1 Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California, 90033; and 2 Department of Medical Physiology, The Panum Institute, DK-2200 Copenhagen N, Denmark
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ABSTRACT |
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Acute hypertension in Sprague-Dawley rats
(SD) provokes a decrease in renal proximal tubule (PT) salt and fluid
reabsorption, redistribution of apical Na/H exchanger isoform 3 (NHE3)
and Na-Pi cotransporter type 2 (NaPi2) out of the brush
border into higher density membranes, and inhibition of renal cortical
Na-K-ATPase (NKA) activity (41). The aims of this study
were to determine 1) whether an increase in arterial
pressure affects distribution or activity of Na transporters in the
spontaneously hypertensive rat (SHR) and 2) whether
development of chronic hypertension in SHR leads to persistent adaptive
changes in NHE3 and NaPi2 distribution and/or NKA activity. Renal
cortex Na transporter protein density distributions and activities were
compared by subcellular fractionation in 1) adult SHR with
an acute increase or decrease in arterial pressure and 2)
young SD (YSD) and young SHR (YSHR) vs. adult SD and SHR. In adult
hypertensive SHR NHE3 was shifted to membranes of higher densities,
analogous to SD with acute hypertension, and there were no further
changes with a further increase or decrease in arterial pressure. There
was no change in total pool size of NHE3 in cortex in YSHR vs. SHR.
NHE3, NaPi2, megalin , NKA -/
-subunit, dipeptidyl peptidase IV
(DPPIV), and villin distributions were the same in YSHR vs. YSD. NHE3,
NaPi2, and megalin shifted to higher densities in adult SHR, but not
SD, with age. Basolateral NKA and apical alkaline phosphatase
activities were 40% greater in YSHR than YSD and decreased to SD
levels in adults. We conclude that there are persistent changes in
Na+ transporter distributions and activity in response to
chronic hypertension in SHR that mimic the responses to acute
hypertension seen in SD rats and that elevated sodium pump activity per
transporter in YSHR may contribute to the generation of hypertension.
basolateral sodium pump; Na/H exchanger isoform 3; Na-Pi cotransporter type 2; membrane trafficking; spontaneously hypertensive rat
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INTRODUCTION |
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IN NORMOTENSIVE Sprague-Dawley rats (SD), an acute increase in arterial pressure evokes a rapid natriuretic and diuretic response in part due to a decrease in NaCl reabsorption in the proximal tubule (PT). The increased flow out of the PT provides the error signal, sensed at the macula densa, to increase afferent arteriole resistance, thus maintaining constant renal blood flow and glomerular filtration rate (10, 11). In spontaneously hypertensive rats (SHR), the natriuresis and diuresis observed with a further increase in arterial pressure is blunted, indicating a functional resetting of the kidney has occurred which sustains the hypertension (31).
Sodium is actively reabsorbed in the PT primarily by the apical Na+/H+ exchanger (NHE3) and extruded through the basolateral sodium pump (Na-K-ATPase). We have previously addressed the cellular mechanisms responsible for the decrease in PT sodium reabsorption during acute hypertension in SD rats. Using subcellular fractionation of renal cortex, we discovered a rapid, reversible redistribution of apical NHE3 and Na-Pi cotransporters (NaPi2) to two regions of heavier density membranes: one that overlapped with the distribution of megalin, a receptor enriched in intermicrovillar cleft and subapical endosomes (4), and another overlapped with the distribution of rabs 4 and 5a, classic markers of endosomes (40). Yip et al. (38) used confocal immunofluorescence microscopy with the same model to verify that this redistribution of NHE3 to heavier membranes was, in fact, redistribution out of the peripheral microvilli to the base of the microvilli and/or subapical domains. We also discovered there was a rapid, reversible inhibition of renal cortex basolateral Na-K-ATPase activity after raising blood pressure (41). Thus it appears that removal of NHE3 and NaPi2 from the apical brush border and inhibition of Na-K-ATPase are molecular explanations for the inhibition of PT Na reabsorption that drives the increased salt delivery to the macula densa to effect autoregulation during hypertension.
Yip et al. (38) also showed, by confocal microscopy, that NHE3 was localized to the base of the microvilli in chronically hypertensive rats, e.g., adult SHR. However, in young, prehypertensive SHR, NHE3 was localized to the brush-border microvilli and redistributed to the base of the microvilli with acute hypertension, indicating that the PT is responsive to perturbations in arterial pressure in the young but not adult SHR.
During the development of chronic hypertension (prior to 8 wk of age), SHR retain more urinary sodium, potassium, and water than age-matched Wistar-Kyoto rats (WKY) (3). This can be explained by increased PT Na/H exchanger transport activity and Na-K-ATPase activity in young SHR compared with age-matched WKY (19, 28). Sodium retention is also favored by decreased glomerular filtration rate (GFR) due to increased tubuloglomerular feedback sensitivity in young SHR (14, 15). With established hypertension in the adult SHR, the differences in transporter activity and GFR between the two strains disappear (8, 15, 18).
