Chronic metabolic acidosis upregulates rat kidney Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters NBCn1 and NBC3 but not NBC1

Tae-Hwan Kwon1,2, Christiaan Fulton1, Weidong Wang1, Ira Kurtz3, Jørgen Frøkiær1, Christian Aalkjær1, and Søren Nielsen1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Several members of the Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBC) family have recently been identified functionally and partly characterized, including rkNBC1, NBCn1, and NBC3. Regulation of these NBCs may play a role in the maintenance of intracellular pH and in the regulation of renal acid-base balance. However, it is unknown whether the expressions of these NBCs are regulated in response to changes in acid-base status. We therefore tested whether chronic metabolic acidosis (CMA) affects the abundance of these NBCs in kidneys using two conventional protocols. In protocol 1, rats were treated with NH4Cl in their drinking water (12 ± 1 mmol · rat-1 · 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<UP><SUB>3</SUB><SUP>−</SUP></UP> uptake in rats with CMA. In conclusion, CMA is associated with a marked increase in the abundance of NBCn1 in the mTAL and NBC3 in intercalated cells, whereas the abundance of NBC1 in the proximal tubule was not altered. The increased abundance of NBCn1 may play a role in the reabsorption of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> in the mTAL and increased NBC3 in reabsorbing HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

acid-base balance; bicarbonate transport; intracellular pH; intercalated cell; thick ascending limb


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE KIDNEY MAINTAINS ACID-BASE balance by H+ secretion and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption. The proximal tubule cells reabsorb HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by the apically expressed Na+/H+ exchanger (NHE3) in conjunction with the basolaterally expressed Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (kNBC1) that plays an important role in mediating electrogenic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux (10, 18, 34, 37). Consistent with this, recent immunocytochemical analyses demonstrated that the electrogenic Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (kNBC1) (35) is present at the basolateral plasma membrane domains of predominantly S1 and S2 segments in proximal tubules of rat kidney (27, 36). Moreover, in rabbit kidney, kNBC1 mRNA expression was demonstrated in the proximal tubule (2).

In addition to kNBC1, several other members of the Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBC) family have recently been identified and functionally characterized in vitro and could potentially play a role in mediating acid-base transport in kidneys or in maintaining intracellular pH levels (2, 6, 11, 32, 47). The electroneutral NBCn1 (GenBank accession no. AF070475) was cloned from rat smooth muscle cells (11), and it has been demonstrated that NBCn1 immunolabeling in normal rat kidney is present in the basolateral domains of thick ascending limb (TAL) cells in the outer medulla (42). Consistent with this, studies of intracellular pH measurement confirmed that the sodium-dependent recovery from acidosis in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and amiloride is present in the TAL (42). In addition, collecting duct intercalated cells in the inner stripe of the outer medulla and in the inner medulla also exhibit NBCn1 immunolabeling (42).

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<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in TALs in slices from rats with chronic metabolic acidosis and those from controls.


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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 wt-1 · 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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration and total CO2 levels (protocol 1). Plasma was also collected for the measurement of sodium, potassium, creatinine, and osmolality.

Membrane 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<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBC1), a synthetic peptide corresponding to COOH-terminal (amino acids 1021-1035, GenBank accession no. AF004017) of rat kidney electrogenic NBC (rkNBC1) (35), was used to generate polyclonal antibodies (27).

Electroneutral Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBCn1), a synthetic peptide corresponding to COOH-terminal (amino acids 1204-1218: NH2-CEDEPSKKYMDAETSL-COOH, GenBank accession no. AF070475) of NBCn1 (11), was used to generate polyclonal antibodies (42).

Electroneutral Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter 3 (NBC3),2 a synthetic peptide corresponding to COOH-terminal (amino acids 1197-1214: NH2-CISFEDEPRKKYVDAETSL-COOH, GenBank accession no. AF047033) of NBC3 (32), was used to generate polyclonal antibodies (23, 33).

