Departments of 1 Medicine and 2 Cellular and Molecular Medicine, Division of Nephrology, University of Ottawa and Ottawa Hospital, Ottawa, Ontario, Canada K1H 8M5
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ABSTRACT |
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To determine the in vivo effects of chronic ANG II type 1 (AT1)-receptor blockade by losartan (Los) on enhanced unidirectional bicarbonate reabsorption (JHCO3) of surviving distal tubules, nephrectomized rats drank either water or a solution of Los, 7 days before microperfusion. JHCO3 was suppressed by 50% after Los without further reduction by 5 nM concanamycin A (Conc), suggesting that Los suppresses all Conc-sensitive H+-ATPase pumping. Indeed, ultrastructural analysis of A-type intercalated cells revealed a 50% reduction of H+-ATPase immunogold labeling of the apical plasma membrane, whereas Western blotting showed that H+-ATPase protein levels were also reduced by one-half by Los treatment. To identify other transporters sustaining JHCO3, we perfused three inhibitors simultaneously [5-(N,N-dimethyl) amiloride hydrochloride, Conc, Schering 28080] with or without prior Los treatment: JHCO3 was unchanged despite marked reduction of water reabsorption. We conclude enhanced distal tubule JHCO3 of surviving nephrons is largely mediated by AT1 receptor-dependent synthesis and insertion of apical H+-ATPase pumps in A-type intercalated cells.
kidney failure; receptors; losartan; proton-transporting adenosine 5'-triphosphate synthase; concanamycin A
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INTRODUCTION |
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WE HAVE RECENTLY REPORTED that when compared with normal kidneys, surviving distal tubules (DT) of remnant kidneys strikingly augment proton secretion and bicarbonate reabsorption in a fashion that depends in part on ANG II and enhanced expression/activation of H+-ATPase (12). Acute, in vivo microperfusion of the ANG II type 1 (AT1)-receptor blocker losartan (Los), resulted in significant, but incomplete suppression of net bidirectional bicarbonate flux. The present studies were devoted to characterizing further the portion of bicarbonate reabsorption not suppressed by acute intratubular perfusion of Los. The rationale underlying our experiments is conveniently explained by addressing three questions.
1) Is the persistent residual proton secretory flux suppressible by chronic AT1-receptor blockade? ANG II, via the AT1 receptor, is reported to be a stimulator of renal tubular cell RNA and protein synthesis (27), and chronic AT1 blockade has been reported to induce changes in rapidly growing kidneys of neonatal rats (23) and after 5/6 nephrectomy (7, 19). Accordingly, for the present studies we theorized that chronic AT1-receptor blockade, induced by drinking Los, may more completely inhibit DT bicarbonate reabsorption, perhaps by impeding synthesis of H+-ATPase.
2) What is the transport basis of the persistent residual
proton secretory flux? We recognized the possibility that the
persistent residual flux may be ANG II independent even after chronic
AT1 blockade. In our previous studies on nephrectomized
(Nx) rats eating a normal diet, 5 nM concanamycin A (Conc), the
specific H+-ATPase inhibitor (17), perfused alone, reduced
the bicarbonate flux by 31% (12 ), leaving a large residual flux
intact. In K-depleted Nx rats, in which bicarbonate reabsorption was
greatly increased and insensitive to Los, the bicarbonate flux
augmentation was again prevented by a 5 nM Conc perfusion with a large
residual flux still persisting. This proton secretory flux was
resistant to inhibition by 105 M
ethylisopropyl amiloride (EIPA) or 10
4 M
5-(N, N-dimethyl) amiloride (DMA) in Nx rats, or
10
5 mM Schering 28080 (Sch-28080) in
K-depleted Nx rats (12, 13).