When differences in sodium transporter characteristics are noted between hypertensive and normotensive strains of rats, it is not always obvious which differences contribute to the development of hypertension and which are adaptive responses to the hypertension. In this study we test the hypothesis that the development of chronic hypertension in SHR provokes adaptive persistent changes in NHE3 distribution and Na-K-ATPase activity that mimic those seen in SD during acute hypertension. The hypothesis was tested with the same strategies used in previous studies to determine the molecular mechanisms responsible for the rapid responses to acute hypertension in SD. We chose to compare SHR vs. SD rather than WKY due to the evidence that SHR and WKY have an extremely high amount of divergence, comparable to the maximum divergence possible between two unrelated humans, and share only 50% of a DNA fingerprint (22, 32). The findings of this study provide evidence for strain-specific differences in Na-K-ATPase activities in YSHR vs. YSD rats which can contribute to the development of hypertension and demonstrate that, during the development of hypertension in SHR, there is a chronic translocation of apical sodium transporters and an inhibition of basolateral Na-K-ATPase that closely mimic the rapid responses of adult SD rats to acute hypertension, and there are no further changes with an acute increase or decrease of arterial pressure in adult SHR.
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EXPERIMENTAL PROCEDURES |
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Animal preparations. Three studies were conducted, and all included the following procedures. Rats had free access to food and water before the experiment. After anesthesia, rats were placed on a thermostatically controlled warming table to maintain body temperature at 37°. Polyethylene catheters were placed into the carotid artery for monitoring blood pressure, into the right jugular vein for infusion of 0.9% NaCl+4% BSA at 50 µl/min during the entire experiment to maintain euvolemia.
In the first study, 12-wk-old male adult SHR were anesthetized with nembutol (30.mg/kg), and an additional catheter was inserted into the ureter for urine collection. To increase blood pressure, the total peripheral resistance was increased as suggested by Roman and Cowley (31), without hormone infusion. Mean arterial pressure was increased 40-50 mmHg over basal levels (141 ± 4 to 183 ± 8 mmHg) for 5 min by constricting the superior mesenteric artery, celiac artery, and abdominal aorta below the renal artery by tightening silk ligatures. Paired sham procedures were performed. N = 10 in each group, of which a subset was used for subcellular fractionation. In the second study, 12-wk-old adult SHR were anesthetized with ketamine (Fort Dodge Laboratories) and xylazine (Miles 1:1, vol/vol) intramuscularly, which lowered blood pressure to 97 ± 5 mmHg (35). Paired sham procedures were performed in which animals were anesthetized with nembutol (30.mg/kg). N = 8 in each group, of which a subset was used for subcellular fractionation. In the third study, four groups of male rats were compared: 1) YSD, n = 8, 3-4 wk old, 79 ± 7 g body wt, 2) YSHR, n = 10, 3-4 wk old, 76 ± 2 g body wt, 3) adult SD, n = 6, 12 wk old, 356 ± 6 g body wt, and 4) adult SHR, 12 wk old, 286 ± 5 g body wt. All rats were anesthetized by intraperitonial injection with nembutol (30 mg/kg). Polyethylene catheters were placed into the carotid artery for monitoring blood pressure, and into the right jugular vein for infusion of 4% bovine serum albumin in 0.9% NaCl at 50 µl/min during the experimental period to maintain euvolemia. Mean arterial pressures were monitored for at least 30 min before removal of kidneys and death by overdose injection of nembutol.Urine collection and endogenous lithium clearance. Urine volume, collected from the ureter catheter, was determined gravimetrically. A blood sample was collected after the kidneys were removed. The concentrations of endogenous lithium in blood and urine samples were measured by flameless atomic absorption spectrophotometry (Perkin-Elmer 5100PC) as described previously (24).
Homogenization, differential sedimentation, and density gradient
centrifugation.
The procedure for subcellular fractionation of the renal cortex
membranes has been described in detail (40). In brief,
kidneys were cooled in situ before excision by flushing the abdominal cavity with ice-cold PBS solution to block membrane trafficking. After
excision, renal corticies were rapidly dissected in isolation buffer
(5% sorbitol, 0.5 mM Na2EDTA, 0.2 mM phenylmethylsulfonly fluoride, 1.4 µM aprotinin, and 5 mM histidine-imidazole buffer, pH
7.5) and homogenized in two rounds with a Tissuemiser (Tekmar Instruments) for 10 min at a thyristor setting of 45 then centrifuged at 2,000 g for 10 min. The two low-speed surpernatants
(So) were pooled and aliquots of cortex So were
stored at 80°C pending assays. Without freezing, cortex
So was loaded at the interface between two hyperbolic
sorbitol gradients (35 and 70% sorbitol), and centrifuged 100,000 g for 5 h. Twelve fractions were collected with a
Buchler AutoDensi Flow apparatus from the top, and each fraction was
diluted with isolation buffer, pelleted by centrifugation (250,000 g for 1.5 h), resuspended in 1 ml isolation buffer, and stored at
80°C, pending assays.
