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,
*
Department of Cell Biology, University of Aarhus, Aarhus,
Denmark
Department of Medical Biochemistry, University of Aarhus, Aarhus,
Denmark
Department of Physiology, University of Aarhus, Aarhus, Denmark
§
Department of Cellular and Molecular Physiology, Yale University School of
Medicine, New Haven, Connecticut.
Correspondence to Dr. Arvid B. Maunsbach, Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark. Phone: +45 8942 3065; Fax: +45 8612 8808; E-mail: maunsbach{at}ana.au.dk 1046-6673/1112-2179
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With the cloning of the electrogenic Na+/HCO3- cotransporter (NBC1) from the kidney (5,6,7,8), it became possible to immunolocalize the NBC1 in the kidney (9). It was shown at the light microscopic level that the NBC1 localizes to the basolateral part of the proximal tubule of rat and rabbit, consistent with the physiologic evidence. Somewhat surprising is that there was only a weak staining of the proximal tubules of the salamander kidney, whereas a strong labeling of the basolateral part of the late distal tubule was found. It was not possible from the immunofluorescence experiments, however, to determine the detailed subcellular localization of NBC1 because no immunoelectron microscopy was made.
In this study, we therefore determined the subcellular localization of NBC1 in rat and salamander kidney using immunolabeling of ultrathin cryosections or Lowicryl sections. The analysis included segments S1, S2, and S3, as defined by electron microscopy in the rat proximal tubule (10) and proximal and distal tubules in salamander kidney. For this purpose, we made a new antibody based on the sequence of rat kidney NBC1 (rkNBC1) and used this together with the previously described antibody MBP-NBC-5 (9).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-rkNBC1-CT108 (identical to MBP-NBC-5: IIFPVMILALVAVRKGMDYLFSQHDLSFLDDVIPEKDKKKKEDEKKKKKKKGSLDSDNDDSDCPYSEKVPSIKIPMDITEQ QPFLSDNKPLDRERSSTFLERHTSC) was raised in rabbits immunized with a fusion protein that consisted of MBP coupled to amino acids 928 to 1035 of rat kidney NBC1, corresponding to the C-terminus of the protein (9).
Membrane Fractionation for Immunoblotting
The kidneys from normal Munich-Wistar rats were divided into cortex, outer
stripe and inner stripe of the outer medulla, and the inner medulla. These
tissues were homogenized in homogenization buffer (HB; 300 mM sucrose, 25 mM
imidazole, 1 mM ethylenediaminetetraacetate [pH 7.2] containing 8.5 µM
leupeptin, 1 mM phenylmethyl sulfonylfluoride) using an ultra-turrax T8
homogenizer (IKA Labortechnik, Staufen, Germany), at maximum speed for 30 s,
and the homogenate was centrifuged in an Eppendorf centrifuge at 4000 x
g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The
supernatant was then centrifuged at 200,000 x g for 1 h to
produce a pellet containing membrane fractions enriched for both plasma
membranes and intracellular vesicles
(12). Gel samples (Laemmli
sample buffer containing 2% sodium dodecyl sulfate) were made of this
pellet.
Kidneys from Ambystoma tigrinum (obtained from Charles Sullivan, Nashville, TN) were divided into a medial portion containing glomeruli and the initial part of the distal tubuli and a lateral portion containing predominantly proximal tubules and the late part of the distal tubuli. The tissue was homogenized in HB containing 10 µM leupeptin and 0.4 mM Pefabloc for 60 s using a Potter-Elvehjelm homogenizer (Bie & Berntsen, Copenhagen, Denmark). The homogenate was centrifuged as above to produce a pellet that contained plasma membranes and intracellular membranes and that was dissolved in gel sample buffer.
Electrophoresis and Immunoblotting
The total protein content of samples of membranes from rat kidney cortex,
outer stripe and inner stripe of the outer medulla, and the inner medulla as
well as whole kidney from Ambystoma was estimated by the Bio-Rad
Protein Assay (Hercules, CA) based on the method of Bradford
(13). Twenty-five µg of
prepared sample was loaded in each well and run on 10 to 20% gradient
polyacrylamide minigels (NOVEX, San Diego, CA) under reducing and nonreducing
conditions. After transfer by electroelution to nitrocellulose membranes,
blots were blocked with 5% milk in phosphate-buffered saline (PBS)-Tris (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
anti-rkNBC1-CT15 and anti-rkNBC1-CT108 both diluted 1:300. To control for
nonspecific reactions, blocking experiments were performed by adding 10 µg
of synthetic peptide to the anti-rkNBC1-CT15 and 10 µg of fusion protein
used for the production of the antibody to anti-rkNBC1-CT108 (approximately
50-fold molar excess) and incubated overnight before immunoblotting. The
labeling was visualized with horseradish peroxidase-conjugated secondary
antibodies (P217; DAKO, Glostrup, Denmark; diluted 1:5,000) using enhanced
chemiluminescence system (Amersham International, Little Chalfont, UK).
