1 Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, California 90089-9142; and 2 Department of Medical Physiology, Division of Renal and Cardiovascular Research, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark
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ABSTRACT |
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Acute hypertension
inhibits proximal tubule (PT) sodium reabsorption. The resultant
increase in NaCl delivery to the macula densa suppresses renin release.
We tested whether the sustained pressure-induced inhibition of PT
sodium reabsorption requires a renin-mediated decrease in ANG II
levels. Plasma ANG II concentration of anesthesized Sprague-Dawley rats
was clamped by simultaneous infusion of the ANG I-converting enzyme
inhibitor captopril (12 µg/min) and ANG II (20 ng · kg1 · min
1). Blood
pressure was increased 50 mmHg for 20 min by arterial constriction ± ANG II clamp or by sham operation. This acute
hypertension increased urine output and endogenous Li+
clearance, and these responses were blunted 40-50% in ANG II clamped rats. Acute hypertension provoked a rapid redistribution of
Na+/H+ exchanger isoform 3 (NHE3) out of apical
brush-border membranes (21 ± 4% decrease of total NHE3
abundance) to endosomal/lysosomal membranes (16 ± 6% increase of
total). In ANG II-clamped rats, acute hypertension also provoked
disappearance of NHE3 from the apical membranes (27 ± 2%
decrease of total), but NHE3 was shifted to membranes enriched in
intermicrovillar cleft and dense apical tubules (step 1)
rather than endosomal/lysosomal membranes (step 2). This
difference was independently confirmed by confocal analysis. In
contrast, the pressure-induced redistribution of
Na+-Pi cotransporter type 2 was not altered by
ANG II clamp. We conclude that the responses to acute hypertension,
including diuresis and redistribution of PT NHE3 into intracellular
membranes, require a responsive renin-angiotensin system and that the
responses may be induced by the sustained increase in NaCl delivery to
the macula densa during acute hypertension.
kidney; tubuloglomerular feedback; sodium transport; blood pressure; sodium-phosphate cotransporter type 2
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INTRODUCTION |
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ACUTE INCREASE IN ARTERIAL pressure rapidly inhibits proximal tubule (PT) sodium reabsorption (8). The resultant increase in chloride delivery to the macula densa provides the error signal for the tubuloglomerular feedback (TGF) mechanism (1) to autoregulate glomerular filtration rate (GFR) and decreases renin release (25), which in turn downregulates the renin-angiotensin system (RAS). It remains unknown what signals mediate and sustain the error signal for the TGF response. In a study that investigated the PT response to acute hypertension in rats, Chou and Marsh (4) excluded nerve activity, high antidiuretic and antinatriuretic hormone levels, and other physical factors affiliated with acute hypertension as potential mediators of the error signal in rats. However, ANG II was suggested as a likely candidate, because a renin-mediated decrease in systemic ANG II concentration ([ANG II]) could reduce the stimulus to PT Na+ reabsorption. Furthermore, Sorensen et al. (26) also demonstrated that clamping systemic [ANG II] improved renal blood flow autoregulation range but abolished the ability to reset renal blood flow autoregulatory range in rats, which is evidence for the importance of an intact RAS for the hemodynamic responses during acute pressure change. However, the specific effects of ANG II on pressure-induced inhibition of PT Na+ reabsorption remain to be established.
It is well documented that the majority of the PT luminal Na+ reabsorption (NaHCO3 and NaCl) is coupled to Na+/H+ exchanger (NHE) activity (7, 22, 23) and that NHE isoform 3 (NHE3) is the primary isoform responsible for PT apical Na+ entry (19, 27). Our laboratory previously demonstrated that acute hypertension induces a rapid and reversible internalization of transport-competent NHE3 from the apical brush border to subapical endosomes that can account, at least in part, for the acute pressure-induced decrease in PT Na+ reabsorption (28, 29, 32). Na+-Pi cotransporter type 2 (NaPi2) also redistributes out of the apical brush border during acute hypertension, but the response does not appear to be reversible (29). Whether the pressure-induced renin-mediated decrease in ANG II production is necessary for this "redistribution" of NHE3 and NaPi2 during acute hypertension is the focus of this study.