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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport rate, we made a separate set of rats with chronic metabolic acidosis (n = 10) and control rats (n = 10). Chronic metabolic acidosis was established by using protocol 1. Kidneys from rats with CMA and controls were quickly removed after the rats were killed with CO2. An approximately 1-mm slice of the kidney (sliced perpendicular to the polar axis) was fixed above a coverslip, which formed the floor in a 10-ml organ bath (42). The bathing solution was physiological salt solution (PSS; for composition, see below). The slice was incubated for ~40 min with 2 µM of the acetoxymethyl ester (AM) form of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) at room temperature. The organ bath was placed on the stage of an inverted microscope for time-resolved fluorescence measurements. For time-resolved measurements, the preparation was excited alternately via a monochromator with 435- and 488-nm light. The emission from the preparation was collected through a band-pass filter (520-560 nm), and the ratio of the emissions at the two excitation wavelengths was determined after subtraction of the background fluorescence, which was always <10% of the signal. The equipment used was a PTI Deltascan fitted to a Leica DM IRB microscope with a Leica ×40/0.55 objective. The PSS contained (in mM) 119 NaCl, 4.7 KCl, 1.8 KH2PO4, 1.17 MgSO4, 25 NaHCO3, 1.6 CaCl2, 0.026 EDTA, 10 HEPES, and 5.5 glucose. The solution was gassed with 5% CO2 in air, and pH was 7.45-7.5. In sodium-free PSS, NaCl was substituted with N-methyl-D-glucamine and NaHCO3 with cholinebicarbonate on an equimolar basis, and the pH was titrated to 7.45 with HCl. In bicarbonate-free solutions, NaHCO3 was substituted with NaCl. The bicarbonate-free solutions were gassed with air.

Statistical 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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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration (19 ± 1 vs. 31 ± 0.4 mmol/l in controls, P < 0.05) and total CO2 levels (21 ± 1 vs. 33 ± 0.5 mmol/l in controls, P < 0.05), consistent with significant chronic metabolic acidosis (Table 1). Urine pH levels also decreased markedly: 5.2 ± 0.04 vs. 6.9 ± 0.1 in control rats (P < 0.05, Table 1).

                              
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Table 1.   Functional data of renal function

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 · min-1 · 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 · min-1 · 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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Fig. 1.   Semiquantitative immunoblotting of membrane fractions of whole kidneys. A and B: immunoblots were reacted with anti-Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBCn1 and revealed a broad band centered at ~180 kDa. CON, control. C and D: densitometric analyses revealed that the abundance of whole kidney NBCn1 in rats treated with NH4Cl in their drinking water [chronic metabolic acidosis (CMA), protocol 1] was dramatically increased to 995 ± 87% of control levels (100 ± 27%, *P < 0.05). Rats treated with NH4Cl in their food (CMA, protocol 2) also had a significantly increased abundance of whole kidney NBCn1: 262 ± 42% of control levels (100 ± 14%, *P < 0.05).



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Fig. 2.   Immunohistochemistry from rat with CMA (A and C) and control rats (B and D) treated according to protocol 1 (A and B) or protocol 2 (C and D). NBCn1 labeling is associated with basolateral plasma membrane domains of the thick ascending limbs (TALs) in the inner stripe of the outer medulla (ISOM; arrowheads in A-D). A and B: in rats with CMA induced by 0.28 M NH4Cl in their drinking water (protocol 1), NBCn1 labeling in the TALs of the ISOM (arrowheads in A) was significantly increased compared with control rats (arrowheads in B). C and D: in rats with CMA induced by protocol 2, NBCn1 labeling in the TALs of the ISOM (arrowheads in C) was also increased compared with control rats (arrowheads in D). Magnification: ×630 (A-D).