Na-K-ATPase and enzymatic marker measurements. Na+-K+-ATPase activity was measured by two methods: the K+-dependent p-nitrophenyl phosphatase (K+-pNPPase) reaction (29) was used to assay both So and fractionated samples, and ouabain-sensitive Na-K-ATPase activity was also measured in the So, as previously described (25) with the modification that samples were preincubated with 2.5 mM ouabain for 30 min. Standard assays were used for alkaline phosphatase activity (27), dipeptidyl-aminopeptidase IV (DPPIV) activity (23), and protein concentration (26).
Immunoblot analysis and antibodies.
A constant volume of sample from each gradient fraction was prepared in
SDS-PAGE sample buffer (final concentration: 2% SDS, 1%
-mercaptoethanol, 0.25 mM Na2EDTA, and 2.5mM
H2PO4-HPO4 buffer, pH 7.0),
incubated for 30 min at 37°C, resolved on 7.5% SDS polyacrylamide gels, and transferred to polyvinylidene fluoride membranes according to
standard methods. The antibody incubation protocol has been detailed
previously (1). An NHE3 monoclonal cell (2B9) culture supernatant, provided by D. Biemesderfer (Yale) (4,
37), was used without dilution on blots of fractionated
membranes, NHE3 in total membranes (Fig. 6) was measured with a
polyclonal (L546) provided by M. Knepper, National Institutes of
Health, and both were detected with an enhanced chemiluminescence kit (Amersham); polyclonal antibody to the Na-Pi cotransporter isoform 2 (NaPi2), provided by H. Murer (13), was used at 1:4,000
dilution. The monoclonal antibody specifically against the rat
Na-K-ATPase
1-subunit (464.6), provided by M. Kashgarian
(Yale Univ.), was used at 1:200 dilution, a polyclonal anti-rat
1-fusion protein, generated in our laboratory, was used
at 1:500 dilution; for detection of
1, samples were
deglycosylated with N-glycanase (PNGase F, Genzyme,
Cambridge, MA) before SDS-PAGE. Monoclonal antibody to villin
(Immunotech, Westbrook, Maine) and polyclonal antisera to DPPIV and
megalin, provided by M. Farquhar (Univer. of California at San Diego),
were all used at 1:1,000 dilutions, and detected with
125I-labeled protein A (ICN; the monoclonals after
incubation with rabbit anti mouse secondary). The resulting
autoradiographic signals were quantified with a Bio-Rad imaging
densitometer with Molecular Analyst software. Multiple volumes were
analyzed in a subset of fractions and multiple exposures of
autoradiograms were analyzed to ensure that signals were within the
linear range of the film.
Quantitation and statistical analysis. Data are expressed as means ± SE. ANOVA was applied to determine whether there was a significant effect of treatment on the overall fractionation pattern of a given parameter. Age, strain, and fraction were the repeated factors. If the interactions between either age and fraction or strain and fraction (or both) was found to be significant (P < 0.05), then it was concluded that either or both had a significant effect. If a significance was established, the location of the difference in the pattern was assessed by two-tailed Student's t-test for paired samples, and differences were regarded significant at P < 0.05, after application of an adjustment for multiple comparisons.
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RESULTS |
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Acute changes in arterial pressure in the SHR.
In the first study, SHR rats were subjected to a further increase in
arterial pressure for 5 min and compared with SD subjected to a 5 min
increase in arterial pressure as previously reported (41).
Figure 1 summarizes the mean arterial
blood pressure, urine output, and endogenous lithium clearance
following constriction of the celiac artery, superior mesenteric
artery, and the abdominal aorta in both SD (41) and SHR.
Arterial blood pressure increased immediately by 40-50 mmHg in
both SD and SHR, from 107 ± 3 to 165 ± 2 and 141 ± 4 to 183 ± 8 mmHg, respectively. These arterial pressures are
within the autoregulatory range for GFR and renal blood flow (RBF) in
both strains (10, 31). In SD, 5-min acute hypertension increased urine output 4.8 ± 0.6-fold and endogenous lithium clearance, an inverse measure of proximal tubule sodium reabsorption (33), 2.9 ± 0.3-fold. In SHR, the
increase in urine output was blunted, increasing to only 2.7 ± 0.3-fold, though lithium clearance increased similarly to that in SD
(3.3 ± 0.4-fold).
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Age- and strain-dependent changes in renal apical Na transporter
distributions.
