Proximal tubule Na transporter responses are the same during acute and chronic hypertension

Clara E. Magyar1, Yibin Zhang1, Niels-H. Holstein-Rathlou2, and Alicia A. McDonough1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -/beta -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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% beta -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 alpha 1-subunit (464.6), provided by M. Kashgarian (Yale Univ.), was used at 1:200 dilution, a polyclonal anti-rat beta 1-fusion protein, generated in our laboratory, was used at 1:500 dilution; for detection of beta 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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Physiological responses to constricting celiac and superior mesenteric arteries and abdominal aorta for 5 min in Sprague-Dawley (SD) and spontaneously hypertensive (SHR) rats. A: arterial pressure, recorded from carotid artery. B: urine output collected over 5-min intervals, expressed as urine weight in µg/min. C: endogenous lithium clearance calculated for 5-min collection period as urine [Li+] × urine output rate/plasma [Li+] in (ml/min), where brackets indicate concentration. Data are expressed as means ± SE; n = 5 for SD, n = 7 for SHR. Note: SD data previously published (41). *P < 0.05 vs. control period, #P < 0.05 vs. SD during acute hypertension both by paired Student's t-test.

Distributions of apical and basolateral sodium transporters were analyzed on sorbitol density gradients. Because renal cortex is heavily enriched in proximal tubules and because these transporters are heavily enriched in proximal tubules, we assumed that distributions of transporters in renal cortex are representative of that in proximal tubules. As previously reported (41), in the SD with an acute increase in arterial pressure, cortical apical NHE3 redistributes from a peak in fractions 4-6 primarily to a peak in heavier density fractions (5-7) with a smaller shoulder appearing at fractions 8-10 (Fig. 2, A and C). In addition, distributions of apical membrane marker activities, alkaline phosphatase and DPPIV also shifted to heavier densities with acute hypertension in the SD (Fig. 3A), while basolateral Na-K-ATPase activity was inhibited by about 30% in fractions 3-5 (Fig. 3A). We then examined these distributions in the chronically hypertensive SHR with a further increase in pressure. Peak distribution was similar to that seen in the acutely pressure challenged SD (fractions 5-7) and NHE3 distribution did not change with a further increase in arterial pressure (Fig. 2, B and C). No further changes in apical alkaline phosphatase, DPPIV, and basolateral Na-K-ATPase distributions or activity levels were observed by a further increase in arterial pressure in the SHR (Fig. 3B).


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Fig. 2.   Density distribution of Na/H exchanger (NHE3) in membrane fractions of renal cortex. A: comparison of NHE3 distribution in cortex from normotensive SD and after 5-min acute hypertension (41). B: comparision of NHE3 distribution in cortex from adult SHR, SHR after a further acute increase in arterial pressure, and SHR after acute arterial pressure reduction via ketamine/xylazine anesthesia. A constant volume of each fraction was resolved by SDS-PAGE, blotted, and probed with NHE3-specific monoclonal antibody. Antibody antigen complexes were detected by enhanced chemiluminescence. NHE3 immunoreactivity in each fraction is expressed as the percentage of total signal in all 12 fractions. Results are expressed as means ± SE, n = 5 in each group. C: representative immunoblots from a typical experiment with NHE3 detected at 80 kDa. *P < 0.05 vs. basal period assessed by multiple comparison ANOVA and followed by paired Student's t-test.



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Fig. 3.   Distribution of enzyme activities of apical and basolateral membrane proteins in membrane fractions of renal cortex from normotensive SD and after 5 min acute hypertension (41) (A), adult SHR and SHR after a further acute increase in arterial pressure (B), and adult SHR and SHR after acute normalizing of blood pressure with ketamine/xylazine (C). Apical alkaline phosphatase activity, apical dipeptidyl-peptidase IV (DPPIV) activity, and basolateral Na-K-ATPase activity are all expressed and corrected for total protein recovered from all 12 fractions. Data are expressed as means ± SE; n = 5. *P < 0.05 vs. control, assessed by multiple comparison ANOVA and followed by paired Student's t-test.

When arterial pressure was acutely decreased in the SHR, neither NHE3 distributions (Fig. 2B), nor apical alkaline phosphatase, DPPIV, and basolateral Na-K-ATPase activity distributions changed (Fig. 3C). Thus apical protein distributions and basolateral Na-K-ATPase activities in adult SHR resemble that in the acutely hypertensive SD and are not altered by a further increase or decrease in arterial pressure. The differences between adult SHR and SD may reflect an adaptation to the chronic hypertension in the SHR or to the fact that SHR have a different genetic background than the SD. To address this question, we examined Na transporter distributions and activities in young prehypertensive SHR compared with age-matched SD, comparing young vs. adult animals.