In the present study, we investigated the hypothesis that the pressure-induced diuretic responses as well as the redistribution of apical Na+ transporters (NHE3 and NaPi2) were dependent on a responsive RAS. The RAS was clamped by simultaneous infusion of the angiotensin-converting enzyme (ACE) inhibitor captopril and ANG II to restore blood pressure (BP) to baseline. This ANG II clamp blunted the diuretic responses to acute hypertension. Furthermore, the ANG II clamp specifically inhibited the pressure-induced internalization of NHE3 but not NaPi2. These results indicate that both the diuretic response and the internalization of PT NHE3 during acute hypertension require an intact responsive RAS.
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METHODS |
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Animal preparation and surgical protocols. Experiments were performed in male Sprague-Dawley rats (300 ± 5 g body wt) that were kept under diurnal light conditions and had free access to food and water. Rats were anesthesized intramuscularly with ketamine (Fort Dodge Laboratories) and xylazine (1:1, vol/vol; Miles) and placed on a thermostatically controlled operating table to maintain body temperature at 37°C. Polyethylene catheters (PE-50) were placed into the carotid artery for BP monitoring and into the jugular vein for infusion of drugs and 4.0% BSA in 0.9% NaCl at 50 µl/min to maintain euvolemia. The same BSA/saline solution was used as the solvent vehicle for all drug infusion. The left ureter was cannulated with a Surflo iv catheter (Terumo) for urine collection. The same rats were used for measurement of urine output, endogenous Li+ clearance (CLi), and NHE3 distribution by subcellular fractionation (n = 4-6 for each treatment group), whereas different sets of rats were used for determination of GFR (n = 3 for control and n = 5 for ANG II clamp) and confocal microscopy analysis. All animal experiments were approved by the University of Southern California Keck School of Medicine and conducted in accord with the Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press, 1996).
ANG II clamp and induction of acute hypertension.
The ANG II level was clamped by inhibition of de novo ANG II synthesis
with captopril, an inhibitor of ACE (2), followed by
infusion of ANG II at a rate that restored baseline BP determined in an
analogous study (10a). Specifically, captopril was infused intravenously at 12 µg/min (9) for 10 min before the
coinfusion of captopril (12 µg/min) and ANG II (20 ng · kg1 · min
1; both from Sigma).
Measurements of GFR. GFR was determined by inulin clearance (13). FITC-inulin (5 mg/ml; Sigma), dissolved in BSA/saline, was infused at 50 µl/min for 60 min before the first urine sample was collected. At this rate, FITC-inulin equilibrated with the plasma by 50 min (data not shown). Urine and plasma samples were collected at 5- to 10-min intervals for FITC-inulin analysis. All samples collected were protected from direct ambient light and diluted with phosphate-buffered saline (pH 7.4), and the fluorescence was measured (excitation = 480 nm and emission = 530 nm) in 1-cm cuvettes with a PerkinElmer LS5 spectrofluorometer. The amount of FITC-inulin in the samples was quantified with known FITC-inulin standards (0.0125 to 0.2 mg/ml). GFR was calculated as urine inulin concentration × urine flow rate/plasma inulin concentration.
Urine output and endogenous CLi. Urine samples were collected from the ureter catheter at 5- or 10-min intervals. Urine volume was determined gravimetrically. A blood sample was collected at the end of each experiment for measurement of plasma Li+ concentration ([Li+]). The [Li+] in the plasma and urine samples was measured by flameless atomic absorption spectrophotometry (PerkinElmer 5100PC) (11). Endogenous CLi, a measure of the volume flow out of the PT, was calculated as [Li+] in urine × urine flow rate/[Li+] in the plasma sample obtained at the end of the experiment.
Homogenization and subcellular fractionation.
