1 The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark; 2 Department of Physiology, School of Medicine, Dongguk University, Kyungju 780-714, Korea; and 3 Division of Nephrology, University of California Los Angeles School of Medicine, Los Angeles, CA 90095-1689
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ABSTRACT |
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Several
members of the Na-HCO1 · day
1) for 2 wk with free access to water (n = 8). Semiquantitative immunoblotting demonstrated that whole kidney abundance of NBCn1 and
NBC3 in rats with CMA was dramatically increased to 995 ± 87 and
224 ± 35%, respectively, of control levels (P < 0.05), whereas whole kidney rkNBC1 was unchanged (88 ± 14%). In
protocol 2, rats were given NH4Cl in their food
(10 ± 1 mmol · rat
1 · day
1) for 7 days, with a fixed daily water intake (n = 6).
Consistent with protocol 1, whole kidney abundances of NBCn1
(262 ± 42%) and NBC3 (160 ± 31%) were significantly
increased compared with controls (n = 6), whereas whole
kidney rkNBC1 was unchanged (84 ± 17%). In both protocols,
immunocytochemistry confirmed upregulation of NBCn1 and NBC3 with no
change in the segmental distribution along the nephron. Consistent with
the increase in NBCn1, measurements of pH transients in medullary thick
ascending limb (mTAL) cells in kidney slices revealed two- to threefold
increases in DIDS- sensitive, Na+-dependent
HCO
acid-base balance; bicarbonate transport; intracellular pH; intercalated cell; thick ascending limb
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INTRODUCTION |
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THE KIDNEY MAINTAINS ACID-BASE balance by
H+ secretion and HCO
In addition to kNBC1, several other members of the
Na-HCO
Electroneutral NBC3,1 which was isolated from the human skeletal muscle cells (32) and is 89-92% identical to NBCn1 (11), is exclusively associated with intercalated cells in connecting tubules and in cortical, outer medullary, and initial inner medullary collecting ducts of rat kidney (23). In particular, NBC3 labeling in connecting tubule and cortical collecting duct is associated with both type-A and type-B intercalated cells. NBC3 colocalizes with the H+-ATPase in the apical domains in the type-A intercalated cells and in the basolateral domains in the type-B intercalated cells (23, 33). Therefore, NBC3 may participate in the H+/base transport in the collecting duct.
It is presently not established whether the expression of these NBCs
undergoes regulation, e.g., in conditions with altered acid-base
status. In the present studies, we therefore examined whether the
expression of kNBC1, NBCn1, and NBC3 in rat kidneys is altered in
response to chronic metabolic acidosis, using two different protocols
and immunoblotting and immunocytochemistry. Moreover, we examined the
rate of sodium-dependent HCO
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METHODS |
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Experimental Animals and Induction of Chronic Metabolic Acidosis
Protocol 1. Experiments were performed using male Munich-Wistar rats (250-300 g, Møllegard Breeding Centre) that were maintained on a standard rodent diet (Altromin, Lage, Germany). Rats subjected to a control group and chronic metabolic acidosis were randomly chosen. To examine whether there are changes in the expression of NBCs in response to chronic metabolic acidosis, rats were treated orally with 0.28 M NH4Cl in the drinking tap water for 2 wk ad libitum (n = 8) (5, 7, 25). Control rats received tap water ad libitum (n = 6). All rats had free access to standard rodent food (Altromin).
Protocol 2.
To avoid the potential effects of high water intake and increased urine
output on the expression of several NBCs (protocol 1), rats
in protocol 2 received fixed amounts of food and water with
the use of a protocol previously described (19, 31). Experiments were performed using male Munich-Wistar rats (250-300 g, Møllegard Breeding Centre). A standard rodent diet (15 g · 220 g body wt1 · day
1,
Altromin) mixed with water (37 ml · 220 g body
wt
1 · day
1) was given to each rat.