Figure 4 summarizes the mean arterial
pressures in young and adult SHR and SD rats. No significant
differences were found between the 3 to 4-wk-old YSD and YSHR, there
was no significant increase in arterial pressure between 3 and 12 wk in
the SD, and, as expected, blood pressure increased 50 mmHg in the SHR
between 3 and 12 wk, evidence of establishment of hypertension.
Although body weights were not different in the YSHR vs. YSD rats
(76 ± 2 g vs. 79 ± 7 g), adult age-matched SHR weighed
significantly less than their age-matched SD (286 ± 5g vs.
356 ± 6 g) as reported previously (36).
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Age- and Strain-Dependent Differences in Renal Na-K-ATPase Activity
We previously demonstrated that Na-K-ATPase activity, measured as K+-pNPPase activity, decreased with acute hypertension in adult SD (Fig. 3A) and that the activity levels returned toward control levels when normotension was restored (41). The peak basolateral membranes in these experiments are found in fractions 4-7, slightly lighter in density than the apical membranes. Figure 8B demonstrates 40% higher basolateral Na-K-ATPase activity in young SHR compared with young SD rats, and that with hypertension, Na-K-ATPase activity decreases to levels observed in adult SD, analogous to the acute decrease in Na-K-ATPase activity measured in adult SD rats subjected to acute hypertension (Fig. 3A). Likewise, Garg and Narang (18) concluded that Na-K-ATPase activity in proximal tubules of young SHR was higher than in adult SHR, and that Na-K-ATPase activity was not different in any nephron segments in adult (12 wk) SHR compared with adult WKY (17). To determine whether the higher activity in young SHR could be accounted for by higher levels of expression of the sodium pumps, activity and abundance were measured in total unfractionated So fraction from young and adult SD and SHR collected as sets of one each (YSD, YSHR, adult SD, adult SHR) and processed together on the same day (Fig. 9). As observed in the fractionated membranes, K+-pNPPase activity was significantly higher in young SHR compared with young SD (P < 0.05); in addition, ouabain-sensitive Na-K-ATPase activity was also higher in young SHR (P < 0.05, Fig. 9A). Activities were not different in adult SHR compared with adult SD. Figure 9B summarizes the immunoblot analysis of Na-K-ATPase
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DISCUSSION |
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The SHR develops hypertension with age, resembling the development of human essential hypertension. SHR have been routinely compared with their normotensive counterparts the WKY, because the SHR was originally bred from a male Wistar rat showing spontaneous hypertension and a female Wistar rat with a blood pressure slightly above average to obtain F1. From the F1 generation, a pair with spontaneous hypertension was selected and mated again until 100% spontaneous hypertension occurred (30). However, there is now a very high degree of divergence between WKY and SHR (22, 32), so we decided to match the SHR to SD for comparison. In support of this comparison, we verified that mean arterial pressures in SHR and SD were not different at 3-4 wk of age, that the SHR developed a significantly higher arterial pressure by 12 wk of age, and that there was no change in blood pressure in the SD between 3 and 12 wk of age. While the weight of the SHR and SD were not different at 3 wk, the SD rats gained more weight than the SHR by 13 wk of age. However, since no differences in sodium transporter distribution or activity were detected in the SD between 3 and 12 wk of age, the outcome of this study would not have been changed by pairing them by weight. Comparison of SHR vs. SD provided a convenient recheck for conclusions based on SHR vs. WKY comparisons, and we verified that Na-K-ATPase activity is higher in the young SHR, and that the activity falls with development and hypertension in SHR compared with either WKY or SD.
As seen with acute hypertension, a further increase in arterial pressure in the SHR produces a comparable decrease in proximal tubule sodium reabsorption (increase in lithium clearance), although the increase in urine output is blunted compared with SD. This suggests that, in addition to the response of a decrease in proximal tubule sodium reabsorption in the SHR, there may be an enhancement of fluid and solute reabsorption distal to the proximal tubule to account for the blunted urine output. Acute increases in arterial pressures of 40-50 mmHg in both strains have been shown to be within the autoregulatory range for GFR and RBF (10, 31), while total sodium excretion and urine output are lower in the SHR compared with WKY at comparable arterial pressures (31). This strengthens the idea that in the SHR, in addition to a decrease in proximal tubule sodium reabsorbtion, more distal reabsorption may be affected.
We previously provided evidence for potential molecular mechanisms involved in the decreased proximal tubule sodium reabsorption and increased urine output with acute hypertension in SD: internalization of apical NHE3 and NaPi2 and an inhibition of basolateral Na-K-ATPase activity (Figs. 2 and 3) (41). Yip and Marsh (39) in a preliminary report, also demonstrated that brush border NHE3 activity decreases due to internalization. This explains, in part, what is happening in the SD with an acute pressure challenge, but not necessarily what is happening in the SHR. We demonstrated that proximal tubule NHE3 distribution in the SHR is like that in the acutely pressure challenged SD (Fig. 3A) and that there is no further internalization of NHE3 or inhibition of Na-K-ATPase with a further increase of arterial pressure (Fig. 3B) also demonstrated by immunofluorescence studies (38). In spite of the lack of change in apical Na transporter distribution, there is a comparable decrease in proximal tubule sodium reabsorption. One possible explanation to this response is that there is an inhibition of apical NHE3 activity, which may be governed by phosphorylation, as demonstrated by Fan et al. (17) and/or other sodium transporters in the brush border.