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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Fig. 4.   Mean arterial pressures of 3 to 4-wk, young, SD and SHR rats, and 12-wk, adult SD and SHR measured via carotid catheter. Values are means ± SE; n = 8 for young SD, n = 10 for young SHR, n = 6 for each adult SD and SHR. *P < 0.05 vs. adult SD by Student's t-test.

To determine which differences in sodium transporter distribution, abundance, and activity between SHR and SD were present before hypertension developed, and which occurred as hypertension developed, we collected and analyzed, at the same time, samples from young SHR vs. young SD, and from adult SHR and SD. Figure 5 summarizes the density distributions of NHE3 and NaPi2, as well as the apical cytoskeletal membrane marker, villin. There was no difference in density distribution patterns of apical transporters or markers in young SD vs. SHR, indicating that distribution of NHE3 at heavier densities observed in adult SHR (Fig. 2) is not present before hypertension develops, thus unlikely to be genetically determined (Fig. 5). NHE3, detected at 80 kDa by immunoblot (5), distributes to a major peak at fractions 4-7 in both YSD and YSHR, containing 75% of the total NHE3, and to a shoulder at fractions 8-10 containing the other 25% (Fig. 5A). With transition to adult and to chronic hypertension in SHR, there is a consistent shift in the immunoreactive pool of NHE3 to heavier fractions compared with adult SD: the NHE3 moves out of fractions 4 and 5 and increases in fractions 7 and 8. This redistribution in SHR with development and ensuing hypertension is identical to the shift in NHE3 when adult SD are subjected to acute hypertension (Fig. 2A, 41). In addition, this comparison revealed there are distinct age-specific changes in NHE3 distribution in SD as there is a 10% increase in the percentage of NHE3 in apical enriched fraction 5. These results indicate that while there is a redistribution of NHE3 out of the fractions enriched in the apical microvilli (fractions 4 and 5) with development of chronic hypertension in SHR, there is a relative enrichment of NHE3 in the apical microvilli with age in the SD. Total pool size of NHE3 in young and adult SHR vs. SD was analyzed by immunoblot of three sets of membranes before fractionation (Fig. 6). There were no consistent differences in pool size of NHE3 between SHR and SD in the young animals, or in the adult rats after hypertension developed in the SHR, indicating that neither chronic hypertension nor development provoke a detectable change in the total pool size of NHE3.


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Fig. 5.   Apical protein density distributions in young vs. adult SD and SHR rats. Top and middle: density distribution of NHE3 (A), NaPi cotransporter isoform 2 (NaPi2; B), and the cytoskeletal protein villin (C) in membrane fractions of renal cortex from young SD (YSD), young SHR (YSHR), adult SD, and adult SHR groups. A constant volume of each fraction was resolved by SDS-PAGE, blotted, and probed with NHE3-specific monoclonal antibody, NaPi2-specific polyclonal antibody, or villin-specific monoclonal antibody. Antibody-antigen complexes were detected by enhanced chemiluminescence for NHE3 and 125I-protein A for NaPi2 and villin. Immunoreactivity in each fraction is expressed as the percentage of total signal in all 12 fractions. Results are expressed as mean ± SE. For NHE3, n = 7 each for YSHR and YSD, n = 6 each for SD and SHR; for NaPi2, n = 3 for YSD, n = 5 for YSHR, n = 6 each for SD and SHR; for villin, n = 3 in each group. For both NHE3 and NaPi2, distributions were not significantly different between YSD and YSHR, while adult SHR distributions are shifted to heavier densities compared with adult SD. Villin distributions were not significantly different in both the young and adult groups. Bottom: representative immunoblots from a typical experiment with NHE3 detected at 80 kDa, NaPi2 detected between 80 and 90 kDa, and villin at 95 kDa. *P < 0.05 vs. YSD or SD, assessed by ANOVA and followed by paired Student's t-test.