Rats were fed once daily in the morning and ate all of the food offered
during the course of the day. NH4Cl was included in the
diet at 7.2 mmol · 220 g body
wt
1 · day
1 and was administered for
7 days (n = 6). Control rats received the same diet but
without NH4Cl (n = 6). Detailed
characterization of the physiological responses of the rats or changes
in the expression of sodium transporters to NH4Cl loading
has been well reported previously (9, 19, 20). The daily
acid load administered in protocol 2 (10 ± 1 mmol · day
1 · rat
1) was
similar to that in protocol 1, which averaged 12 ± 1 mmol · day
1 · rat
1.
Blood-Gas Analyses and Clearance Studies
The rats were maintained in the metabolic cages, allowing quantitative urine collections and measurements of water intake. Urine volume, osmolality, creatinine, sodium, and potassium concentration as well as urine pH were measured. Arterial blood was collected from the abdominal aorta at the time of death for measuring plasma HCOMembrane Fractionation for Immunoblotting
All rats were killed under light halothane anesthesia, and their kidneys were rapidly removed. The whole kidney samples were minced finely and homogenized in dissection buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) using an ultra-turrax T8 homogenizer (IKA Labortechnik, Staufen, Germany). This homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was then centrifuged at 200,000 g for 1 h to produce a pellet containing membrane fractions enriched for both plasma membranes and intracellular vesicles (26). Gel samples (Laemmli sample buffer containing 2% SDS) were made of this pellet.Primary Antibodies
For semiquantitative immunoblotting and immunocytochemistry, we used previously characterized polyclonal antibodies as follows.Electrogenic Na-HCO
Electroneutral Na-HCO
Electroneutral Na-HCO
Electrophoresis and Immunoblotting
Samples of membrane fractions from total kidney were run on 6-16% gradient SDS-polyacrylamide minigels for NBC1, NBCn1, and NBC3. For each gel, an identical gel was run in parallel and subjected to Coomassie staining to ensure identical loading (40). Proteins were transferred to nitrocellulose paper by electroelution. After transfer by electroelution, blots were blocked with 5% milk in 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5 for 1 h and incubated overnight at 4°C with primary antibodies (see above). The labeling was visualized with horseradish peroxidase-conjugated secondary antibodies (P448, DAKO, Glostrup, Denmark, diluted 1:3,000) using the enhanced chemiluminescence system (Amersham Phamacia Biotech, Buckinghamshire, UK). Enhanced chemiluminescence films with bands within the linear range were scanned (26) using an AGFA scanner (ARCUS II) and Corel Photopaint Software to control the scanner. The labeling density was corrected by densitometry of the Coomassie-stained gels.Immunocytochemistry
Kidneys from rats with CMA (n = 4 in protocol 1 and protocol 2, respectively) and control rats (n = 4 in protocol 1 and protocol 2, respectively) were fixed by retrograde perfusion via the aorta with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4 (23, 46). The kidneys were removed, postfixed for 1 h, and either 1) cryoprotected overnight in 25% sucrose and rapidly frozen in CO2 or 2) dehydrated in graded ethanols followed by xylene and finally embedded in paraffin. Cryostat sections (10 µm) were incubated overnight at 4°C with primary antibodies, and labeling was visualized with horseradish peroxidase-conjugated goat anti-rabbit immunoglobin (P448, 1:100, DAKO). The paraffin-embedded tissue were cut at 2 µm on a rotary microtome (Leica), and sections were dewaxed and rehydrated. For immunoperoxidase labeling, endogenous peroxidase were blocked by 0.5% H2O2 in absolute methanol for 10 min at room temperature. To reveal antigens, sections were treated with 1 mmol/l Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and heated for 10 min using a microwave oven. Nonspecific binding of immunoglobulin was prevented by incubating the sections in 50 mM NH4Cl for 30 min followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with primary antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100 after being rinsed with PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin for 3 × 10 min. For immunoperoxidase labeling, the sections were washed and followed by incubation in horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (P448, diluted 1:100. DAKO) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. The microscopic examination was carried out using a Leica DMRE light microscope.pH Measurements in Medullary TAL
