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Institut National de la Santé et de la Recherche Médicale Unités 356 and 430, Université Pierre et Marie Curie, and Hôpital Broussais, Paris, France; and University of Alabama at Birmingham, Birmingham, Alabama 35294-0007
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Four Na+/H+ exchangers (NHE1 to NHE4) have been detected in the kidney. Renal NHE2 expression sites have not been fully established. We have raised rabbit antisera against an oligopeptide related to the amino acids 652 to 661 of rat NHE2. Western blot analysis of plasma membrane fractions isolated from rat renal cortex showed that affinity-purified anti-NHE2 antibody detected an 85-kDa protein in apical but not in basolateral membranes. The labeling of this 85-kDa protein was specifically blocked by preincubation of the antibody with its monomeric peptide, indicating specific recognition. Indirect immunolabeling was performed on sections of paraformaldehyde-fixed rat kidney embedded in paraffin. Strong staining was seen in the apical membrane of cortical thick ascending limbs, distal convoluted tubules, and connecting tubules. Much weaker apical staining was found in medullary thick ascending limbs of Henle. In the inner medulla, some thin limbs were intensively labeled by the anti-NHE2 antibody. No staining could be detected in any segments of the proximal tubule and collecting duct.
sodium/hydrogen exchanger; apical membrane
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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THE
Na+/H+
exchangers (NHE) comprise a family of structurally related plasma
membrane proteins, NHE1 to NHE5. One or more of the NHE isoforms have
been documented in virtually all mammalian cells studied to date. They
have varying cellular and subcellular distributions, consistent with
the broad range of cellular functions of
Na+/H+
exchange. Indeed, aside from its role in the maintenance of
intracellular pH in most animal cells,
Na+/H+
exchange activity is also involved in transepithelial transport of
Na+ and
HCO
3 and in cell volume regulation.
NHE1 messages are expressed in virtually all tissues (18, 23). In the
kidney, NHE1 was shown to localize to the basolateral membrane of
multiple nephron segments (3, 5). NHE3 transcripts are specifically
expressed in epithelial tissues, with highest levels found in kidney
and intestine (18, 22). Immunologic studies have localized NHE3 to the
apical membrane of cells in the proximal tubule and the thick ascending
limb of Henle (2-4). Therefore, NHE3 is believed to participate in
both NaCl and NaHCO3 absorption in
the proximal tubule and HCO3
absorption in the thick ascending limb. Recent studies showed that NHE3
plays an important role in regulation of systemic acid-base balance in
both proximal tubule and thick ascending limb (1, 17, 29).
As for NHE3, distributions of NHE2 and NHE4 messages are more restricted than that of NHE1. The NHE4 message appears to be most highly expressed in stomach but is detectable in small intestine, colon, kidney, brain, uterus, and skeletal muscle (18). Whole- kidney in situ hybridization studies show that NHE4 mRNA is highly expressed in outer and inner medulla, whereas it appears at a lower level in tubules dispersed throughout the renal cortex (6). Recent immunohistochemical analysis has shown that in rat renal cortex, NHE4 is predominantly expressed in distal tubules, where it is localized to the basolateral membrane (8). In rat, NHE2 is expressed predominantly in gastrointestinal tract with much lower levels in skeletal muscle, kidney, brain, and lung (28). Recent immunohistochemical studies have demonstrated that NHE2 colocalizes in brush-border membranes of most human and rabbit intestinal cells (13). Apical localization of NHE2 has also been suggested based on Western blot analysis of apical membranes from rat renal cortex (13). Few studies have dealt with the renal expression sites of the NHE2-related mRNA (12, 20), and none, to our knowledge, have rendered a complete description of the NHE2 protein distribution along the rat nephron. We investigated NHE2 expression from glomeruli down to inner medullary collecting duct cells immunohistochemically by using a polyclonal antibody that we have raised against a 10-amino acid internal peptide of the rat NHE2 protein.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Production of Polyclonal Antibodies to NHE2 Exchanger Isoform
