
and may be responsible for sodium retention and hypertension (3). In the present study, phenol injury-induced chronic
hypertension is associated with a 44% fall in total NHE3 abundance in
the renal cortex. When one considers the fact that NHE3 distribution in WI roughly doubles (from 12.7 ± 2.7 to 25.3 ± 3%), this 44% drop in total cortical NHE3 abundance would return the
total amount of NHE3 in apical membranes (WI) close to that
seen before phenol injection. Therefore, this marked reduction may be
the major molecular mechanism responsible for adaptation to chronic
stimulation of SNS activity and may contribute to resetting the
pressure-natriuresis relationship. It remains to be determined whether
this decrease in NHE3 is due to decreased synthesis or increased
degradation. Proximal tubule NHE3 abundance is also reduced 50% in CRF
induced by
In summary, the persistent redistribution of NHE3, NaPi2, and Na-K-ATPase to the plasma membranes may contribute to the generation and/or maintenance of chronic hypertension induced by phenol injury. In parallel, the decrease in total cortical NHE3 abundance in the chronic phase of phenol-induced hypertension is evidence for a coincident escape mechanism in the same region of the nephron that could counteract the effect of redistributing a larger fraction of sodium transporters to the apical cell surface.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Shaohua Ye for guidance and advice regarding chronic phenol model implementation. Michaela MacVeigh provided assistance in confocal microscopy.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34316 and DK-48522 as well as fellowship support from the American Heart Association, Western States Affiliate (to L. E. Yang and P. K. K. Leong).
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 (E-mail: mcdonoug{at}hsc.usc.edu).
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 January 28, 2003;10.1152/ajprenal.00317.2002
Received 5 September 2002; accepted in final form 10 January 2003.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Azuma, KK,
Balkovetz DF,
Magyar CE,
Lescale-Matys L,
Zhang Y,
Chambrey R,
Warnock DG,
and
McDonough AA.
Renal Na+/H+ exchanger isoforms and their regulation by thyroid hormone.
Am J Physiol Cell Physiol
270:
C585-C592,
1996
2.
Bertorello, AM,
Ridge KM,
Chibalin AV,
Katz AI,
and
Sznajder JI.
Isoproterenol increases Na+-K+-ATPase activity by membrane insertion of -subunits in lung alveolar cells.
Am J Physiol Lung Cell Mol Physiol
276:
L20-L27,
1999
3.
Bickel, CA,
Verbalis JG,
Knepper MA,
and
Ecelbarger CA.
Increased renal Na-K-ATPase, NCC, and -ENaC abundance in obese Zucker rats.
Am J Physiol Renal Physiol
281:
F639-F648,
2001
4.
Biemesderfer, D,
Pizzonia J,
Abu-Alfa A,
Exner M,
Reilly R,
Igarashi P,
and
Aronson PS.
NHE3: a Na+/H+ exchanger isoform of renal brush border.
Am J Physiol Renal Fluid Electrolyte Physiol
265:
F736-F742,
1993
5.
Bigazzi, R,
Kogosov E,
and
Campese VM.
Altered norepinephrine turnover in the brain of rats with chronic renal failure.
J Am Soc Nephrol
4:
1901-1907,
1994[Abstract].
6.
Briggs, JP,
and
Schnermann JB.
Whys and wherefores of juxtaglomerular apparatus function.
Kidney Int
49:
1724-1726,
1996[ISI][Medline].
7.
Brown, D,
Lydon J,
McLaughlin M,
Stuart-Tilley A,
Tyszkowski R,
and
Alper S.
Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS).
Histochem Cell Biol
105:
261-267,
1996[ISI][Medline].
8.
Campese, VM.
Neurogenic factors and hypertension in renal disease.
Kidney Int Suppl
75:
S2-S6,
2000[Medline].
9.
Chou, CL,
and
Marsh DJ.
Role of proximal convoluted tubule in pressure diuresis in the rat.
Am J Physiol Renal Fluid Electrolyte Physiol
251:
F283-F289,
1986
10.
Chou, CL,
and
Marsh DJ.
Time course of proximal tubule response to acute arterial hypertension in the rat.
Am J Physiol Renal Fluid Electrolyte Physiol
254:
F601-F607,
1988
11.
Converse, RL, Jr,
Jacobsen TN,
Toto RD,
Jost CM,
Cosentino F,
Fouad-Tarazi F,
and
Victor RG.
Sympathetic overactivity in patients with chronic renal failure.
N Engl J Med
327:
1912-1918,
1992[Abstract].
12.
Feraille, E,
and
Doucet A.
Sodium-potassium-adenosine triphosphatase-dependent sodium transport in the kidney: hormonal control.
Physiol Rev
81:
345-418,
2001
13.
Garg, LC,
and
Narang N.
Differences in renal tubular Na-K-adenosine triphosphatase in spontaneously hypertensive and normotensive rats.
J Cardiovasc Pharmacol
8:
186-189,
1986[ISI][Medline].
14.
Girardi, AC,
Degray BC,
Nagy T,
Biemesderfer D,
and
Aronson PS.
Association of Na+-H+ exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule.
J Biol Chem
276:
46671-46677,
2001
15.
Guyton, AC.
Blood pressure controlspecial role of the kidneys and body fluids.
Science
252:
1813-1816,
1991[ISI][Medline].
16.
Hayashi, M,
Yoshida T,
Monkawa T,
Yamaji Y,
Sato S,
and
Saruta T.
Na+/H+-exchanger 3 activity and its gene in the spontaneously hypertensive rat kidney.
J Hypertens
15:
43-48,
1997[ISI][Medline].
17.