For the measurement of the sodium-dependent HCOStatistical Analyses
Values are presented as means ± SE. Comparisons between groups were made by unpaired t-test. P values < 0.05 were considered significant. ![]() |
RESULTS |
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Rats Treated With NH4Cl Were Associated With Chronic Metabolic Acidosis
Rats with ad libitum access to 0.28M NH4Cl in their drinking water for 2 wk (protocol 1) had a significantly reduced plasma HCO
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Rats treated with NH4Cl in their drinking water ad libitum
for 2 wk (protocol 1) had significantly increased water
intake (138 ± 6 vs. 118 ± 3 µl · min1 · kg
1 in
control rats, P < 0.05). In parallel, the increase in
water intake was accompanied by a significantly increase in urine
output (85 ± 6 vs. 48 ± 4 µl · min
1 · kg
1 in
control rats, P < 0.05). However, the rats treated
with NH4Cl had similar urine osmolality compared with
control rats [1,697 ± 98 vs. 1,480 ± 56 mosmol/kgH2O in control rats, not significant (NS), Table
1], despite the high urine output and water intake due to osmotic diuresis.
To avoid the potential effects of high urine output and water intake
(protocol 1) on the expression of NBCs, we used a separate rat model with chronic metabolic acidosis (protocol 2), in
which rats received fixed amounts of food and water using a model
previously described (19, 20, 31). Urine pH levels were
significantly lower in the acidotic rats compared with control rats
(5.9 ± 0.1 vs. 8.1 ± 0.3, P < 0.05, Table
1). Rats treated with NH4Cl and control rats had similar
urine output due to the fixed amount of water intake (66 ± 3 vs.
62 ± 3 µl · min1 · kg
1 in
controls, NS).
Rats Treated With NH4Cl Had Significant Upregulation of Whole Kidney NBCn1 Levels
Semiquantitative immunoblotting demonstrated that rats with chronic metabolic acidosis (protocol 1) had a dramatically increased abundance of whole kidney NBCn1: 995 ± 87 vs. 100 ± 27% in control rats (P < 0.05, Fig. 1, A and C). Consistent with this, immunoperoxidase microscopy revealed that NBCn1 labeling in the medullary TAL (mTAL) cells was significantly increased in rats with chronic metabolic acidosis compared with control rats (arrowheads in Fig. 2, A and B). Rats treated with NH4Cl in their food (protocol 2) also had a significantly increased abundance of whole kidney NBCn1: 262 ± 42 vs. 100 ± 14% in control rats (P < 0.05, Fig. 1, B and D). This was also confirmed by immunohistochemistry showing marked increase in the NBCn1 labeling in mTAL from acidotic rats (arrowheads in Fig. 2, C and D).
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Rats Treated With NH4Cl Had Enhanced DIDS-Sensitive,
Sodium-Dependent HCO
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Rats Treated With NH4Cl Had Significant Upregulation of Whole Kidney NBC3 Levels
Electroneutral NBC3, cloned from human skeletal muscle and functionally characterized (32, 33, 47), is present in the intercalated cells in connecting tubules and cortical, outer medullary, and inner medullary collecting ducts of rat kidney (23). Rats with chronic metabolic acidosis (protocol 1) had a significantly increased abundance of whole kidney NBC3: 226 ± 35 vs. 100 ± 16% in control rats (P < 0.05, Fig. 5, A and C). Consistent with this, immunohistochemistry revealed that NBC3 labeling in collecting duct intercalated cells was significantly enhanced in metabolic acidosis (arrows in Fig. 6, A and B). Rats treated with NH4Cl in their food (protocol 2) also had a significantly increased abundance of whole kidney NBC3: 160 ± 31 vs. 100 ± 31% in control rats (P < 0.05, Fig. 5, B and D). This was confirmed by immunohistochemistry showing a marked increase in the NBC3 labeling in the intercalated cells from acidotic rats (Fig. 6, C and D). In both acidotic and control rats, NBC3 labeling3 was seen in both type-A and type-B intercalated cells in the cortical collecting duct, as described previously. Immunoperoxidase microscopy revealed that NBC3 labeling in the apical domains of type-A intercalated cells in the cortical collecting ducts was increased (arrows in Fig. 6, A and C) compared with control rats (arrows in Fig. 6, B and D), whereas cytoplasmic and basal labeling in type-B intercalated cells was maintained (not shown). NBC3 labeling in the outer medullary and inner medullary intercalated cells was consistently increased in rats with chronic metabolic acidosis (not shown).