Rats Treated With NH4Cl Had Enhanced DIDS-Sensitive, Sodium-Dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Uptake in TAL Cells

The rate of pH recovery was assessed in the mTAL with the use of the NH<UP><SUB>4</SUB><SUP>+</SUP></UP> prepulse technique in the presence of 1 mM amiloride to block Na/H exchange (Fig. 3). We have previously shown, using confocal imaging, that the recovery of the fluorescence ratio after NH4Cl washout in the presence of amiloride reflects HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in the mTAL (42). In rats having NH4Cl in their drinking water, the rate of recovery in the presence of sodium and bicarbonate was >100% faster than in control rats (Fig. 4). In the absence of sodium or bicarbonate, there was no recovery and the recovery was partly inhibited by 400 µM DIDS. One possible reason for the different rates of recovery is a difference in buffering capacity between the two groups of rats. We addressed this possibility by checking the fluorescence ratios immediately before and after washout of NH4Cl into sodium-free PSS. These values [(ratio units)/s] × 103 were 1.46 ± 0.05 and 1.42 ± 0.05 before washout in control and CMA, respectively, and 0.35 ± 0.03 and 0.34 ± 0.03 after washout in control and CMA, respectively, and not significantly different between the two groups of rats. In preliminary experiments, we used 5 µM nigericin to calibrate the fluorescence ratio and found no difference in the calibration between the two groups of rats. These findings indicate that there is no difference in buffering capacity and that the difference in the rate of fluorescence recovery reflects a difference in acid equivalents transported across the membrane.


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Fig. 3.   Bicarbonate transport in mTAL. A: trace from an experiment illustrating the general protocol used. For simplicity, the entire trace is only shown for the washout to Na+-free solution (-Na+); the trace marked +Na+ (top trace segment) shows recovery in the presence of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.



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Fig. 4.   HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in medullary TAL (mTAL). This shows the geometric means of the recovery rates in the solutions indicated. Open bars, rats with CMA; solid bars, control rats.

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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Fig. 5.   Semiquantitative immunoblotting of membrane fractions of whole kidneys. A and B: immunoblots were reacted with anti-NBC3 and revealed an ~200-kDa band. C and D: densitometric analyses revealed that whole kidney NBC3 abundance was significantly increased in rats treated with NH4Cl in their drinking water (CMA, protocol 1) to 226 ± 35% of control levels (100 ± 16%, *P < 0.05). Rats treated with NH4Cl in their food (CMA, protocol 2) also had a significantly increased abundance of whole kidney NBC3: 160 ± 31% of control levels (100 ± 31%, *P < 0.05).



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Fig. 6.   Immunohistochemistry from rats with CMA (A and C) and control rats (B and D) treated according to protocol 1 (A and B) or protocol 2 (C and D). NBC3 labeling is associated with intercalated cells in the cortical collecting duct (arrows in A-D). A and B: in rats with CMA, NBC3 labeling in intercalated cells of the cortical collecting duct (arrows in A) was significantly increased compared with control rats (arrows in B). C and D: in rats with CMA, NBC3 labeling in intercalated cells of the cortical collecting duct (arrows in C) was also increased compared with control rats (arrows in D). Magnification: ×1,000 (A-D).

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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Fig. 7.   Semiquantitative immunoblotting of membrane fractions of whole kidneys. A and B: immunoblots were reacted with anti-rkNBC1 and revealed an ~140-kDa band. C and D: densitometric analyses 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% of control levels [100 ± 19%, not significant (NS)]. In protocol 2, rats treated with NH4Cl in their food also had a similar abundance of whole kidney rkNBC1 compared with control rats: 84 ± 17% of control levels (100 ± 15%, NS).



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Fig. 8.   Immunohistochemistry from rats with chronic metabolic acidosis (A) and control rats (B) treated according to protocol 1. rkNBC1 labeling is associated with basolateral plasma membrane of the proximal tubules (arrowheads). In A and B, rkNBC1 labeling in proximal tubule cells in rats with chronic metabolic acidosis (arrowheads in A) appeared not to be changed compared with control rats (arrowheads in B). Magnification: ×1,000 (A and B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in mTAL and 2) a significantly increased abundance of the NBC3 in the intercalated cells of the collecting ducts. In contrast, the abundance of rkNBC1 in proximal tubule cells was not altered. The role of sodium-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport outside the proximal tubules is still uncertain; however, the marked increase in the abundance of the NBCn1 and NBC3 in response to chronic metabolic acidosis suggests that electroneutral sodium-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport through these NBCs is significantly enhanced in chronic metabolic acidosis.