In contrast, in intact rat kidneys subjected to Cl depletion metabolic
alkalosis and hypokalemia (25), we demonstrated that 3 mM amiloride and
107 M bafilomycin completely supressed
the DT bicarbonate flux, and, in acid-loaded intact rats,
10
5 M Sch-28080 reduced it by one-half
(14). Furthermore, Wang et al. (25) showed that
10
5 M Sch-28080 and
10
5 M EIPA also significantly reduced
distal tubular bicarbonate reabsorption after 8 days of dietary K
depletion, indicating inhibitory effects on the two proton secretory
transporters, H+-K+-ATPase and the
Na+/H+ antiporter. Of particular importance to
the present studies, and showing marked differences between DT
bicarbonate fluxes in intact vs. Nx rats, are an additional three
studies. We first showed that in intact rat kidneys, late DT
bicarbonate reabsorption was enhanced by endogenous ANG II and
inhibitable by 10
6 M Los (11).
Subsequently, Wang and Giebisch (24) reported ANG II stimulation only
in early DT that could be abolished by 10
3 M amiloride. Furthermore,
Barreto-Chaves and Mello-Aires (1), by measuring bicarbonate
reabsorption by continuous intratubular pH measurements, made four
additional observations: 1) ANG II stimulated proton secretion
in both early and late DT; 2) this was completely abolished by
10
6 M Los as we showed; 3) this
was greatly suppressed by 10
4 M
5-(N,N-hexamethylene) amiloride; and
4) bafilomycin (2 × 10
7 M) suppressed the flux by only
33% in late, but not early, DT.
How, then, can we explain the resistance of surviving DT to inhibitors
that are able, in most studies, to greatly reduce proton secretion in
DT of non-Nx rats? One group of possibilities is that DT epithelium in
surviving nephrons differs in cell type, transporter density, or
transporter isoforms, as we have suggested previously (12). In the case
of H+-ATPase, both a novel isoform, and cytoplasmic
stimulators of the pump, have been described (18, 28). With respect to
Na+/H+ antiporter, Tse et al. (22) have
described a recombinant NHE3 resistant to amiloride. Another
possibility is that there is a higher density of the colonic
-isoform of the H+-K+-ATPase, the message of
which is upregulated in kidneys in rats subjected to chronic
hypokalemia (2-4) and is Sch insensitive when expressed in a
heterologous system (6, 9). Accordingly, it is possible that the
Sch-resistant colonic H+- K+-ATPase
-isoform
is active in surviving DT of Nx rats, and, in fact we have already
shown that the Sch-sensitive gastric
-isoform is not upregulated to
detectable levels in the Nx kidney (2-4, 13). Thus isoforms to one
or more of the three major transporters, resistant to commonly used
inhibitors, may be a characteristic of surviving DT.
Notwithstanding these considerations, we believed a simpler hypothesis should be tested first: it is conceivable that when luminal H+-ATPase activity is blocked by Conc, a NHE3 may sustain a larger portion of the bicarbonate reabsorptive flux. Conversely, perhaps during perfusion with DMA or Sch, proton secretion is diverted to, and sustained by, an H+-ATPase. It seemed, therefore, appropriate to evaluate the effects of not only chronic Los administration but also of three of the above-mentioned inhibitors, simultaneously perfused, and at doses known to inhibit proton transport in intact rats.
3) Can part of the persistent residual net bicarbonate flux be attributed to a bicarbonate secretory component, rather than resistance to inhibition of unidirectional reabsorption? In our previous studies, which measured changes in net bidirectional bicarbonate reabsorption, it was conceivable that a portion of the residual flux was due to persistence of unidirectional secretion, rather than a failure of inhibition of unidirectional reabsorption. Moreover, it is possible that Los itself might stimulate a secretory component. In the rabbit cortical collecting duct, Weiner et al. (26) reported that ANG II stimulates luminal alkalinization through a basolateral AT1 receptor. Therefore, for these experiments, to more carefully examine mechanisms of bicarbonate reabsorption in surviving DT, a zero-Cl solution was used to minimize the secretory flux whereas unidirectional bicarbonate reabsorption, JHCO3, was measured as we have previously reported in normal rats (14, 16) .