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Rats Treated With NH4Cl Did Not Experience Changes in the Abundance of Whole Kidney rkNBC1
As shown in Fig. 7, A and C, semiquantitative immunoblotting revealed that whole kidney rkNBC1 abundance was not significantly altered in rats treated with NH4Cl in their drinking water (protocol 1) compared with control rats (88 ± 14 vs. 100 ± 19% in control rats, NS). Consistent with this, rats treated with NH4Cl in their food (protocol 2) also had a similar abundance of whole kidney rkNBC1 compared with control rats: 84 ± 17 vs. 100 ± 15% (Fig. 7, B and D; NS). This is consistent with a previous observation demonstrating unchanged renal cortical rkNBC1 mRNA expression in rats with NH4Cl-induced metabolic acidosis (10). Immunoperoxidase microscopy confirmed that rkNBC1 labeling in the proximal tubular basolateral plasma membrane was not altered in rats with chronic metabolic acidosis compared with controls in both protocol 1 (Fig. 8) and protocol 2 (not shown). Importantly, rkNBC1 labeling remained restricted to S1 and S2 proximal tubular segments with no appearance of rkNBC1 labeling in S3 proximal tubule (not shown).
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DISCUSSION |
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We demonstrated that chronic metabolic acidosis (chronic
NH4Cl loading) in rats was associated with 1) a
significantly increased abundance of the NBCn1 as well as enhanced
HCO
Chronic Metabolic Acidosis is Associated With a Dramatic Increase
in NBCn1 Abundance in mTALs and Increased TAL HCO
In the present study, semiquantitative immunoblotting and
immunocytochemistry revealed that the protein abundance of NBCn1 is
dramatically enhanced in response to chronic metabolic acidosis induced
by oral NH4Cl loading. This supports our previous study demonstrating a DIDS-sensitive and sodium- and bicarbonate-dependent recovery from acidosis in kidney slices from the inner stripe of the
outer medulla (42). Nevertheless, the electroneutral Na-HCO
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Conversely, it may be possible to speculate that increased basolateral
NBCn1 activity in response to chronic metabolic acidosis may contribute
to the increase in intracellular Na+ that, in turn,
decreases the inward gradient for the apical Na-K-2Cl cotransporter;
hence it may reduce NH
In the present study, the inhibition with DIDS (~70%) was much greater than in the cloning studies (~25% in Ref. 11). However, it should be pointed out that in smooth muscle cells (1) and cardiac myocytes (13), where electroneutral NBC was first demonstrated, the inhibition induced by DIDS was at least 70%. The smaller effect of DIDS on the NBCn1-B expressed in oocyotes may have several explanations. It cannot be excluded that another DIDS-sensitive electroneutral NBC isoform, distinct from NBCn1, may also be expressed and may play a major role in the basolateral plasma membrane of TAL cells. Further studies are therefore warranted to define this.