Chronic Metabolic Acidosis is Associated With a Dramatic Increase in NBCn1 Abundance in mTALs and Increased TAL HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Influx

It is well known that chronic metabolic acidosis significantly increases urinary net acid excretion, associated with a number of adaptive changes in renal tubules that contribute to increased urinary acidification. These adaptations include an increase in the activity of Na+/H+ exchange and bicarbonate reabsorptive capacity in the proximal tubules (4, 22, 30), an increased capacity for net bicarbonate reabsorption in the cortical and inner medullary collecting ducts (28, 44), and enhanced NH<UP><SUB>4</SUB><SUP>+</SUP></UP> production and secretion in the proximal tubule (17). Moreover, in the mTALs of the rat kidney, chronic metabolic acidosis induced by oral NH4Cl loading in the drinking water significantly increased bicarbonate reabsorption as well as net NH<UP><SUB>4</SUB><SUP>+</SUP></UP> absorption (16).

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<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity in rat mTAL is not well understood. Moreover, the dramatic increase in the expression of basolateral NBCn1 and uptake of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in chronic metabolic acidosis seems unexpected. One possible explanation may be that NBCn1 plays a role in supporting NH<UP><SUB>4</SUB><SUP>+</SUP></UP> reabsorption in the mTAL (Fig. 9), which is significantly enhanced in response to chronic metabolic acidosis as an adaptive change (16). A major fraction of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is reabsorbed at the TALs mainly through the Na-K-2Cl cotransporter, presumably by the substitution for K+ (15, 21). Then, in TAL cells, lipid soluble ammonia (NH3) from NH<UP><SUB>4</SUB><SUP>+</SUP></UP> can passively diffuse into the interstitium through the basolateral plasma membrane, whereas H+ from NH<UP><SUB>4</SUB><SUP>+</SUP></UP> combines intracellulary with bicarbonate to form H2CO3 (carbonic acid). This is converted to CO2 and H2O, and CO2 diffuses into the interstitium where it combines with H2O to yield H+ and bicarbonate. In the medullary interstitium, H+ combines with NH3 to form NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, whereas bicarbonate is transported into the cells though basolaterally expressed NBCn1. Consistent with both the increase in the abundance and functional activity of NBCn1, chronic metabolic acidosis has been known to be associated with increased urinary excretion of total ammonia (NH<UP><SUB>4</SUB><SUP>+</SUP></UP>/NH3). Upregulation of NH4Cl reabsorption in chronic metabolic acidosis (16) would therefore be expected, in this case, to be associated with an increase in NBCn1 activity in the basolateral membrane. It can also be noted that in this model the basolateral influx of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> does not lead to a net transport of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> toward the lumen. Thus NBCn1 possibly plays an important role in 1) NH<UP><SUB>4</SUB><SUP>+</SUP></UP> reabsorption, medullary accumulation, and urinary excretion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and 2) basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport into TAL cells.


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Fig. 9.   The possible role of NBCn1 in NH<UP><SUB>4</SUB><SUP>+</SUP></UP> reabsorption in the mTAL. NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is mainly reabsorbed at mTAL (MTAL in the figure) through the Na-K-2Cl cotransporter (open circle ) presumably by substituting for K+. Then, in mTAL cells, lipid soluble NH3 from NH<UP><SUB>4</SUB><SUP>+</SUP></UP> can passively diffuse into the interstitium through the basolateral plasma membrane. H+ from NH<UP><SUB>4</SUB><SUP>+</SUP></UP> combines intracellulary with HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to form H2CO3 (carbonic acid), which is converted to CO2 and H2O. CO2 diffuses into the interstitium, where it combines with H2O to yield H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. In the medullary interstitium, H+ combines with NH3 to form NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, whereas HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is transported into the cells though NBCn1 (), which is located in the basolateral plasma membrane with Na+. Thus NBCn1 may play an important role in 1) NH<UP><SUB>4</SUB><SUP>+</SUP></UP> reabsorption, medullary accumulation, and urinary excretion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>; and 2) the basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport into the TAL cells. mTAL cells also express apical and basolateral Na/H exchangers as well as basolateral Na-K-ATPase.