The procedure for homogenization of whole renal cortex and subcellular
fractionation of total cortical membranes has been described in detail
previously (29, 30). In brief, after being subjected to
centrifugation at 100,000 g for 5 h in a hyperbolic sorbitol gradient, the cortical membranes were collected into 12 fractions for the density gradient distribution analysis of NHE3 and
NaPi2. After collection, the gradient fractions with similar membrane
characteristics were pooled into three "windows" to simplify
analyses. Our laboratory previously reported the following membrane
assignment for the three windows (28): window I
(Win I, fractions 3-5) is enriched in apical
membrane markers alkaline phosphatase and dipeptidyl-peptidase IV;
window II (Win II, fractions 6-8)
contains a mixed pool of the apical membrane markers alkaline phosphatase and dipeptidyl-peptidase IV and the intermicrovillar cleft
and dense apical tubule membrane marker megalin; and window III (Win III, fractions 9-11) is
enriched in endosomal (rab5a) and lysosomal (-hexosaminidase)
membrane markers as well as the intermicrovillar cleft marker megalin.
Immunoblot analysis and antibodies.
For analysis of the relative distribution of NHE3 and NaPi2 in the
three pooled membrane windows, a constant volume from Win I-III was prepared in SDS-PAGE sample buffer
(final concentration: 2% SDS, 1% -mercaptoethanol, 0.25 mM
disodium EDTA, and 2.5 mM H2PO4/HPO4, pH 7.0), denatured at
37°C for 30 min, resolved on the same 7.5% SDS-PAGE gels (Criterion
precast gel, Bio-Rad), and transferred to polyvinylidene difluoride
membranes (Immobilon-P, Millipore) according to standard methods. For
detection of NHE3, we generated a polyclonal antibody NHE3-C00 against
amino acids 809-831 of the rat NHE3 (28).
Polyclonal anti-NaPi2 antibody (L697) was generously provided by Dr. M. Knepper (National Institutes of Health) and used at 1:2,000 dilution.
All antibody/antigen complexes were detected by enhanced
chemiluminescence (ECL kit, Amersham Pharmacia Biotech), and the
autoradiographic signals were quantified with a Bio-Rad Imaging
Densitometer equipped with Molecular Analyst software. Selected samples
were run at one-half volume on each blot to ensure that signals were in
the linear range of detection. Multiple time exposures of
autoradiograms were analyzed to avoid signals beyond the linear range
of the film (Biomax MR, Eastman Kodak). Within each experiment, the
arbitrary density in each window was divided by the total density in
all three windows, defined as 100%, before all the experiments in a
set were summarized. To illustrate the redistribution more clearly for
the report, individual fractions 4-11 from control,
hypertension, and ANG II clamp hypertension were resolved on the same
26-well gel and included in the figures. Fraction 3 was not
included in these illustrations (although it was included in the
analysis of the three windows) because of the limited number of wells
and because our laboratory's recent experience with the gradients indicated that there was no detectable NHE3 or NaPi2 in fraction 3 (28).
Indirect immunofluorescence. Confocal microscopy analysis was conducted as previously described (28). Before they were excised from the rats, one kidney from each rat was placed in a kidney-shaped Plexiglas cup and chemically fixed in situ (i.e., without perfusion) in 2% paraformaldehyde, 75 mM lysine, and 10 mM Na-periodate, pH 7.4 (PLP fixative) for 20 min. During fixation, there was no interruption of the continuing in vivo procedures, such as ANG II clamp and induced hypertension. The excised kidneys were cut in half along the midsagittal plane and postfixed in PLP fixative for 4-6 h, rinsed twice with PBS, cryoprotected by overnight incubation in 30% sucrose in PBS, embedded in Tissue-Tek OCT Compound (Sakura Finetek), and frozen in liquid nitrogen, and cryosections (5 µm) were prepared. Immunofluorescence labeling was conducted as previously described (28). Sections were rehydrated in PBS, washed with 50 mM NH4Cl in PBS and then in 1% SDS in PBS, and washed twice in PBS and then blocked with 1% BSA. Double labeling of sections was performed by incubation with the anti-NHE3 polyclonal antibody NHE3-C00 and the anti-villin monoclonal antibody (Immunotech), both at 1:100 dilution in 1% BSA in PBS for 1.5 h at room temperature. After three 5-min washes in PBS, the sections were incubated with a mixture of FITC-conjugated goat anti-rabbit and Alexa 568-conjugated goat anti-mouse secondary antibody (Accurate Chemical & Scientific), both at 1:100 dilution in 1% BSA in PBS for 1 h followed by three 5-min PBS washes. The sections were then mounted in Prolong Antifade (Molecular Probes) and air 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 captured with Simple PCI C-Imaging hardware and Quantitative Measuring software and processed with Adobe PhotoDeluxe 1.0 (Adobe Systems).