Deglycosylation
For N-glycosidase F (PNGase F) digestion, 100 µl of membrane fractions
from rat cortex was incubated under native conditions at room temperature for
6 h in the presence of 5 units of PNGase F obtained from Boehringer Mannheim
(Mannheim, Germany). Boiling the suspensions in Laemmli sample buffer stopped
the enzymatic reactions, and samples were analyzed by immunoblotting.
Immunohistochemistry
Kidneys from normal Munich-Wistar rats were fixed by retrograde perfusion
via the aorta with periodate-lysine-paraformaldehyde (10 mM NaIO4,
75 mM lysine, 2% paraformaldehyde, in 37.5 mM Na2HPO4
buffer [pH 6.2]). For preparation of cryostat sections, tissue was
cryoprotected in 25% sucrose. Cryostat sections (10 µm) were incubated
overnight at 4°C with anti-rkNBC1-CT15 (diluted 1:50), and labeling was
visualized with HRP-conjugated secondary antibody (P448, 1:100, DAKO)
(14).
Immunoelectron Microscopy
Kidneys from rats were perfusion-fixed retrograde via the aorta with 4%
paraformaldehyde in 100 mM sodium cacodylate buffer (pH 7.2). Tissue blocks
were trimmed from the cortex as well as from the outer stripe of the outer
medulla. The tissue was postfixed in the same fixative for 2 h, cryoprotected
with 2.3 M sucrose containing 2% paraformaldehyde, mounted on holders, and
frozen in liquid nitrogen. Kidneys from Ambystoma tigrinum were fixed
by perfusion through the renal portal vein
(15), with
periodate-lysine-paraformaldehyde adjusted for Ambystoma (8 mM
NaIO4, 60 mM lysine, 4% formaldehyde, in 30 mM
Na2HPO4 buffer [pH 6.2]). Small tissue blocks from the
lateral portion of the kidneys containing proximal and late distal tubules
were dissected out, postfixed for 4 h, cryoprotected in 2.3 M sucrose
containing 2% paraformaldehyde, and frozen in liquid nitrogen as the rat renal
tissue.
Immunoelectron microscopy was performed on either thin (80 nm) cryosections prepared from the frozen tissue on a Reichert Ultracut S (Leica, Vienna, Austria) or on tissue that was cryosubstituted in a Reichert AFS freeze-substitution unit (Leica) and embedded in Lowicryl HM20 (16) as described previously (17). Briefly, the samples were sequentially equilibrated over 3 d in methanol containing 0.5% uranyl acetate at temperatures gradually increasing from -90°C to -70°C, and then rinsed in pure methanol for 24 h while increasing the temperature from -70°C to -45°C. At -45°C, the samples were infiltrated with Lowicryl HM20 and methanol 1:1, 2:1, and, finally, pure Lowicryl HM20 before UV-polymerization for 2 d at -45°C and 2 d at 0°C. Ultrathin (50 nm) Lowicryl sections were prepared in a Reichert Ultracut S at room temperature. For immunoelectron microscopy, the ultrathin cryosections or Lowicryl sections were first preincubated in PBS (10 mM sodium phosphate buffer containing 150 mM sodium chloride [pH 7.4]) containing 0.1% skimmed milk powder and 50 mM glycine. The sections were then incubated for 1 h at room temperature with rabbit anti-rkNBC1-CT15 or anti-rkNBCT-CT108 antibody diluted 1:100 in PBS containing 0.1% skimmed milk powder. The primary antibody was visualized using goat anti-rabbit IgG conjugated to 10 nm colloidal gold particles (GAR.EM1O, Bio-Cell Research Laboratories, Cardiff, UK) diluted 1:50 in PBS with 0.1% skimmed milk powder and polyethyleneglycol (5 mg/ml). The Lowicryl sections were stained with uranyl acetate, and the ultrathin cryosections were stained with 0.3% uranyl acetate in 1.8% methyl-cellulose for 10 min before examination in a Philips 208 electron microscope (Eindhoven, The Netherlands).
The following immunolabeling controls were used at the electron microscopic level: (1) The primary antibody was substituted with nonimmune rabbit IgG, (2) absorption controls were made by incubation with purified rkNBC1 peptide (approximately 50-fold molar excess), and (3) incubation without the use of primary antibody. All controls showed absence of labeling.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the protein was glycosylated, we determined its sensitivity to PNGase F treatment, which cleaves N-linked saccharides. As demonstrated in Figure 2, treatment of membranes from rat kidney cortex with PNGase F caused a significant reduction in molecular weight to approximately 120 kD.