The lack of response of NHE3 distribution and Na-K-ATPase activity in the proximal tubule to a further increase of arterial pressure in the SHR could indicate that this facet of regulation in the face of chronic hypertension is already maximal, although there is still gain in the urine output and lithium clearance response. To further examine the responsiveness of the chronically hypertensive SHR kidney, we acutely decreased arterial pressure in the SHR for approximately 1 h which also did not change NHE3 distributions or Na-K-ATPase activties (Fig. 3C). Since this was an immediate response to the anesthesia, we were unable to quantitate a change in lithium clearance or urine output. We previously showed that when arterial pressure was restored (decreased from 150-100 mmHg) for 20 min in the SD, there was the expected increase in proximal tubule sodium reabsorption and decrease in urine output and lithium clearance to basal levels. This was accompanied by a return of apical NHE3 and NaPi2 distributions and basolateral Na-K-ATPase activity levels to that observed before the acute pressure challenge (41). The observations support the hypothesis that hypertension, whether chronic or acute, leads to an internalization of apical NHE3, and that chronic hypertension may effect the normal responsiveness of kidney to changes in arterial pressure. A long-term normalizing of arterial pressure may be necessary to restore the normal responsesiveness of the SHR kidney.
In the proximal tubule apical membrane before hypertension develops, the NHE3, NaPi2, and villin density distributions were indistinguishable in SHR compared with age-matched SD. In addition, there was no significant difference in the absolute NHE3 protein abundance in total membranes (or, not shown, in the peak fractions) between the two strains at 3 wk of age, indicating that the characteristics of the apical membrane are similar in the two strains. Biemesderfer (5) has shown, by electron microscopy, that NHE3 is localized to the apical brush border as well as to subapical endosomes in adult normotensive rats. With established hypertension in adult SHR, NHE3, and NaPi2 shift to heavier density membranes (relative to that in adult SD) enriched in markers of the intermicrovillar cleft (megalin, Fig. 7) and endosomes (rabs 4 and 5a, Ref. 40). This response is directly parallel to what we have previously reported for redistribution of NHE3 and NaPi2 in adult SD subjected to acute hypertension. By confocal microscopy, NHE3 distribution in adult SHR and in SD subjected to acute hypertension also appears to be localized mainly at the base of the brush border, or in subapical stores (38). Taken together, these studies provide biochemical as well as microscopic evidence that increased arterial pressure, whether chronic or acute, provokes a redistribution of apical sodium transporters out of the microvilli into intermicrovillar cleft and/or subapical endosomes. This response likely contributes to the observed decrease in proximal tubule sodium reabsorption during acute hypertension.
The density distribution patterns of NHE3 and NaPi2 are very distinct in young vs. adult rats, but very similar between the two strains: in young SHR and SD the apical peak (fractions 4-7) contains more than 70% of the NHE3 and only about 30% of the NaPi, with 70% of the NaPi2 in the intracellular pools (fractions 8-12). A very recent study of the ontogeny of NaPi2 in the kidney by Traebert et al. (34) demonstrates, by confocal immunofluorescence microscopy, that there is, indeed, extensive intracellular staining of NaPi2 in 3-wk-old rats of the Wistar strain, and that this staining decreases with development. They suggest that these pools could be recruited to the membrane during development. This observation of a decrease of intracellular pools of NaPi2 with development concurs with our finding of a decrease in the percentage of NaPi2 found in fractions 8-12 in adult vs. young SHR in both strains, further validating identification of fractions 8-12 as intracellular membranes.
NHE3 has recently been shown, by immunoprecipitation, to exist in two states in the proximal tubule: a meglin-free form, sedimentation coefficient of 9.6 S, and a megalin bound form, sedimentation coefficient of 21 S (6). Megalin, a scavenger receptor, is localized primarily to the intermicrovillar cleft, clathrin-coated pits and endosomes (4). It is not surprising, then, that there is also a shift in megalin distribution with the development of hypertension in the SHR. The shift in megalin and NHE3 suggests colocalization to the base of the villi and further shift to endosomal populations. NaPi2 does not immunoprecipitate with megalin (6), which may indicate a different trafficking route or mechanism.