Accordingly, the objectives of the present study were to determine, in surviving DT, the components of JHCO3, in the presence of multiple inhibitors with and without chronic AT1 blockade. Our results show that 1) 7 days of Los drinking does not impair remnant kidney gross function but suppresses the markedly elevated DT JHCO3 by ~50% without further reduction by the proton pump inhibitor Conc; 2) both Western blot analysis and immunogold labeling show suppression of H+-ATPase protein levels after chronic AT1 blockade; and 3) despite multiple inhibitor perfusions during chronic AT1 blockade, a substantial bicarbonate reabsorptive flux persists despite marked reduction in water reabsorption (Jv).
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METHODS |
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Renal Ablation
Adult male Sprague-Dawley rats, born and raised in a climate-controlled facility at the University of Ottawa and weighing between 200 and 270 g, underwent right nephrectomy and polectomy of the left kidney resulting in ~2/3-5/6 nephrectomy (Nx), as previously described (12). The rats were allowed 13-16 days recovery after surgery before microperfusion.Microperfusion Studies
The rats were given either a Los drink (180 mg/l) 7 days before micropuncture or water to drink ad libitum. They were maintained on standard laboratory rat chow (Ralston-Purina, Woodstock, ON). The rats were housed in individual stainless steel metabolic cages for 16 h (overnight) before microperfusion, allowing measurement of ingested food and drink and collection of urine under oil, with the use of thymol as a preservative. The following morning, the animals were anesthetized with 100 mg/kg thiobutabarbital sodium (Inactin; Research Biochemicals International, Natick, MA) and prepared for microperfusion, as described previously (12). Briefly, the rat was placed on a heated operating table, and a tracheostomy was performed by using PE-240 tubing. The left carotid artery was cannulated for continuous blood pressure measurement and collection of blood for acid-base and electrolyte analyses, whereas the left jugular vein was cannulated with three lines for infusion of fluid, pentobarbital sodium anesthetic (Somnotol; MTC Pharmaceuticals, Cambridge, ON), and 10% Lissamine green. The left kidney was exposed by flank incision, carefully dissected from the adrenal gland, and immobilized in a stainless steel cup covered with mineral oil. The ureter was catheterized with PE-50 tubing to ensure proper urine flow.To replace surgical fluid losses, the rats were infused via the jugular vein at 1% body wt/h for 30 min with donor plasma from a control rat (non-Nx and having received no Los). The animal was then maintained on 0.9% saline at 1% body wt/h for the remainder of the experiment.
Perfusable surface two-loop DT were identified by injecting a 0.02-ml bolus of 1% Lissamine green (Steinhausen) into surface proximal loops and observing its passage through the nephron. DT were perfused at 15 nl/min with hypotonic solution containing (in mM) 28 HCO3, 0 Cl, 56 Na, 2 K, 1.8 Ca, 16.6 urea, and 30 gluconate. FD & C green no. 3 dye (0.05%; Keystone, Chicago, IL) and bovine serum albumin (0.1%; Intergen, Purchase, NY) were also added to the perfusate. [3H]inulin (Mandel Canada, Guelph, ON) was added to the perfusate as a marker of Jv. The perfusion rate of 15 nl/min was chosen on the basis of preliminary experiments in Nx rats, which showed early distal free-flow rates of 13.3 ± 0.7 nl/min (n = 12). The perfused bicarbonate load, higher than in free flow, was chosen to more easily reveal effects of inhibitors. Sample collections were quantitative and timed. A 10-min preperfusion period preceded all collections. Perfused tubules that provided samples were backfilled from the collection site with latex (Microfil; Flow Tech, Carver, MA) to confirm surface direction of flow and to provide a hardened cast that, when removed from the digested kidney, allows measurement of the length of the perfused segment.