Rabbit antisera were produced against a synthetic oligopeptide DA-10, which is related to an internal sequence (amino acids 652 to 661) of the rat NHE2 protein. The multiple antigenic peptide system was used for the preparation of antipeptide site-specific antibodies. This approach uses a small polypeptidic core matrix bearing eight radially branching synthetic peptides as described previously (21). The specific multiple antigenic peptide (MAP DA-10) was synthesized by Research Genetics (Huntsville, AL). Rabbits chosen for immunization had preimmune sera showing virtually no reactivity toward the specific MAP as determined by ELISA using the MAP DA-10 to coat the plates. Before injection, 2 mg/ml of the MAP in PBS were mixed with complete Freund's adjuvant (ratio 1:1). New Zealand White rabbits were immunized intramuscularly with 1 ml emulsified antigen. Two booster intramuscular injections were given 3 and 6 wk later with 1 mg emulsified antigen in incomplete Freund's adjuvant (ratio 1:1), and blood samples were collected from the ear artery 10 days after the second booster immunization. Postimmune sera were selected that showed high antibody titers as determined by ELISA. In a study of the cross-reactivity of the hepta-lysyl backbone, which is common to the MAP system, we found no cross-reactivity in ELISA between the MAP DA-10 and the MAP RK-10, which is an internal sequence of 10 residues related to the amino acids 687 to 696 of rat NHE3. Polyclonal antibodies were purified from rabbit antiserum on a peptide affinity column, synthesized by coupling the oligopeptide used as the antigen to a support column (Affigel) by Research Genetics (Huntsville, AL). The column was equilibrated with the Immunopure Ag/Ab binding buffer from Pierce (Rockford, IL), and antiserum was passed over the column at least four times. The column was then extensively washed with the binding buffer, and reacting antibodies were eluted from the column using the Immunopure Ab/Ag gentle elution buffer from Pierce. Eluted fractions were dialyzed against Tris-buffered saline solution (TBS), pH 7.3, containing 0.02% NaN3, for 24 h at 4°C and concentrated using the Centriprep-30 concentrator (Amicon, Beverly, MA).Membrane Preparations
Membranes were prepared from male Sprague-Dawley rats (220-240 g) anesthetized by intraperitoneal injection of 50 mg/kg body wt of pentobarbital sodium. Apical membrane fractions were isolated from whole renal cortex using the divalent-cation aggregation method as described previously (10). Differential and Percoll gradient centrifugations were used for isolating basolateral membrane fractions from whole renal cortex (9). Protease inhibitors were included in all buffers. Protein contents were determined using the Bradford protein assay.SDS-PAGE and Immunoblotting
Membrane fractions were solubilized and separated by SDS-PAGE using 7.5% polyacrylamide gels according to Laemmli (16). For immunoblotting, proteins were transferred electrophoretically for 1 h at 4°C from the gel to nitrocellulose membranes and stained with 0.5% Ponceau S in acetic acid. Immunoblotting was performed as follows. Strips of nitrocellulose were incubated first in 10% nonfat dry milk in PBS, pH 7.4, for 1 h at room temperature to block nonspecific binding of antibody, followed by an overnight incubation at 4°C with a 1:500 dilution (9 µg/ml) of the affinity-purified rabbit anti-NHE2 peptide antibody. Membranes were then washed four times with PBS containing 0.1% Tween-20 for 5 min each and once with PBS for 5 min before incubation with a 1:3,000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA). Blots were washed as above, and luminol-based enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) was used to visualize bound antibodies on Polaroid film. Photographs of immunoblots were digitized under NIH image software. In addition, adsorption controls were performed. The peptide antigen or an irrelevant peptide (100 µg/ml) was incubated with a 1:500 dilution of the affinity-purified antibody (4°C, overnight), and the resulting media were used in place of the antibody.Immunohistochemistry