We recently demonstrated that in vivo treatment of SD with parathyroid hormone (PTH) also causes a decrease in proximal tubule sodium reabsorption associated with redistribution of apical NHE3 and NaPi2 to heavier density membranes, very similar to the response to acute hypertension (42). Another in vivo study by Fan et al. (17) also found that PTH treatment decreased the amount of NHE3 present in the brush border membrane fraction. Because of the similarity of the responses to PTH (42) and acute hypertension (40, 41), we postulate that the redistribution and proximal tubule natriuresis during hypertension also depends on the generation of cAMP-protein kinase A (PKA).
Multiple apical proteins shift to higher densities in the sorbitol gradient with the development of hypertension, which led to the question of whether there was an overall change in the density of membranes associated with the cytoskeleton of the villi. Drueke et al. (16) concluded there was a patchy 10-15% loss of microvilli in the 12 to 14-wk-old SHR, but no differences in the abundance of villin, a cytoskeletal bundling protein. We found that villin density distribution remained the same during development of hypertension in SHR, suggesting that villi structure was not disrupted.
In addition to redistribution of apical sodium transporters and
membrane markers, there is inhibition of renal cortex Na-K-ATPase activity when SD are subjected to acute hypertension (41, Fig. 3), a
decrease in Na-K-ATPase activity in the transition from young,
prehypertensive SHR to hypertensive SHR adult (Fig. 8), and no further
change when adult SHR are subjected to further hypertension, or if
blood pressure is normalized (Fig. 3C). These findings lead
us to conclude that the proximal tubule basolateral Na-K-ATPase
activity is similarly regulated during acute and chronic hypertension.
There were no differences in Na-K-ATPase 1 or
1 abundance levels in either young or adult SD or SHR
strains (not shown), indicating that the changes in activity observed
with either acute or chronic hypertension are due to changes in the activity per protein rather than to absolute changes in protein levels,
and that hypertension does not change sodium pump subunit expression.
Both Na-K-ATPase activity and alkaline phosphatase activities were elevated in the young SHR compared with the young SD. In addition, NHE3 activity has been reported to be higher in young, prehypertensive SHR vs. WKY (21, 28). Since we saw no evidence for higher protein levels of Na-K-ATPase or NHE3 in SHR, we tentatively conclude that there is higher activity per NHE3 and per Na-K-ATPase transporters in young SHR vs. young SD. This elevation in activity may be related to the events triggering the increase in blood pressure. Young, prehypertensive SHR are in a state of positive Na balance (3) and volume expansion, (36) both of which contribute to the generation of hypertension. Numerous potential mechanisms that may effect elevated Na reabsorption during development of hypertension have been reported. Relevant to the proximal tubule, the increased Na-K-ATPase activities observed in young SHR vs. age-matched WKY may be attributed to increased plasma renin activity and aldosterone concentration (8), increased expression of type 1 angiotensin II receptors (9), and increased adrenergic stimulation (2), all of which diminished with age and establishment of hypertension in the SHR. Garg et al. (18, 19) measured higher Na-K-ATPase activity in proximal tubule and lower Na-K-ATPase activity in thick ascending loop of Henle in young SHR. Our comparisons of Na-K-ATPase activity in the SHR vs. SD complement these observations: basolateral Na-K-ATPase activity in young SHR renal cortex was 40% greater than that seen in age-matched SD, and, in fact, thick ascending loop of Henle Na-K-ATPase activity in the same kidneys was 25% lower in young SHR compared to young SD (not shown).
In conclusion, the results of this study support the hypothesis that an increase in arterial pressure, whether chronic (as in SHR) or acute (as in the acute hypertension SD model), leads to a translocation of apical Na-transporters (NHE3, NaPi2) and an inhibition of basolateral Na-K-ATPase activity without a change in protein levels. The higher Na-K-ATPase activity in the young prehypertensive SHR vs. age- matched SD provides further evidence that strain-specific difference in sodium pump activity can drive the development of hypertension. The increased cortical Na-K-ATPase activity and increased NHE activity may lead to positive sodium balance and increase arterial pressure which would provoke pressure natriuresis to combat the increased salt and volume load. During the development and maintenance of hypertension, the decrease in Na-K-ATPase activity and translocation of apical sodium transporters to internal stores may be responsible, at least in part, for changing the set point of the kidney's natriuretic response to elevated arterial pressure.
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ACKNOWLEDGEMENTS |
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We acknowledge the technical advice and assistance of Dr. Li Yang and Dr. Jon Armstrong.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-34316 to A.A. McDonough, and HL-45623 to N-H. Holstein-Rathlou. Portions of this work were presented at the 1996 and 1997 Annual Meetings of the American Society of Nephrology. Y. Zhang was supported by a Fellowship award 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 School of Medicine, 1333 San Pablo St., Los Angeles, CA 90033.
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. §1734 solely to indicate this fact.