Statistical analysis. Data are expressed as means ± SE. ANOVA was used for multiple-group comparisons. If a significant difference among groups was concluded by ANOVA, further pairwise comparisons were assessed by two-tailed Student's t-test with the application of Bonferroni's adjustment to correct for multiple comparisons. Comparisons between two data groups were also assessed by two-tailed Student's t-test. P value <0.05 was considered significant.
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RESULTS |
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Effect of ANG II clamp on BP and GFR.
Arterial constriction induced a rapid increase in mean arterial
pressure (BP) by 50-60 mmHg that remained significant for >60 min
in control rats (Fig. 1A). In
the ANG II-clamped rats, captopril infusion (<5 min) lowered
BP from 102 ± 4 to 79 ± 8 mmHg (Fig. 1B).
Subsequent infusion of ANG II at 20 ng · kg1 · min
1 restored BP
to basal levels. This infusion rate was previously shown to restore
baseline BP (10a). The magnitude of hypertension induced by arterial
constriction in the ANG II-clamped rats was similar to that seen in
controls (Fig. 1B).
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Effect of ANG II clamp on urine output and endogenous
CLi.
Acute hypertension induced a rapid increase in urine output in both
controls and ANG II-clamped rats (Fig.
2A). However, there was a
significant blunting of the diuretic response from 0.14 ± 0.02 ml/min in controls to 0.08 ± 0.02 ml/min in ANG II-clamped rats.
This blunting became more pronounced with time despite the fact that BP
was at least as high as in the controls during acute hypertension.
Similar to urine output, acute hypertension induced a significant but
blunted increase in CLi in ANG II-clamped rats (0.43 ± 0.08 ml/min) compared with controls (0.75 ± 0.43 ml/min) (Fig.
2B) and this difference was more pronounced at 10-20
min.
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Effect of ANG II clamp on the regulation of the subcellular
distribution of renal NHE3 and NaPi2 during acute hypertension.
Our laboratory has previously established that acute hypertension
provokes a rapid redistribution of NHE3 from the apical membranes to
higher density subapical and endosomal pools (28). As
confirmed in the present study, acute hypertension induced a decrease
in total NHE3 immunoreactivity in the apical membranes (Win
I) from 31 ± 2.8 to 10 ± 3.2% and an increase in NHE3
in the endosome/lysosome membranes (Win III) from 6.0 ± 1.4% in controls to 22 ± 6.0% (Fig. 3, A and
B). Clamping of ANG II levels
per se had no effect on the overall distribution pattern of cortical NHE3 at basal arterial pressure (Fig. 3C). However, during
ANG II clamp, the redistribution of NHE3 in response to acute
hypertension was significantly altered. NHE3 left Win I as
in acute hypertension alone (from 31 ± 2.8% in controls to
4.0 ± 2.8% of total), but there was enhanced redistribution to
Win II (enriched in apical, intermicrovillar cleft, and
dense apical tubule membranes) from 63 ± 1.7% in controls and
68 ± 3.9% in acute hypertension alone to 83 ± 1.7% of
total NHE3 in acute hypertension with ANG II clamp, and there was no
significant redistribution to Win III (6.0 ± 1.4% in
controls and 13 ± 1.6% in acute hypertension with ANG II clamp).