Tissue preparation. Male Sprague-Dawley rats purchased from IFFA-CREDO (L'Arbresle, France), weighing 250-300 g, were prepared for retrograde vascular perfusion under anesthesia provided by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). The abdominal aorta was exposed via laparotomy. The aorta was clamped above and below the renal arteries, and a cannula was inserted into it to perfuse the kidneys with freshly made cold 4% paraformaldehyde in Dulbecco's modified Eagle's-Ham's F-12 medium. Prior to perfusing the kidneys, we cut the inferior vena cava. The kidneys were then removed. Coronal kidney sections were incubated overnight at 4°C in 4% paraformaldehyde and then embedded in paraffin. Subsequently, 4-µm sections of the paraffin block were deparaffinized with toluene, washed in graded ethanol, and rehydrated in TBS, then labeled using a three-layer method that utilizes a biotinylated secondary IgG in conjunction with alkaline phosphatase- and/or horseradish peroxidase-conjugated streptavidin.Immunolabeling. Immunolabelings for NHE2 and for the Tamm-Horsfall glycoprotein (THP) were performed in two consecutive 4-µm thick sections. To reduce nonspecific binding, sections were treated with either 10% goat serum (Vector Laboratories, Burlingame, CA), in TBS, pH 7.6 or background reducing buffer from Dako (Copenhagen, Denmark) for NHE2 and THP labeling, respectively. This was blotted away after 10 min, and the sections were incubated with either a 1:200 dilution of the anti-NHE2 peptide antibody or a 1:1,000 dilution of goat anti-THP antibody (Cappel, ICN pharmaceuticals, Aurora, OH) overnight in a humid chamber at room temperature. Sections were then washed in TBS (three 5-min periods) and incubated with 1:200 dilution of goat biotinylated anti-rabbit IgG (Vector Laboratories) or donkey biotinylated anti-goat Ig (Amersham) followed by the Immunopure peroxidase suppressor (Pierce Chemicals, Rockford, IL) to block endogenous peroxidase activity, each for 30 min at room temperature with three TBS washes in between. After three 5-min washes in TBS, sections were incubated with a 1:400 dilution of horseradish peroxidase-conjugated streptavidin (Amersham) or a 1:50 dilution of alkaline phosphatase-conjugated streptavidin (Amersham) for 30 min at room temperature for NHE2 and THP labelings, respectively. Peroxidase activity was revealed with 3-amino-9-ethylcarbazole (AEC) or 3,3-diamonobenzidine (DAB) and H2O2, which give red-brown or brown precipitates, respectively. Alkaline phosphatase activity was revealed with either Fast Red (Dako) or Vector Blue (Vector Laboratories) as enzyme substrates.
To ascertain the presence of NHE2 in distal convoluted tubules, immunostainings of the same cells with antibodies to NHE2 and to the thiazide-sensitive NaCl cotransport (NCC) were performed in two consecutive 4-µm thick sections. Labeling for NCC was performed using the same method as for NHE2 except that the section was incubated with a 1:200 dilution of a rabbit anti-NCC antibody containing serum in place of the anti-NHE2 peptide antibody.
Sections were also doubly labeled with anti-NHE2 antibody and antibody to THP. Sections were first incubated with the anti-THP antibody for 30 min at room temperature. Sections were then washed in TBS (three 5-min washes) and incubated with a 1:200 dilution of donkey biotinylated anti-goat Ig (Amersham) followed by a 1:50 dilution of alkaline phosphatase-conjugated streptavidin (Amersham), each for 30 min at room temperature with three washes in between. Alkaline phosphatase activity was revealed with Vector Blue (Vector Laboratories) as the chromogen. Sections were washed in TBS before proceeding with protocol to stain the NHE2 antigen as described above.