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Immunohistochemical Localization of rkNBC1 in Rat Kidney
To determine the cellular localization of NBC1, we performed
immunohistochemistry using cryostat sections from perfusion-fixed rat kidneys
using the anti-rkNBC1-CT15 (Figure
3). In the cortex, strong labeling was associated with proximal
tubules (Figure 3A) and the
labeling was exclusively present in the basolateral domains (arrows in
Figure 3, B and C). In
contrast, apical domains were unlabeled, which is especially apparent in
supranuclear regions that are completely devoid of NBC1 labeling (arrowheads
in Figure 3, B and C). In the
proximal tubules, basolateral plasma membrane domains of segments S1 and S2
were strongly labeled (Figure 3, A through
C), whereas no labeling was observed in late segments of proximal
tubules (Figure 3E). The
transition between the labeled S2 and the unlabeled S3 segment is abrupt, with
labeled and unlabeled cells sometimes seen side by side in the same tubule
(Figure 3D). No immunolabeling
was associated with glomerulus (Figure 3, A
and B), distal convoluted tubule
(Figure 3C), collecting ducts
(Figure 3E), descending thin
limbs, ascending thick limbs (Figure
3F), or vascular structures. Thus, NBC1 immunolabeling was found
associated only with basolateral domains of segment S1 and segment S2 proximal
tubule cells in rat kidney.
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Immunoelectron Microscopic Localization of rkNBC1
In segments S1 and S2 of the rat proximal tubule, NBC1 immunoreactivity was
closely associated with the basolateral plasma membrane, including both the
lateral and the basal parts (Figure
4). In places where the plasma membrane was cut at a right angle,
the colloidal gold particles were distinctly associated with the inner leaflet
of the plasma membrane (Figure
4, arrows). No immunoreactivity was present in mitochondria or
other cell organelles, including the apical endocytic organelles and dense
apical tubules (Figure 4A), and
immunolabeling was not associated with vesicular or endocytic/exocytic
profiles along basolateral membranes. Important is that the apical plasma
membrane and brush border were unlabeled
(Figure 4A). The same labeling
pattern was observed with the present anti-rkNBC1-CT15 antibody and with the
previously described anti-rkNBC1-CT108 (identical to MBP-NBC-5;
Figure 5). No labeling was
observed in the S3 segment (Figure
6). Immunolabeling controls were negative
(Figure 7).
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In the kidney of Ambystoma tigrinum, labeling was observed in the proximal tubule and the late distal segment both with anti-rkNBC1-CT15 and anti-rkNBC1-CT108, but no labeling was seen in the early distal tubule. In the proximal tubule, a weak labeling was observed along the lateral plasma membrane and the basal cytoplasmic folds that project into the so-called basal extracellular labyrinth (arrows in Figure 8A) which is located between the cell and the basement membrane. In the late distal segment, labeling was observed along the basolateral membrane (arrows in Figure 8B), both close to the basement membrane and in the mid-regions of the tubule cells. No labeling was observed in the cytoplasm or associated with cell organelles.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunoblotting of cortical membranes revealed a strong band of approximately 280 kD, which disappeared under strongly reducing conditions. This suggests that the NBC1 exists in a dimeric form in the membrane. For the Cl/HCO3- cotransporter, which has approximately 40% homology dimerization, has also been documented (22,23) and suggested to be a form present in the cell membrane. It will be interesting to find out whether dimers or higher oligomers of the NBC1 also exists and are of importance for the transport function.
NBC1 in Rat Kidney
In this study, the distribution of the NBC1 in the rat kidney was
investigated using a new affinity-purified NBC1 antibody (anti-rkNBC1-CT15) to
be confined to S1 and S2 of the proximal tubule and also to the proximal
tubule and the distal tubule in the Ambystoma kidney. At the
subcellular level, labeling in these segments was distinctly associated with
the cytoplasmic side of the basolateral membrane, consistent with an internal
position of the C-terminus of the transporter. We could not detect any NBC1
immunoreactivity in the S3 segment of the rat kidney proximal tubule. These
findings were obtained with high-resolution immunoelectron microscopy in
combination with immunoblot analyses and indirect immunoperoxidase microscopy.
They extend to the subcellular level the observations made in a previous
immunofluorescence study, which reported the lack of NBC1 staining in segment
3 of rat and rabbit proximal tubules, using anti-(MBP-NBC-3) and
anti-(MBP-NBC-5) sera (9).