Two-way ANOVA of the subcellular distribution pattern of NHE3 provides
evidence for three significantly distinct distribution patterns among
the three treatment groups. To illustrate the redistribution in the
prepooled gradient fractions, a constant volume of sorbitol gradient
fractions 4-11 from the same paired set of control,
hypertension, and hypertension with ANG II clamp were all run on the
same 26-well gel and blotted (fraction 3 was not included on
this blot because of the limited number of gel lanes, and NHE3 was
undetectable in lane 3). Figure 3B illustrates
the pressure-induced reduction of NHE3 immunoreactivity in the apical
membranes during acute hypertension in both groups, enhanced
redistribution to Win II (fractions 6-8)
during ANG II clamp and no redistribution to Win III
(fractions 9-11) in ANG II-clamped rats. The
redistribution of NHE3 among membrane windows was not due to a change
in NHE3 protein abundance, because neither acute hypertension per se
nor 20-min hypertension with ANG II clamp had a significant effect on
the total abundance of NHE3 in whole renal cortex (data not shown).
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Immunocytochemistry evidence for the blunting of pressure-induced
NHE3 internalization by ANG II clamp.
Confocal immunofluorescence analysis of PT NHE3 redistribution for the
three groups was conducted as an independent strategy to evaluate the
conclusions of the membrane fractionation analysis (Fig.
5). The actin bundling protein villin
labels the brush-border microvilli as well as the microvillar core
rootlets in the terminal web just under the cell membrane where villin
is also present (24). In controls, NHE3 (green) is
present primarily in the brush border where it colocalizes with villin
(red) as evidenced by the yellow stain, which denotes red and green
overlay (Fig. 5, top). Acute hypertension provokes the
redistribution of NHE3 out of the apical brush border, revealing
red-stained villin, into a subapical compartment where it formed a
green-stained layer below the villin distribution (Fig. 5,
middle). In the ANG II-clamped rats, acute hypertension also
provoked the redistribution of NHE3 out of the brush border, revealing
red-stained villin (Fig. 5, bottom). However, NHE3 moved to
stores at the base of the microvilli where it colocalized with villin
(indicated by yellow stain), and little if any NHE3 is evident below
the villin staining where it would appear green (Fig. 5,
bottom). These observations were consistent with the
conclusion of the membrane fractionation analysis that ANG II clamp did
not prevent the pressure-induced redistribution of NHE3 out of the
apical microvillar membranes but did prevent significant redistribution
of NHE3 into endosomal pools that extend below the microvilli and
subapical terminal web (Fig. 3, A and B).
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DISCUSSION |
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Acute hypertension rapidly inhibits PT fluid and NaCl reabsorption (8) associated with an internalization of PT apical NHE3 (28). The resultant increase in end proximal flow rate provides the error signals to mediate the TGF response (1) and to decrease renin release and ANG II production (14, 25). We tested whether the increase in end proximal flow rate and internalization of NHE3 during hypertension requires a responsive RAS by clamping plasma ANG II levels. The study revealed that the diuretic response to acute hypertension was blunted when the plasma [ANG II] was clamped and that at the molecular level, clamping ANG II inhibited the second step of a two-step internalization route for NHE3 (28); NHE3 moved out of the apical microvillar membranes (Win I) but not into the endosomal stores beneath the apical membranes (Win III).
The ANG II infusion rate used in this study (20 ng · kg1 · min
1) was
determined empirically by setting a dose that would restore baseline BP
after captopril. A dose-response of ANG II infusion after captopril has
recently been reported with the same model (10a). Mattson and Roman
(18) reported that this same infusion rate increased
plasma [ANG II] to ~200 pg/ml, which is two- to fourfold higher
than the baseline levels reported by others (21, 33). Is
it possible that the blunting of diuresis, and the block of
internalization are due to the two- to fourfold elevated levels of ANG
II? Our laboratory's recently published results would argue against
that conclusion. When rats were treated with a bradykinin receptor
antagonist to block the effects of the buildup of bradykinins that
occurs after captopril treatment, the ANG II clamp infusion rate to
restore BP was 10 times lower (2 ng · kg
1 · min
1) yet there
was a similar blunting of the diuresis and CLi as in this
study (10a).