Groups
Nx rats were divided into five groups as shown in Table 1, one Nx control group (group 1) and four experimental groups (groups 2-5), designed to assess the role of the AT1 receptor and/or inhibitors in decreasing DT unidirectional JHCO3 in rats with reduced renal mass. Group 2 rats were given 180 mg/l Los to drink for 7 days before microperfusion. Next, group 3, after receiving 7 days of the Los drink, was perfused with 5 nM concanamycin A (Sigma-Aldrich Canada, Mississauga, ON) dissolved in 0.1 mM DMSO (final concentration DMSO in perfusate was 0.1%) to inhibit V-type H+-ATPase activity. Group 4 (without Los) was perfused with three transport inhibitors: 5 nM Conc dissolved in DMSO (final concentration DMSO in perfusate was 0.1%); DMA (Research Biochemicals International), an amiloride analog, solubilized in distilled water at a concentration of 0.075 mM; and 10
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Analyses
Whole blood and urine pH and PCO2 were measured quantitatively by electrodes (IL 1610 blood-gas system; Instrumentation Laboratory, Milano, Italy), and bicarbonate concentrations were calculated. Plasma and urine Na and K concentrations were measured by flame photometry (IL 943 flame photometer; Instrumentation Laboratory), and Cl concentrations were measured by electrotitration (CMT 10 chloride titrator; London Scientific, London, ON). Plasma total protein concentrations and urine specific gravity were measured by refractometry (10400A TS meter; Cambridge Instruments, Buffalo, NY), and hematocrits were determined by microcapillary reader (International Equipment, Needham Heights, MA). Urine osmolalities were determined by freezing-point osmometry (advanced model 3MOplus MicroOsmometer, Advanced Instruments, Norwood, MA). Plasma creatinine concentrations were measured by the kinetic Jaffé method, without deproteinization (BM/Hitachi System 717; Boehringer Mannheim, Laval, PQ). Perfusates and samples were counted by using the Beckman model 3801 liquid scintillation system (Beckman Instruments Canada, Mississauga, ON).Perfusate and sample total carbon dioxide (tCO2) concentrations were measured by microcalorimetry, as previously described (12). A standard curve (10, 20, 30, and 40 mM NaHCO3) was run before sample analysis, and standards bracketed the determination of sample and perfusate tCO2 concentration. Perfusate and sample Cl concentration were determined by constant-current electrotitration with potentiometric end-point sensing, as described previously (12). A series of four standards (20, 40, 60, and 80 mM NaCl) was run before perfusate and sample analysis to generate a regresssion line, and, in analytes suspected to have a low Cl concentration, a midcurve standard was added to the sample or perfusate before titration.
Calculations
The perfusion rate (RP) was calculated as the product of the collected rate (RC) and the ratio of sample inulin concentration over perfusate inulin concentration. The difference between the calculated perfusion rate and the measured collected rate (RP
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Electron Microscopy and H+-ATPase Immunogold Labeling
Kidneys from Nx and Nx+Los rats were removed after abdominal aortic perfusion of cold PBS, pH 7.4, followed by cold 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, and 1- to 2-mm freehand sagittal sections were cut with a razor blade. Slices were immediately immersed in fixative and then 1-mm2 pieces of cortex were excised, immersed in fixative for 3-4 h, and rinsed with 0.1 M phosphate buffer. Residual aldehyde groups were quenched with 50 mM glycine in 0.1 M phosphate buffer for 30 min. The tissue was then rinsed with phosphate buffer and treated with freshly made 0.1 M NH4Cl for 1 h. Specimens were dehydrated sequentially in 30, 50, 70, and 90% ethanol and infiltrated with 1:1 90% ethanol-LR White resin (Marivac), followed by 1:2 and 1:3 mixtures, and finally by pure LR White resin. The above procedures were done at 4°C. The tissue samples were then placed in gelatin capsules, subsequently filled with LR White resin, and made airtight with a cover. Polymerization was done at 50°C for 6 h in a vacuum oven.Silver-gold sections were cut with a diamond knife (Diatome) and collected on formvar-coated 150-mesh nickel grids (Marivac). Sections were floated on 1% casein-5% normal goat serum in PBS for 30 min at room temperature to block the nonspecific binding sites, then rinsed with PBS and treated overnight at 4°C with either 1) H+-ATPase antiserum diluted 1:200 with PBS or 2) preimmune serum diluted 1:200 with PBS. The H+-ATPase antiserum was raised in a rabbit against the COOH-terminal decapeptide of the 31-kDa subunit of the bovine V-type H+-ATPase, as described by Sullivan et al. (20 ). Grids were then thoroughly washed with PBS and incubated for 1 h at room temperature with diluted (1:20) goat anti-rabbit IgG coated with 18-nm gold particles (Bio/Ca Scientific, Mississauga, ON). Sections were lightly stained with 0.25% uranyl acetate and lead citrate and examined with a Philips 300 electron microscope. Images were recorded on Eastman 35-mm film at a magnification of ×4,125 for morphometric analysis or ×16,600 for illustrations, as previously described (13).