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Fig. 6.   NHE3 protein levels in total membranes of renal cortex from YSD, YSHR, adult SD, and adult SHR collected and analyzed as sets of 1 each, n = 5 sets. Top: summarizes the immunoblot analysis of all sets. Density, measured in arbitrary units, was normalized to that of the YSD sample, defined as 1.0. Values shown are means ± SE. No significant differences were noted. Bottom: shows all 5 sets of samples run at 20 µg/lane and probed with the anti-NHE3 antibody L546 followed by enhanced chemiluminescence. A subset of the samples was run at 10 µg/lane to verify the linearity of the detection system.

Since we had previously observed that not only NHE3, but also NaPi2, the apical isoform of the Na-Pi cotransporter (13), shifts to heavier densities with acute hypertension in adult SD, we examined the distribution of NaPi2 in the young vs. adult SD and SHR. As with NHE3, NaPi2 distributions are not different in the young animals (Fig. 5B). While they are both apical antigens, they have distinct distributions: there is a much higher percentage of NaPi2 in fractions 8-12, corresponding to intracellular stores, than there is of NHE3. In both SD and SHR, the percentage of NaPi2 found in fractions 8-12 decreased with age while that in fractions 4-7 increased with age. This shift remarkably reproduces a decrease in intracellular stores of NaPi2 in Wistar rat proximal tubules between 3 and 6 wk of age, detected by confocal microscopy, reported in a very recent study of NaPi2 during postnatal ontogeny (34). This provides additional support for designating fractions 8-12 as intracellular membranes. In the adult SHR, the peak of NaPi2 is focussed in fractions 6-7, heavier densities compared with the NaPi2 peak in normotensive SD, focussed in fractions 5-7, and analogous to the relative distributions of NHE3 in SHR or SD during acute hypertension vs. control SD (Fig. 5).

To verify that the shifts in apical Na transporters with development are not due to an overall difference in the density of the microvillar membranes themselves with the development of hypertension in SHR, we examined the distribution of membranes associated with villin, a 95-kDa microvillar cytoskeletal bundling protein. Figure 5C illustrates that villin is broadly distributed in fractions 5-11 and is not different between the two strains at either age. The preservation of the villin density distribution indicates that overall microvillar membrane density does not change with age, is not different between the two strains, and does not change with hypertension, which indicates that the differences in both NHE3 and NaPi2 distributions with acute or chronic hypertension are not secondary to an overall change in density of microvilli.

To ascertain the specificity of the redistribution of NHE3 and NaPi2 with development of hypertension, additional apical markers were examined. The apical membrane of the proximal tubule can be roughly divided into two domains, the microvilli and the intermicrovillar cleft regions. DPPIV and alkaline phosphatase are markers of the apical microvillar membrane while megalin, also known as gp330 (330 kDa), a scavenger receptor, is found mainly in the intermicrovillar cleft and clathrin coated pits (4, 12). In this fractionation scheme both DPPIV and megalin have peaks at fraction 6, overlapping with the peaks in both NHE3 and NaPi2, and considerable amounts in fractions 8-12 in the adult normotensive SD. Interestingly, megalin is shifted to higher density membranes in the adult SHR compared with adult SD (Fig. 7), and there is an apparent shift in the pattern of DPPIV which did not reach statistical significance by ANOVA. This coordinated shift is not totally unexpected, as Girardi et al. have recently reported (20) that a significant fraction of NHE3 exists as a multimeric complex with megalin and that NHE3 also forms a multimer with DPPIV (20). The higher density fractions 9-11 contain rab5a and rab4, the monomeric GTPases associated with endocytosis and exocytosis (40). The appearance of increased NHE3 (Fig. 5) and megalin (Fig. 7B) in the heavier density shoulder (fraction 9-11) with chronic hypertension suggests redistribution of both to endosomal populations.


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Fig. 7.   Density distribution of apical microvillar and intermicrovillar markers in membrane fractions of renal cortex from adult SD and adult SHR groups. A: distribution of DPPIV, a marker for the apical microvillar membrane, was detected by immunoblot with a constant volume of each fraction resolved by SDS-PAGE, blotted, and probed with a DPPIV-specific polyclonal antibody. Antibody-antigen complexes were detected with 125I-protein A. DPPIV immunoreactivity in each fraction is expressed as the percentage of total signal from all 12 fractions. B: distribution of megalin, a marker found in the intermicrovillar cleft and endosomes, was detected by immunoblot with a constant volume of each fraction resolved by SDS-PAGE, blotted, and probed with megalin-specific polyclonal antibody. Antibody-antigen complexes were detected with 125I-protein A. Megalin immunoreactivity in each fraction is expressed as the percentage of total signal from all 12 fractions. Results are expressed as means ± SE; n = 6 each for SD and SHR. There is less megalin in the lighter fractions in the SHR compared with SD and more in the heavier densities. Results are expressed as means ± SE; n = 6 each for SD and SHR. C: representative immunoblots with megalin detected at 330 kDa and DPPIV detected at 105 kDa.