To distinguish thin limb segments from vascular structures in the inner stripe of the outer medulla and in the inner medulla, sections were also dually labeled with anti-NHE2 antibody and rat endothelial cell antibody (RECA; Serotec, Oxford, UK), which specifically binds to rat endothelial cells. Sections that had been sequentially incubated with RECA, horse biotinylated anti-mouse IgG (Vector Laboratories), alkaline phosphatase-conjugated streptavidin (Amersham), and Vector Blue (Vector Laboratories) as enzyme substrate were then stained for NHE2 as described above.
After staining, sections were washed briefly in distilled water. Glass coverslips were mounted after applying liquid-phase Glycergel solution (Dako) to the tissue sections. Sections were examined with a Zeiss photomicroscope and photographed using Kodak T64 film.
Controls. Control procedures included sections in which the rabbit anti-NHE2 antibody was replaced by a nonrelevant rabbit anti-CD3 antibody (Dako). In addition, a 1:200 dilution of the anti-NHE2 antibody was preadsorbed with the purified antigen, and the resulting medium was used as negative control.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Immunoblotting of Renal Cortical Membrane Fractions
The ability of affinity-purified anti-NHE2 peptide antibodies to recognize the cognate protein from which they were derived was first assessed by Western blotting experiments against apical membrane preparation isolated from rat kidney cortex. In immunoblots performed with proteins from apical membrane-enriched preparations shown in Fig. 1A, the rabbit anti-rat NHE2 peptide antibody recognized a single protein with an apparent molecular mass of ~85 kDa (Fig. 1A, lane 1). The 85-kDa band was not detected when antibody was preincubated in the presence of excess monomeric peptide antigen prior to immunoblotting apical membrane proteins (Fig. 1A, lane 2). Because three consecutive amino acids of the rat NHE2 peptide antigen sequence (R-E-R) are identical to the corresponding residues in another NHE isoform sequence (NHE3, see Ref. 28), incubations were also carried out in the presence of a synthetic NHE3 decapeptide related to the corresponding sequence of the rat NHE2 peptide antigen. As illustrated in Fig. 1A, lane 3, the 85-kDa band could still be detected in the presence of excess NHE3 peptide, indicating that the anti-NHE2 peptide 652-661 antibody specifically recognizes the NHE2 Na+/H+ exchanger without cross-reactivity against the NHE3 isoform. Western blot experiments were next performed against membrane preparations from rat renal cortex that were appropriately enriched in the Na+/H+ exchanger, NHE1 (Fig. 1B), and the sodium-phosphate cotransporter, NaPi-2 (Fig. 1C), which serve as markers for basolateral and apical membranes, respectively. As shown previously (5), the anti-NHE1 antibody reacted with a 95- to 110-kDa protein that was enriched in basolateral membranes compared with apical membranes. In contrast, the 90- to 80-kDa polypeptides detected by antibody to the NaPi-2 (11) were relatively enriched in apical membranes but poorly detectable in basolateral membranes. As for NaPi-2, NHE2 identified as the 85-kDa protein was enriched in apical membranes (Fig. 1A) and completely absent from basolateral membranes (Fig. 1A). Altogether, these results demonstrate that NHE2 is an 85-kDa protein expressed in apical membranes or in a membrane domain that coenriches with the apical membrane but not in basolateral membranes of rat kidney cortex.