Analysis of electron microscope images was performed on a Power Macintosh 7100/80 computer by using the public domain National Institutes of Health (NIH) Image program (developed at NIH and available at http://rsb. info.nih.gov/nih-image/). The 35-mm negatives were digitized with a Polaroid PrintScan 35 at 675 dpi, and the reversed image was displayed on the computer screen.
Measurements of H+-ATPase immunogold labeling were determined from film negatives at a magnification of ×40,300 by passing the cursor along the boundary of the apical cell membrane including the surface of those microplicae continuous with the cell surface. The number of immunogold particles lying along that distance of membrane was expressed as gold particles per micrometer. Immunogold particle deposition was determined on a total of 135 A-type intercalated cells, representing 67 cells from 3 Nx rats or 68 cells from 3 Nx+Los rats. The extent of background or nonspecific immunogold labeling was determined from 30 A-type intercalated cells from 3 Nx+Los rats incubated with preimmune serum.
Western Blotting Analysis
Five Nx (group 1) and six Nx+Los (group 2) rats were anesthetized by intraperitoneal injection of 100 mg/kg pentobarbital sodium (Somnotol; MTC Pharmaceuticals, Cambridge, ON) and perfused via the abdominal aorta with cold PBS. The remnant kidney was removed, and portions of the microdissected cortex were sonicated in SDS sample buffer as previously described (13). Protein concentration was determined with the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) according to the manufacturer's protocol, as modified for microplate reader (6). Cortical proteins (50 µg) were separated on a 12.5% SDS-PAGE slab gel and electrophoretically transferred to nitrocellulose (21). Immunoblotting to detect the proton pump, followed by quantitation of immunoreactive bands by laser scanning densitometry, were performed exactly as previously described (12, 13).Statistics
All data are expressed as means ± SE. Balance, blood, and urine data summarized in Table 2 represent pooled data from all Nx rats and all Nx+Los rats. Comparisons between two groups were done by two-tailed unpaired Student's t-test, whereas comparisons among more than two groups were carried out by ANOVA with posttesting by either Dunnett's test (when comparisons were made within all possible pairings) or the Newman-Keuls test (when each group vs. a single control was compared). When raw data failed statistical tests for normality and/or homoscedasticity, the corresponding nonparametric test was employed. P < 0.05 indicated a statistically significant difference between groups.
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RESULTS |
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Balance, Blood, and Urine Data
Compared with control Nx rats, after 7 days of drinking 180 mg/l Los there were no differences in body weight, kidney weight, systemic and urine acid base status, and serum K values (Table 2). Systolic blood pressures recorded during the experiment were also not different (Nx, 131 ± 4 mmHg vs. Nx+Los, 132 ± 5 mmHg, P > 0.05).
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Microperfusion Data
As already detailed above, we have shown in two separate recent studies (12, 13) that 5 nM Conc alone significantly suppresses DT bicarbonate reabsorption in Nx rats, and therefore this group was not repeated. Figure 1 shows results from group 1, control Nx rats; group 2, after 7 days Los; and group 3 after 7 days Los plus acute intratubular Conc perfusion. There was a 50% decrease in JHCO3 of control Nx rats (group 1) after Los blockade (group 2): 82 ± 10 vs. 33 ± 10 pmol · minElectron Microscopy and Immunogold Labeling
Quantitation of H+-ATPase
immunogold labeling of A-type intercalated cells.
H+-ATPase immunogold labeling of A-type intercalated cell
apical plasma membrane was suppressed by 7 days of Los administered to
Nx rats when compared with Nx controls: 2.96 ± 0.21 vs. 1.64 ± 0.14 gold particles/µm, P < 0.01 (Fig. 3). The extent of
background or nonspecific immunogold labeling determined with preimmune
serum for Nx+Los rats was negligible: 0.05 ± 0.004 gold
particles/µm or 3.1%.