The major peak of the classical apical membrane marker alkaline phosphatase activity is located in fractions 5-8, overlapping with NHE3, NaPi2, and DPPIV distributions (Figs. 3 and 8). Peak activity of alkaline phosphatase is 40% higher in the young SHR compared with young SD, and decreases, as hypertension develops, to the same activity measured in adult SD. Total alkaline phosphatase activity, measured in renal cortical membranes before fractionation, was 37% higher in young SHR compared with young SD (2.84 ± 0.35 vs. 2.07 ± 0.11 µmol Pi · mg protein-1 · h-1 P < 0.05), but was not different in adult SHR vs. SD (2.10 ± 0.45 vs 2.05 ± 0.45 µmol Pi · mg protein-1 · h-1), illustrating that the alkaline phosphatase activity in SHR fell significantly with age (P < 0.05). Likewise, other investigators have not seen a difference in alkaline phosphatase activity in adult hypertensive SHR vs. normotensive WKY (7, 16). The pattern of a decrease in alkaline phosphatase activity with chronic hypertension is the same as the rapid decrease in activity during acute hypertension in the adult SD (Fig. 3A), even though the activities are higher in the chronically hypertensive SHR (Fig. 8).


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Fig. 8.   Density distribution of apical alkaline phosphatase and basolateral Na-K-ATPase activity in membrane fractions of renal cortex from YSD, YSHR, adult SD, and adult SHR groups following density gradient fractionation. Left: apical alkaline phosphatase was significantly higher in YSHR than YSD, but not in adult SHR vs. SD. Right: Na-K-ATPase-specific activity, assessed by measuring K+-dependent p-nitrophenylphosphatase (K+-pNPPase) activity as described in EXPERIMENTAL PROCEDURES, was significantly higher in YSHR than YSD in the basolateral membrane peak fractions, whereas there was no difference in the adult SHR vs. SD. Results are expressed as means ± SE, n = 8 for YSD, n = 10 for YSHR, and n = 3 for SD and SHR. *P < 0.05 vs. YSD or SD, assessed by ANOVA and followed by Student's paired t-test.

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 alpha 1 and beta 1 abundance measured in the total So fraction (n = 4). Figure 9C shows two sets of typical blots and illustrates the linearity of the detection system by analysis of two amounts of a subset of the samples. Prior to detection of beta 1, the samples were deglycosylated to remove the possibility that differences in the sugar groups between the two strains could influence immunoreactivity. As summarized in Fig. 9B, the levels of alpha 1 and beta 1 were not higher in YSHR compared with YSD, in the same samples where Na-K-ATPase enzymatic activity was significantly higher, consistent with the interpretation that there is higher Na-K-ATPase activity per transporter in young SHR. There was an insignificant tendency for alpha 1 and beta 1 to be higher in adult SD compared with YSD, but no significant difference in adult SHR vs. SD.


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Fig. 9.   Na-K-ATPase activity and subunit levels in total membranes of renal cortex from YSD, YSHR, adult SD, and adult SHR collected and analyzed as sets of 1 each, n = 4 sets. A: K+-pNPPase activity and ouabain-sensitive Na-K-ATPase activity expressed as µmol inorganic phosphate liberated per mg protein per hour. YSHR is elevated compared with YSD (P < 0.05) by paired Student's t-test. B: Summary of immunoblot analysis of the four sets. Density, measured in arbitrary units, was normalized to that of the YSD sample, defined as 1.0 [note: variance of YSD could be determined for the beta 1 sample (sets on same blot), but not for alpha 1 (sets on different blots)]. Values shown are mean ± SE. C: Two sets of samples probed with anti-alpha 1 or anti-beta 1 antibodies and 125I-protein A (note: same two sets are shown for each antibody). A subset of the samples were run at two protein levels to verify linearity of the detection system.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 1 or beta 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.


    ACKNOWLEDGEMENTS

We acknowledge the technical advice and assistance of Dr. Li Yang and Dr. Jon Armstrong.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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