Chronic Metabolic Acidosis Is Associated With a Dramatic Increase in NBC3 Expression in Intercalated Cells
It was recently demonstrated that, in rat kidney, NBC3 is specifically localized in the connecting tubule and in cortical, outer medullary, and initial inner medullary collecting duct (23). High-resolution immunocytochemistry and immunoelectron microscopy demonstrated that both type-A and type-B intercalated cells in the connecting tubule and cortical collecting duct exhibit strong NBC3 immunolabeling, whereas principal cells are unlabeled. In type-A intercalated cells, NBC3 immunolabeling is confined to the apical plasma membrane and subapical intracellular vesicles and tubulocisternal profiles. In contrast, in type-B intercalated cells, NBC3 is abundantly expressed in the basolateral plasma membrane and is absent in the apical plasma membrane domains. Moreover, in rabbit kidney, we also have demonstrated colocalization of the apical membrane of type-A intercalated cells with NBC3 and E11, the 31-kDa subunit of the vacuolar H+-ATPase (33). Until now, a sodium-coupled HCOOur data revealed that whole kidney NBC3 abundance is significantly
increased in response to chronic metabolic acidosis. In particular,
NBC3 abundance was also markedly increased in rats with chronic
metabolic acidosis that received fixed amounts of food and water intake
(protocol 2). This suggests that NBC3 expression is enhanced
in response to chronic metabolic acidosis per se. This is consistent
with a recent study demonstrating that the intracellular pH recovery
rate due to apical NBC3 in the intercalated cells of the outer medulla
is approximately four times higher than apical H+-ATPase-
and H+-K+-ATPase-dependent recovery rates
(47). Thus the increased expression of NBC3 selectively in
type-A cells in response to chronic metabolic acidosis is likely to
play an important role in maintaining intracellular pH levels in these
cells and aid in HCO
Chronic Metabolic Acidosis Is Not Associated With Altered Abundance of Whole Kidney rkNBC1
Na-HCOSeveral previous studies demonstrated that systemic pH levels may play
a significant role in the regulation of HCO
Summary
Chronic metabolic acidosis is associated with a marked increase in the abundance of NBCn1 in the mTAL and NBC3 in the intercalated cells, whereas the abundance of NBC1 in the proximal tubule was not altered. The role of sodium-dependent HCO ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Helle Høyer, Merete Pedersen, Inger Merete Paulsen, Zhila Nikrozi, Mette Vistisen, and Gitte Christensen for expert technical assistance.
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FOOTNOTES |
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The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). Support for this study was provided by the Karen Elise Jensen Foundation, the Human Frontier Science Program, the European Commission (European Union-Biotech and KA 3.1.2 programs), the Novo Nordic Foundation, the Danish Medical Research Council, the University of Aarhus Research Foundation, the University of Aarhus, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58563 (I. Kurtz), and Dongguk University.
2 As discussed at the beginning of this study, NBCn1 immunolabeling in normal rat kidney is present in the basolateral domains of TAL cells in the outer medulla as well as of intercalated cells in the inner medulla. NBC3, 89-92% identical to NBCn1, is exclusively associated with intercalated cells in connecting tubules and in cortical, outer medullary, and initial inner medullary collecting ducts of normal rat kidney.
3 In response to acidosis, there is a marginal appearance of NBC3 labeling of TAL cells (basolateral), corresponding to limited cross-reactivity, likely with NBCn1. This is predicted due to a very high degree of homology between the two transporters with respect to the amino acid sequence used to produce the antigenic peptide. In contrast, NBCn1 labeling exclusively occurred in mTAL cells and in intercalated cells of the inner medullary collecting duct but never in the apical domain of type-A intercalated cells or in the intercalated cells in the inner medullary collecting duct. Thus this demonstrates the absence of cross-reactivity in NBCn1 labeling in response to acidosis.
Address for reprint requests and other correspondence: S. Nielsen, The Water and Salt Research Center, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: sn{at}ana.au.dk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Recently, Amlal et al. (6) reported an NBC-like partial clone, which was also called NBC3. An ~4.4-kb transcript was highly expressed in the brain and spinal cord.
First published September 21, 2001; 10.1152/ajprenal.00104.2001
Received 26 March 2001; accepted in final form 10 October 2001.
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