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Western Analysis
Western blotting for 31-kDa
H+-ATPase in control (Nx) and
experimental (Nx+Los) rats.
As seen in Fig. 4, the levels of the 31-kDa immunoreactive band were
reduced in samples of total cortical tissue prepared from the
experimental animals. Laser scanning densitometry was performed to
determine whether these changes were consistent and statistically
significant. The average integrated intensity of the control
immunoreactive bands was 0.44 ± 0.12, whereas that of the
experimental bands was 0.20 ± 0.04 (P < 0.004), suggesting the H+-ATPase protein levels were reduced to 45% of
control levels by chronic Los treatment.
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DISCUSSION |
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These in vivo microperfusion studies on surviving DT, combined with
Western and ultrastructural analyses of H+-ATPase, examined
for the first time unidirectional bicarbonate reabsorption, i.e.,
JHCO3, after chronic
AT1-receptor blockade with and without multiple inhibitor
microperfusion. The brisk JHCO3
associated with Nx falls by 50% after 7 days of Los drinking, without
impeding compensatory kidney growth, or changing systemic acid base or
K homeostasis. Addition of 5 nM Conc, the specific H+-ATPase inhibitor (17), to the perfusate did not further
reduce JHCO3, strongly suggesting that
Los suppresses H+-ATPase pumping. However, it is also
possible a portion of the JHCO3
reduction by Los is attributable to inhibition of
Na+/H+ exchange: in the early DT of the non-Nx
rat, Na+/H+ exchange is stimulated by ANG II
and blocked by Los (1), but not by 0.01 mM EIPA during perfusion of
whole surviving DT (12). Ultrastructural analysis of A-type
intercalated cells after Los treatment revealed a 55% reduction of
H+-ATPase immunogold labeling of the apical plasma membrane
(2.96 ± 0.21 vs. 1.64 ± 0.14 particles/µm, P < 0.01 vs. Nx). Furthermore, Western analysis was also consistent
in that proton pump protein levels in extracts of total cortical tissue
were reduced by one-half after Los treatment. Finally, we show that
JHCO3 is not further suppressed by the
simultaneous perfusion of three inhibitors: the H+-ATPase
inhibitor 5 nM Conc; the NHE inhibitor 0.075 mM DMA; or the
H+-K+-ATPase inhibitor
105 M Sch-28080. These triple inhibitor
perfusions were undertaken with and without chronic AT1
blockade and were sufficiently potent to markedly suppress
Jv.
The Perfusion Solutions
These solutions were designed to minimize bicarbonate secretion, to maximize bicarbonate reabsorption to reveal differences between groups, and to perfuse three inhibitors simultaneously, while ensuring that collections were reliable in fast-flowing DT from these remnant kidneys. These experimental design requirements, not unexpectedly, led to gross departures from "normal" in vivo tubular fluid concentrations and flow rates, an issue that we have addressed in detail in the past (10). Consequently, the removal of Cl, as used by us and others in the past (14, 15), led to a gluconate substitution, which could conceivably alter intraluminal Ca concentrations. Furthermore, attempting to achieve appropriate concentrations of three inhibitors perfused simultaneously led to differences in osmolality between groups as a result of DMSO addition to attain adequate solubility. However, as Table 3 shows, because all groups were perfused with 30 mM gluconate, it is unlikely there were differences between groups in perfused Ca concentrations. With respect to the possible effects of DMSO and osmolality, because neither Jv nor JHCO3 was significantly different in groups 2 and 3 with the lower osmolality perfusates, when compared with groups 4 and 5, it is also unlikely there were important effects caused by DMSO or osmolality differences. Indeed, although not significant, JHCO3 tended to be higher in groups 4 and 5, although, of course, significantly lower than in group 1.AT1 Receptor Effects in Kidney Tissue