It is important to mention that the application of captopril to inhibit ACE in the present study not only prevents de novo synthesis of ANG II but also allows the buildup of the vasodilator bradykinin (2) that may contribute to the renal effects of ANG II clamp during acute hypertension. Our laboratory addressed this issue in another ANG II clamp study in which the rats were pretreated with the bradykinin B2 receptor blocker HOE-140 to avoid the complication of bradykinin buildup. Our laboratory observed a similar 50% blunting of the pressure-induced increase in urine output and CLi as observed in the present study (10a), indicating that bradykinin buildup does not contribute significantly to the renal effects induced by ANG II clamp during acute hypertension. Could the bradykinin buildup contribute to the blocking of the NHE3 internalization? There are two central arguments against this possibility: 1) kallikreins are primarily located in the distal and connecting tubules downstream of the PT (5); and 2) kinins are known to have diuretic and natriuretic effects (17), whereas the effect seen with the protocol was blunting of diuresis.
A previous time course analysis provided evidence for a two-step process for the internalization of NHE3 in rats during acute hypertension (28). Within 5 min, NHE3 redistributed from membranes enriched in apical brush border (Win I) to membranes enriched in intermicrovillar clefts and dense apical tubules (Win II), and then by 30 min NHE3 internalized to membranes enriched in endosomal/lysosomal membranes (Win III). In this present study, membrane fractionation analysis demonstrated that without ANG II clamp, NHE3 redistributed to Win III membranes after 20 min of induced acute hypertension: NHE3 immunoreactivity decreased from 31 to 10% in Win I and increased from 6 to 22% in Win III. In comparison, in the ANG II-clamped rats, acute hypertension induced NHE3 to traffic out of Win I (27% decrease in total protein) into Win II (20% increase in total protein) with no significant increase in Win III. These findings are supported by the confocal microscopy results demonstrating that with ANG II clamp, acute hypertension induces the shift of NHE3 out of the apical brush border (i.e., revealing the red-stained villin) to the base of the microvilli, where NHE3 remains colocalized with villin (yellow stain; step 1) but did not traffic to membrane pools below the base of the microvilli, where it would appear green (step 2). These combined results indicate that ANG II clamp during acute hypertension has no effects on step 1 of NHE3 redistribution out of the microvillar membranes to intermicrovillar cleft membranes but prevents step 2 of NHE3 from Win II to Win III, which likely represents a blocking of NHE3 internalization.
Can we conclude from fractionation and confocal microscopy results that ANG II clamp blunts NHE3 internalization? In the present study, villin was used primarily as a microvillar marker. However, because villin is also present in the microvillar core rootlets that extend into the terminal web below the microvilli where dense apical tubules as well as some endosomes reside (24), this may potentially complicate the interpretation of the confocal analysis. In a detailed electron microscopic study investigating the in vivo endocytotic route of the insulin receptor complex in the PT, Nielsen (20) concluded that the receptor complex first migrated laterally along the microvilli to the intermicrovillar cleft region into invaginations that pinched off to form endocytic vesicles and vacuoles. Our membrane fractionation and confocal analyses support similar endocytotic routes for NHE3 during acute hypertension and the idea that this pressure-induced NHE3 internalization into endocytic vesicles is significantly blunted by the ANG II clamp. Alternatively, it is possible that ANG II clamp does not blunt the pressure-induced internalization of NHE3 into endocytic vesicles that coincide with the villin labeling of the microvillar core rootlets just under the apical membrane (such as the dense apical tubules), which is not resolved in our confocal analysis, but blunts the trafficking from these vesicles to those further into the cell below the villin stain. We believe it is unlikely that NHE3 is internalized from the pinched off intermicrovillar invaginations to dense apical tubules found directly below the apical membranes, because the invaginations are significantly larger than the small dense apical tubules that Nielsen identifies in this vicinity.