Ibarra, F,
Aperia A,
Svensson LB,
Eklof AC,
and
Greengard P.
Bidirectional regulation of Na+,K+-ATPase activity by dopamine and an -adrenergic agonist.
Proc Natl Acad Sci USA
90:
21-24,
1993[Abstract].
18.
Knepper, MA.
Proteomics and the kidney.
J Am Soc Nephrol
13:
1398-1408,
2002
19.
Kwon, TH,
Frøkiær J,
Fernandez-Llama P,
Maunsbach AB,
Knepper MA,
and
Nielsen S.
Altered expression of Na transporters NHE-3, NaPi-II, Na-K-ATPase, BSC-1, and TSC in CRF rat kidneys.
Am J Physiol Renal Physiol
277:
F257-F270,
1999
20.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
21.
Magyar, CE,
Zhang Y,
Holstein-Rathlou NH,
and
McDonough AA.
Proximal tubule Na transporter responses are the same during acute and chronic hypertension.
Am J Physiol Renal Physiol
279:
F358-F369,
2000
22.
Magyar, CE,
Zhang Y,
Holstein-Rathlou NH,
and
McDonough AA.
Downstream shift in sodium pump activity along the nephron during acute hypertension.
J Am Soc Nephrol
12:
2231-2240,
2001
23.
Majid, DS,
and
Navar LG.
Blockade of distal nephron sodium transport attenuates pressure natriuresis in dogs.
Hypertension
23:
1040-1045,
1994[Abstract].
24.
Menegaz, RG,
Kapusta DR,
Mauad H,
and
de Melo Cabral A.
Activation of 2-receptors in the rostral ventrolateral medulla evokes natriuresis by a renal nerve mechanism.
Am J Physiol Regul Integr Comp Physiol
281:
R98-R107,
2001
25.
Mircheff, AK,
and
Wright EM.
Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na, K-ATPase rich membranes and the distribution of enzyme activities.
J Membr Biol
28:
309-333,
1976[ISI][Medline].
26.
Morduchowicz, GA,
Sheikh-Hamad D,
Jo OD,
Nord EP,
Lee DB,
and
Yanagawa N.
Increased Na+/H+ antiport activity in the renal brush border membrane of SHR.
Kidney Int
36:
576-581,
1989[ISI][Medline].
27.
Murer, H,
Ammann E,
Biber J,
and
Hopfer U.
The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase.
Biochim Biophys Acta
433:
509-519,
1976[ISI][Medline].
28.
Shenolikar, S,
and
Weinman EJ.
NHERF: targeting and trafficking membrane proteins.
Am J Physiol Renal Physiol
280:
F389-F395,
2001
29.
Singh, H,
and
Linas SL.
Role of protein kinase C in 2-adrenoceptor function in cultured rat proximal tubule epithelial cells.
Am J Physiol Renal Physiol
273:
F193-F199,
1997
30.
Wang, XY,
Masilamani S,
Nielsen J,
Kwon TH,
Brooks HL,
Nielsen S,
and
Knepper MA.
The renal thiazide-sensitive Na-Cl cotransporter as mediator of the aldosterone-escape phenomenon.
J Clin Invest
108:
215-222,
2001
31.
Yang, L,
Leong PK,
Chen JO,
Patel N,
Hamm-Alvarez SF,
and
McDonough AA.
Acute hypertension provokes internalization of proximal tubule NHE3 without inhibition of transport activity.
Am J Physiol Renal Physiol
282:
F730-F740,
2002
32.
Yang, LE,
Leong PK,
Ye S,
Campese VM,
and
McDonough AA.
Responses of proximal tubule sodium transporters to acute injury-induced hypertension.
Am J Physiol Renal Physiol
284:
F313-F322,
2003
33.
Ye, S,
Gamburd M,
Mozayeni P,
Koss M,
and
Campese VM.
A limited renal injury may cause a permanent form of neurogenic hypertension.
Am J Hypertens
11:
723-728,
1998[ISI][Medline].
34.
Ye, S,
Ozgur B,
and
Campese VM.
Renal afferent impulses, the posterior hypothalamus, and hypertension in rats with chronic renal failure.
Kidney Int
51:
722-727,
1997[ISI][Medline].
35.
Ye, S,
Zhong H,
Yanamadala V,
and
Campese VM.
Renal injury caused by intrarenal injection of phenol increases afferent and efferent renal sympathetic nerve activity.
Am J Hypertens
15:
717-724,
2002[ISI][Medline].
36.
Yip, KP,
Tse CM,
McDonough AA,
and
Marsh DJ.
Redistribution of Na+/H+ exchanger isoform NHE3 in proximal tubules induced by acute and chronic hypertension.
Am J Physiol Renal Physiol
275:
F565-F575,
1998
37.
Zhang, Y,
Magyar CE,
Norian JM,
Holstein-Rathlou NH,
Mircheff AK,
and
McDonough AA.
Reversible effects of acute hypertension on proximal tubule sodium transporters.
Am J Physiol Cell Physiol
274:
C1090-C1100,
1998
38.
Zhang, Y,
Mircheff AK,
Hensley CB,
Magyar CE,
Warnock DG,
Chambrey R,
Yip KP,
Marsh DJ,
Holstein-Rathlou NH,
and
McDonough AA.
Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F1004-F1014,
1996
39.
Zhang, YB,
Magyar CE,
Holstein-Rathlou NH,
and
McDonough AA.
The cytochrome P-450 inhibitor cobalt chloride prevents inhibition of renal Na,K-ATPase and redistribution of apical NHE-3 during acute hypertension.
J Am Soc Nephrol
9:
531-537,
1998[Abstract].