The presence of strong and specific NBC1 staining in segments S1 and S2 clearly shows that the NBC protein can be detected reliably in this tissue with our approaches. It is not obvious which specific factors could account for a hypothetical false-negative immunolabeling exclusively in the S3 but not in S1 or S2. Thus, the most simple interpretation of the consistent absence of NBC1 staining in the S3 segment is that the NBC1 protein is indeed absent from this segment or present only at insignificant levels.
Our findings, therefore, suggest that the rat S3 segment does not contain an electrogenic cotransporter that could provide a basolateral HCO3- exit pathway. The antibodies used in this and a previous study (9) recognize not only the major renal electrogenic NBC1 but also all other electrogenic NBC isoforms known (19,21,24,25,26). In contrast, the antibodies do not recognize a cloned electroneutral NBC isoform (27,28), which promotes the uptake of HCO3- into the cytoplasm, not the efflux. Therefore, such a transporter probably would not be able to provide a mechanism for basolateral HCO3- exit in the S3 segment.
The absence of NBC1 protein from the S3 segment in rat is interesting with respect to some open questions in the physiology of proximal tubular bicarbonate reabsorption. Thus, it is not clear whether there actually is any net reabsorption of HCO3- in the S3 segment under normal conditions in vivo. It is well established that the transport rates for HCO3- decrease continuously from the S1 toward more distal segments of the proximal tubule (for review, see reference 29). Unfortunately, no direct data are available about the net transport rates of bicarbonate in the S3 segment in vivo, because the S3 segment is not accessible to study by micropuncture techniques in the rat. However, incorporation of the available data from S1 and S2 into mathematical models and subsequent extrapolation has suggested that net reabsorption of HCO3- has actually dropped to zero by the time the S3 segment is reached (29,30). In keeping with this interpretation, two functional studies in rabbits in which the S3 was probed directly by electrophysiologic methods reported evidence against the presence of electrogenic Na+/HCO3- cotransport in the S3 segment (31,32). In addition, these two studies ruled out the existence of an "HCO3- channel" in the basolateral membrane of the S3 segment. However, in parallel and later investigations, electrogenic Na/HCO3 cotransport has been reported in the rabbit S3 segment (33,34,35,36). In addition, in situ hybridization studies revealed the presence of low levels of NBC1 mRNA in the rabbit S3 segment (36), in contrast to its absence in the rat S3 segment (7). This suggests that a species difference exists between rat and rabbit with respect to the presence of NBC1 in the S3 segment.
With respect to the question of whether there is net reabsorption of HCO3- in the rat S3 segment, our findings pointing to the absence of the electrogenic Na+/HCO3- cotransporter NBC1 (and related NBC1 proteins) leave us without a candidate transporter that could provide a mechanism for basolateral HCO3- exit in this segment.
NBC1 in Ambystoma Kidney
Na+/HCO3- cotransport activity was first
described in the Ambystoma kidney
(1), and the first cloning of
NBC1 was made from Ambystoma kidney
(5). Furthermore, in the
Ambystoma, the HCO3- reabsorption is somewhat
different from HCO3- reabsorption in the mammalian
kidney in that less than 50% of the reabsorption of
HCO3- occurs in the proximal tubule with high rates of
reabsorption in the late distal tubule
(37). For these reasons, it is
of interest to investigate the cellular and subcellular distribution of NBC1
in the Ambystoma kidney in addition to the rat kidney.
It is noteworthy that the cell architecture is distinctly different in proximal tubules of rat and in Ambystoma. In the interdigitating rat cells, most of the basolateral membrane faces predominantly the narrow lateral intercellular space. In Ambystoma, however, 75% of the surface area of the basolateral membrane is basal and forms folds and processes projecting into the basal extracellular labyrinth (15). This complex compartment is located between the basement membrane and bulk of the cell body and communicates with the peritubular extracellular space by way of slits between basal cell processes. Thus, the cellular distribution of NBC1 is much different in cells of mammalian and salamander proximal tubules, suggesting that reabsorption pathways also are different. Na/K-ATPase, the sodium pump, has a similar polarized distribution in Ambystoma proximal tubule cells and is abundantly expressed in the plasma membrane of the folds and projections surrounding the basal extracellular labyrinth (38).
In conclusion, we have demonstrated at the ultrastructural level that NBC1 is present in both the basal and the lateral cell membrane of proximal tubule cells in segments S1 and S2 of the rat kidney but absent in segment S3 proximal tubule cells. Moreover, NBC1 labeling is associated with the basal cell membrane facing the basal extracellular labyrinth in the proximal tubule and the basolateral cell membrane in the late distal tubule of the salamander Ambystoma tigrinum. These results are consistent with the available functional studies of electrogenic Na+/HCO3- cotransport in these tubule segments.
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