The effects of ANG II clamp on the redistribution of NHE3 provide evidence for two different and sequential signaling pathways mediating NHE3 internalization during acute hypertension. The step 1 redistribution out of the brush border to the intermicrovillar cleft is independent of a responsive RAS, whereas the step 2 internalization to endosomes is RAS dependent. In their studies of parathyroid hormone (PTH)-mediated inhibition of NHE activity, Hensley et al. (10) also suggested a two-step internalization from microvilli to intermicrovillar cleft to internal membranes. It is noteworthy that the RAS dependence of the step 2 redistribution of NHE3 during acute hypertension is transporter specific, because ANG II clamp did not affect the pressure-induced redistribution pattern of NaPi2. The finding is not unexpected because NHE3 and NaPi2 follow different internalization and recycling routes in the kidney. Our laboratory previously observed that acute hypertension induces the internalization of both NHE3 and NaPi2 to endosomal membranes but that restoration to normotension returned NHE3 but not NaPi2 to the Win I brush-border membranes (29). NHE3 and NaPi2 also undergo distinctly different PTH-induced retrieval pathways with differential fates. In response to PTH treatment, NHE3 undergoes slow microtubule-dependent endocytosis and slow degradation (6), whereas NaPi2 demonstrates quick microtubule-independent endocytosis and rapid degradation (15, 16).
We postulate that the cAMP-PKA pathway could be central to the blunting
effect of ANG II clamp on both pressure-diuresis and -natriuresis and
internalization of NHE3 to Win III. Our laboratory previously determined that activation of cAMP-PKA is necessary for
PTH-driven NHE3 internalization in rat renal cortices by using PTH
analogs that activate distinct signaling pathways (31). Liu and Cogan (12) established that ANG II stimulates PT
NHE activity through the Gi-mediated inhibition of
adenylyl cyclase, which reverses the inhibitory effect of cAMP on
NHE3. Thus it is plausible that ANG II clamp blunts the
pressure-induced internalization of NHE3 by preventing the TGF-mediated
drop in [ANG II] during acute hypertension and preventing the
associated activation of the cAMP-PKA pathway.
In the absence of ANG II clamp, acute hypertension markedly increased the volume flow out of the PT, determined by endogenous CLi. It is well documented that CLi can be influenced by NaCl reabsorption in the PT (3, 4) and changes in GFR. Although we measured a significant transient increase in GFR during acute hypertension, a similar increase (although not statistically significant because of higher data variability) in GFR of comparable magnitude was also observed in the ANG II-clamped rats. Thus it is unlikely that a difference in GFR among the treatment groups accounts for blunting of CLi during acute hypertension in the ANG II-clamped rats. We therefore postulate that the blunting of the pressure-induced increase in PT volume flow in the ANG II-clamped rats was likely due to a weaker pressure-induced inhibition of PT fluid and NaCl reabsorption rather than to a difference in the GFR. By 20 min of acute hypertension in the ANG II- clamped rats, the CLi and urine output fell to levels that were not statistically different from baseline levels (albeit the means were still higher), indicating that the molecular mechanisms responsible for pressure-induced inhibition of PT fluid and NaCl reabsorption cannot be sustained during ANG II clamp. These results suggest that PT luminal sodium reabsorption, which is coupled to NHE3-mediated NHE activity (27), returns toward basal values at 20-min acute hypertension during ANG II clamp and, together with the confocal microscopy and subcellular fractionation evidence, suggest that NHE3 is transport competent in the Win II membranes at the base of the microvilli in the ANG II clamped rats.
In summary, the present study demonstrates that internalization of PT NHE3 during acute hypertension requires a responsive RAS. Preventing a fall in ANG II by the ANG II clamp did not affect the immediate redistribution of NHE3 or NaPi2 out of the brush border into the intermicrovillar cleft but did inhibit the pressure-induced internalization of NHE3 (not NaPi2). These results provide evidence for two distinct steps to NHE3 internalization governed by separate signaling mechanisms. The first step, independent of a fall in plasma ANG II, is likely driven by the sensing of increase in perfusion pressure. The second step is likely driven by a drop in ANG II levels and allows further internalization of NHE3 from the intermicrovillar cleft to dense apical tubules and endosomes.
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ACKNOWLEDGEMENTS |
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We are grateful to Michaela MacVeigh for advice regarding 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 (A. A. McDonough), and confocal microscopy was supported by the Core Center Grant DK-48522. P. K. K. Leong and L. E. Yang were supported by fellowship awards from the American Heart Association, Greater Los Angeles Affiliate.
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.
July 16, 2002;10.1152/ajprenal.00178.2002
Received 7 May 2002; accepted in final form 10 July 2002.
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