For the normal kidney, we have already shown modulation of DT bicarbonate reabsorption by ANG II and AT1-receptor blockade (11). Because the AT1 receptor is located in the DT and several other nephron segments (5), it is not surprising that several recent reports have documented the importance of AT1-receptor activity in renal transport and growth. First, it appears that AT1-receptor activity is critical for normal growth and possibly, therefore, for compensatory growth: chronic Los administered to newborn rat pups (23) induces severe renal growth abnormalities. In contrast, after Nx, Los blockade blunts pathological growth effects: remnant rat kidneys show decreased interstitial fibrosis and glomerulosclerosis (19), which could be effected by suppressing the potent growth factor transforming growth factor-Evidence for Reduced H+-ATPase Activity After Chronic AT1 Blockade
Our measurements of JHCO3 in surviving DT of Nx rats are ~25% higher than the net flux we previously reported in Nx rats (12), consistent with our objective of minimizing the unidirectional secretory component, as previously discussed (16). After 7 days of chronic AT1 blockade, this reduction of JHCO3 was not associated with changes in whole-animal parameters as shown in Table 2 and RESULTS: body weight, kidney weight, blood pressure, systemic and urine acid base, and K values were not different from control Nx rats, and could not account for changes in DT function. Thus we show for the first time, in surviving nephrons, a downregulation of proton pump activity induced by chronic Los treatment. Consistent with this interpretation is the failure of 5 nM Conc, a highly specific H+-ATPase inhibitor, to further suppress JHCO3, despite its clear effectiveness in identical preparations, not subject to chronic AT1 blockade (12, 13). We recognize it is still possible that a portion of the residual flux (see also below) may be sustained by the surviving DT, conceivably producing an H+-ATPase with a different subunit structure (18) or, possibly, by a partially Conc-resistant proton pump, upregulated by a cytosolic factor (28).Resistance of DT JHCO3 to Multi-Inhibitor Perfusions
Surviving DT undergo brisk compensatory growth and augmentation of net bicarbonate reabsorption from zero, in sham-operated rats, to >60 pmol · minNotwithstanding the foregoing, our present results suggest, that with
the exception of the appearance of a colonic
H+-K+ ATPase (see introduction and Refs. 10 and
3), it is unlikely that a significant portion of proton secretion
sustaining the JHCO3 is diverted from
one inhibited transporter to another. That is, the residual flux of
~40
pmol · min1 · mm
1
after Conc is unlikely to have been supported by a DMA-sensitive NHE
isoform. We continue to speculate that the rapidly growing neonatal,
remnant, and the K-depleted remnant kidney, as well as the normal
kidney subjected to severe acid loading, all may have unique transport
systems resistant to inhibitors.
In summary, we show for the first time, by combined in vivo microperfusion studies, ultrastructural immunogold and Western analyses, that chronic AT1 blockade of the remnant kidney reduces by one-half the brisk DT JHCO3, at least in large part, by downregulating H+-ATPase activity and protein levels. It is possible that downregulation of Na+/H+-mediated early distal bicarbonate reabsorption also contributes to the Los effect. By means of multi-inhibitor perfusions we demonstrate persistence of a significant residual flux, conceivably sustained by inhibitor-resistant proton transporters.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the expert assistance of Kim Yates, surgical animal technician with the University of Ottawa Animal Care and Surgical Service.
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FOOTNOTES |
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This work was supported by grants from The Medical Research Council of Canada (D. Z. Levine), The Kidney Foundation of Canada (D. Z. Levine), and The Atkinson Charitable Foundation (M. T. Hincke). Generous donations of Sch-28080 and losartan were made by Schering Canada, Inc., of Pointe Claire, Quebec, Canada, and The DuPont Merck Pharmaceutical Company of Wilmington, DE, respectively.
Abstracts referring to portions of this work were presented at the 1998 meetings of the Canadian Society of Nephrology (Toronto, ON), and the American Society of Nephrology (Philadelphia, PA).
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.
Address for reprint requests and other correspondence: D. Z. Levine, Dept. of Medicine, Div. of Nephrology, Health Science Bldg., 451 Smyth Road, Rm. 1333, Ottawa, Ontario, Canada K1H 8M5.
Received 23 June 1999; accepted in final form 12 October 1999.
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