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<UP><SUB>4</SUB><SUP>+</SUP></UP> reabsorption in the TAL. However, chronic metabolic acidosis is known to inhibit NaCl and fluid reabsorption in the proximal tubule and increase NaCl delivery to the loop of Henle (12). Moreover, a chronic increase in sodium delivery mediates an adaptive increase in bicarbonate and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> reabsorption in the mTAL independently of changes in acid-base balance, presumably by increasing both apical Na-K-2Cl cotransporter (14) as well as basolateral membrane Na-K-ATPase activity (16, 24, 43). Therefore, the increased ability of the mTAL to absorb NH<UP><SUB>4</SUB><SUP>+</SUP></UP> during chronic metabolic acidosis is associated with fine regulation of mTAL NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport via coordinated effects on various apical and basolateral transporters (Fig. 9). It should be emphasized that the model presented in Fig. 9 remains hypothetical, and additional studies are warranted to define the role of each transporter in NH<UP><SUB>4</SUB><SUP>+</SUP></UP> metabolism in response to acidosis.

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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporter in collecting duct intercalated cells has not been well understood. Type-A intercalated cells are thought to secrete protons and absorb HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> via an apical vacuolar H+-ATPase and H+-K+-ATPase (29, 41). HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is then transported across the basolateral membrane via the basolateral AE1 anion exchanger. Type-A intercalated cells are not, however, believed to mediate transepithelial Na+ transport. It is of interest that previous studies have demonstrated the presence of a basolateral Na+/H+ exchanger in this cell (45). The finding that net transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport is Na+ independent in the outer medullary collecting duct (inner stripe) (39) suggests rather that apical NBC3 and basolateral Na+/H+ exchange in type-A intercalated cells may play an important role in mediating H+/base transport across their respective membranes (intracellular pH regulation). Whether luminal NBC3 contributes to passive transepithelial Na+ transport in this segment is unknown. The potential contribution of apical NBC3 and basolateral Na+/H+ exchange to passive transepithelial Na+ transport in the outer medullary collecting duct will require further study.

Our 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<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption.

Chronic Metabolic Acidosis Is Not Associated With Altered Abundance of Whole Kidney rkNBC1

Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport was initially localized by functional studies to the basolateral membrane of the proximal tubule, where it plays a role in mediating electrogenic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux (8). Recently, kNBC1 has been localized by in situ hybridization to the proximal tubule in rabbit kidneys (2), and immunohistochemical analyses have revealed its presence in the basolateral plasma membrane of rat proximal tubule, predominantly in S1 and S2 segments (27, 36). This strongly indicates that kNBC1 mediates basolateral proximal tubule HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux.

Several previous studies demonstrated that systemic pH levels may play a significant role in the regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the proximal tubule (4, 38). In vitro microperfusion studies in the proximal tubule of rat kidney revealed that activities of the apically expressed sodium-hydrogen exchanger (NHE) and basolaterally expressed NBC are upregulated in response to metabolic acidosis (30). Akiba et al. (3) demonstrated that the stimulatory effect of metabolic acidosis was due to an increase in the maximal transport rate of the transporters and that the maximal transport rate of the Na+/H+ antiporter and Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter were inversely related to plasma HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration. A previous study demonstrated that renal cortical rkNBC1 mRNA expression in rats was not altered in response to NH4Cl-induced metabolic acidosis (10). Moreover, our data demonstrated that protein abundance of rkNBC1 remained unchanged in response to chronic metabolic acidosis, suggesting that its functional upregulation in response to metabolic acidosis may be a posttranslational upregulation.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> transport outside the proximal tubules is still uncertain. However, the significant increase in protein abundance of NBCn1 and NBC3 suggests that electroneutral sodium-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport through these NBCs is significantly enhanced in response to chronic metabolic acidosis. Hence this may play an important role in maintaining intracellular pH levels and renal regulation of the acid-base balance in the kidney.


    ACKNOWLEDGEMENTS

The authors thank Helle Høyer, Merete Pedersen, Inger Merete Paulsen, Zhila Nikrozi, Mette Vistisen, and Gitte Christensen for expert technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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