Received 4 January 2000; accepted in final form 22 March 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Azuma, KK,
Balkovetz DF,
Magyar CE,
Lescale-Matys L,
Zhang Y,
Chambrey R,
Warnock DG,
and
McDonough AA.
Renal Na/H exchanger isofroms and their regulation by thyoid hormone.
Am J Physiol Cell Physiol
270:
C585-C592,
1996
2.
Beach, RE,
and
DuBose TD, Jr.
Adrenergic regulation of (Na+, K+)-ATPase activity in proximal tubules of spontaneously hypertensive rats.
Kidney Int
38:
402-408,
1990[ISI][Medline].
3.
Beierwaltes, WH,
Arendshorst WJ,
and
Klemmer PJ.
Electrolyte and water balance in young spontaneously hypertensive rats.
Hypertension
4:
908-915,
1982[Abstract].
4.
Biemesderfer, D,
Dekan G,
Aronson PS,
and
Farquhar MG.
Assembly of distinctive coated pit and microvillar microdomains in the renal brush border.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F55-F67,
1992
5.
Biemesderfer, D,
Rutherford PA,
Nagy T,
Pizzonia JH,
Abu-Alfa AK,
and
Aronson PS.
Monoclonal antibodies for high-resolution localization of NHE-3 in adult and neonatal rat kidney.
Am J Physiol Renal Physiol
273:
F289-F299,
1997
6.
Biemesderfer, D,
Nagy T,
DeGray B,
and
Aronson PS.
Specific association of megalin and the Na/H exchanger isoform NHE3 in the proximal tubule.
J Biol Chem
274:
17518-17524,
1999
7.
Blakeborough, P,
Neville SG,
and
Rolls BA.
The effect of diets adequate and deficient in calcium on blood pressures and the activities of intestinal and kidney plasma membrane enzymes in normotensive and spontaneously hypertensive rats.
Br J Nutr
63:
65-78,
1990[ISI][Medline].
8.
Cangiano, JL,
Rodriguez-Sargent C,
Opava-Stitzer S,
and
Martinez-Maldonado M.
Renal Na-K-ATPase in weanling and adult spontaneously hypertensive rats.
Proc Soc Exp Biol Med
177:
240-246,
1984[Abstract].
9.
Cheng, H-F,
Wang-L J,
Vinson GP,
and
Harris RC.
Young SHR express increased type 1 angiotensin II receptors in renal proximal tubule.
Am J Physiol Renal Physiol
274:
F10-F17,
1998
10.
Chou, C-L,
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
11.
Chou, C-L,
and
Marsh DJ.
Time course of proximal tubule response to acute hypertension in the rat.
Am J Physiol Renal Fluid Electrolyte Physiol
254:
F601-F607,
1988
12.
Christensen, EI,
Nielsen S,
Moestrup SK,
Borre C,
Maunsbach AB,
Heer ED,
Ronco P,
Hammond TG,
and
Verroust P.
Segmental distribution of the endocytosis receptor gp330 in renal proximal tubules.
Eur J Cell Biol
66:
349-364,
1995[ISI][Medline].
13.
Custer, M,
Lotscher M,
Biber J,
Murer H,
and
Kaissling B.
Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F767-F774,
1994
14.
Dilley, JR,
and
Arendshorst WJ.
Enhanced tubuloglomerular feedback activity in rats developing spontaneous hypertension.
Am J Physiol Renal Fluid Electrolyte Physiol
247:
F672-F679,
1984[ISI][Medline].
15.
Dilley, JR,
Steir CT,
and
Arendhorst WJ.
Abnormalities in glomerular function in rats developing spontaneous hypertension.
Am J Physiol Renal Fluid Electrolyte Physiol
246:
F12-F20,
1984[ISI][Medline].
16.
Drueke, TB,
Hennessen U,
Lucas PA,
Nabarra B,
Ben Nasr L,
Thomasset M,
Lacour B,
Coudrier E,
and
McCarron DA.
Epithelial abnormalities in intestine and kidney of the spontaneously hypertensive rat.
Am J Hypertens
3:
185S-201S,
1990.
17.
Fan, L,
Wiederkehr MR,
Collazo R,
Wang H,
Crowder LA,
and
Moe OW.
Dual mechanisms of regulation of Na/H exchanger NHE-3 by parathyroid hormone in rat kidney.
J Biol Chem
274:
11289-11295,
1999
18.
Garg, LC,
and
Narang N.
Differences in renal tubular Na-K-adensosine triphosphatase in spontaneously hypertensive and normotensive rats.
J Cardiovasc Pharmacol
8:
186-189,
1986[ISI][Medline].
19.
Garg, LC,
Narang N,
and
McArdle S.
Na-K-ATPase in nephron segments of rats developing spontaneous hypertension.
Am J Physiol Renal Fluid Electrolyte Physiol
249:
F863-F869,
1985[ISI][Medline].
20.
Girardi, ACC,
DeGray B,
Nagy T,
Biemesderfer D,
and
Aronson PS.
Association of Na-H exchanger and dipeptidlypeptidase IV (DPPIV) in renal brush border membrane (Abstract).
J Am Soc Nephrol
10:
4A,
1999.
21.
Hayashi Yoshida, MT,
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].
22.
Johnson, ML,
Ely DL,
and
Turner ME.
Genetic divergence between the Wistar-Kyoto rat and the spontaneously hypertensive rat.
Hypertension
19:
425-427,
1992[Abstract].
23.
Kato, T,
Nagatsu T,
Kimura T,
and
Sakakibara S.
Fluorescence assay of X-prolyl dipeptidyl-aminopeptidase activity with a new fluorogenic substrate.
Biochem Med
19:
351-359,
1978[ISI][Medline].
24.
Leyssac, PP,
and
Christensen P.
A comparison between endogenous and exogenous lithium clearance in the anesthetized rat.
Acta Physiol Scand
151:
173-179,
1994[ISI][Medline].
25.
Lo, CS,
August TR,
Lieberman UA,
and
Edelman ES.
Dependence of renal Na-K-ATPase activity on thyroid status.
J Biol Chem
251:
7826-7833,
1976[Abstract].
26.
Lowry, OH,
Rosebrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem
193:
265-175,
1951
27.
Mircheff, AK,
and
Wright EM.
Analytical isolation of plasma membrane 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].
28.
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].
29.
Murer, H,
Ammann E,
Biber J,
and
Hopfer U.
The surface membranes of the small intestinal epithelial cell. I. Localization of adenyl cyclase.
Biochim Biophys Acta
433:
509-519,
1976[ISI][Medline].
30.
Okamoto, K,
and
Aoki K.
Development of a strain of spontaneously hypertensive rats.
Jpn Circ J
27:
282-293,
1963.
31.
Roman, RJ,
and
Cowley AW, Jr.
Abnormal pressure-diuresis-natriuresis response in spontaneously hypertensive rats.
Am J Physiol Renal Fluid Electrolyte Physiol
248:
F199-F205,
1985[ISI][Medline].
32.
St. Lezin, E,
Simonet L,
Pravenec M,
and
Kurtz TW.
Hypertensive strains and normotensive 'control' strains. How closely are they related?
Hypertension
19:
419-424,
1992[Abstract].
33.
Thomsen, K.
Lithium clearance: a new method for determining proximal and distal tubular reabsorption of sodium and water.
Nephron
3:
217-223,
1984.
34.
Traebert Lotscher, MM,
Aschwanden R,
Ritthaler T,
Biber J,
Murer H,
and
Kaissling B.
Distribution of the sodium/phosphate transporter during postnatal ontogeny of the rat kidney.
J Am Soc Nephrol
10:
1407-1415,
1999
35.
van Gorp, AW,
van Ingen Schenau DS,
Hoeks AP,
Struijker Boudier HA,
Reneman RS,
and
De Mey JG.
Aortic wall properties in normotensive and hypertensive rats of various ages in vivo.
Hypertension
26:
363-368,
1995
36.
Von Dreele, MM.
Age-related changes in body fluid volumes in young spontaneously hypertensive rats.
Am J Physiol Renal Fluid Electrolyte Physiol
255:
F953-F956,
1988
37.
Wu, MS,
Biemesderfer D,
Giebisch G,
and
Aronson PS.
Role of NHE3 in mediating renal brush-border Na/H exchange-adaptation to metabolic acidosis.
J Biol Chem
271:
32749-32752,
1996
38.
Yip, K-P,
Tse-M C,
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
39.
Yip, K-P,
and
Marsh DJ.
Acute arterial hypertension regulates Na/H exchangers (NHE) activity in proximal tubules via protein trafficking (Abstract).
J Am Soc Nephrol
9:
14A,
1998.
40.
Zhang Mircheff, YAK,
Hensley CB,
Magyar CE,
Warnock DG,
Chambrey R,
Yip-P K,
Marsh DJ,
Holstein-Rathlou-H N,
and
McDonough AA.
Rapid redistribtuion and inhibtion of renal sodium transporters during pressure natriuresis.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F1004-F1014,
1996
41.
Zhang, Y,
Magyar CE,
Norian JM,
Holstein-Rathlou-H N,
Mircheff AK,
and
McDonough AA.
Reversible effects of acute hypertension on proximal tubule sodium transporters.
Am J Physiol Cell Physiol
274:
C1090-C1100,
1998
42.
Zhang, Y,
Norian JM,
Magyar CE,
Holstein-Rathlou-H N,
Mircheff AK,
and
McDonough AA.
In vivo PTH provokes apical NHE3 and NaPi2 redistriubtion and Na-K-ATPase inhibition.
Am J Physiol Renal Physiol
276:
F711-F719,
1999