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Immunolocalization of NHE2 Along the Rat Nephron
The renal expression sites of the NHE2 protein were revealed by indirect immunoperoxidase light microscopy. Strongest staining for NHE2 was found in the apical membrane of some tubules within the cortex (Fig. 2, A, C, and E). Higher magnification revealed it to be present in tubules lacking a brush border (Fig. 2G). Immunolabeling of consecutive serial sections with the anti-NHE2 peptide antibody and antibodies to THP, which is specifically localized to the epithelial cells of the thick ascending limbs of the Henle's loop and the most proximal part of the distal convoluted tubules (15), showed that, in the cortical labyrinth, the anti-NHE2 peptide antibody-labeled tubules exhibiting heterogeneous staining for THP (Fig. 2, A and B). Apical labeling with the anti-NHE2 antibody in tubules located in the cortical labyrinth, showing weak or absent staining for THP, was confirmed when sections were dually labeled with antibodies against THP and anti-NHE2 antibody (Fig. 2C). Immunostaining of two consecutive serial sections with antibodies to NHE2 and to the thiazide-sensitive NCC were next performed. In the kidney, this latter transporter has been shown to be exclusively present in apical membranes of distal convoluted tubules. As shown in Figs. 2, E and F, antibody to NHE2 stained the apical membranes of the same distal convoluted tubules as did the antibody to NCC. Other tubules within the cortical labyrinth showing a characteristic profile of connecting tubules exhibited distinct apical staining for NHE2 (Fig. 2H). In the medullary rays of the cortex, only THP-positive tubules showed NHE2 labeling (Fig. 3, A and B). Much weaker apical membrane labeling for NHE2 was found in tubular segments situated in the outer stripe of the outer medulla, which were identified as thick ascending limbs by THP labeling (Fig. 3, C and D). No staining was observed in any segments of the proximal tubule, identified as brush-border-containing tubules within the cortical labyrinths (Fig. 2, A, C, E, G, and H), medullary rays (Fig. 2A), and outer stripe of the outer medulla (Fig. 3A). Staining for NHE2 was also observed in some thick ascending limb segments (THP-positive tubules) within the inner stripe of the outer medulla (Fig. 3, C and D). Some thin limbs of Henle's loop, which were identified as nonvascular structures, RECA-negative structures, and small-diameter tubule segments with flattened epithelial cells within the inner medulla, demonstrated strong labeling intensity for NHE2 (Fig. 3, E and F). Collecting ducts, which were identified in this region of the kidney by their wide diameter, however, were unstained.
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Immunohistochemical Controls
When the anti-NHE2 peptide antibody was incubated with the peptide antigen before application to section (Fig. 2I) or replaced by nonrelevant antibody (Fig. 2D), no binding of anti-NHE2 antibody was observed. However, faint background stainings were repeatedly observed in collecting ducts within the medullary rays with most of the antibodies used in this study (Fig. 2, A and B).| |
DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In this study, we document that the NHE2 isoform of the Na+/H+ exchanger is expressed on the apical membrane of specific segments of the rat nephron. This conclusion is based on localization of the NHE2 protein by indirect immunoperoxidase labeling performed on rat kidney sections using polyclonal antibodies against a unique 10-amino acid peptide for the NHE2 transporter and immunoblotting of highly purified apical and basolateral membrane fractions isolated from whole renal cortex.
The immunochemical characteristic of rat renal NHE2 as an 85-kDa protein is in perfect agreement with that previously reported by Tse et al. (24) for the O-glycosylated form of the rabbit NHE2 protein in PS120 fibroblasts transfected with the rabbit NHE2 cDNA, the unglycosylated form of which was demonstrated to be 75 kDa. Specificity of labeling of the 85-kDa protein was demonstrated by the ability of the antigenic peptide to block the reaction. Moreover, in our immunohistochemical study, the lack of binding of our anti-NHE2 peptide antibody to the apical membrane of proximal tubules demonstrates an absence of cross-reactivity of the anti-NHE2 peptide antibody to NHE3 protein, which is known to be expressed along the apical membrane of these tubular cells (2, 4). Similarly, it is unlikely that the labeled protein corresponds to either NHE1 or NHE4 isoforms, both of which have a basolateral location in several nephron segments (5, 8).
In our immunohistochemical study, in which proximal tubules were easily identified by their brush border, whereas cortical distal tubules were identified as tubule segments lacking a brush border, we found that cortical distal tubules displayed strong apical labeling intensities for NHE2 compared with proximal tubules, which showed no staining. Admittedly, the inability of the anti-NHE2 antibody to stain proximal tubules could reflect insufficient sensitivity of the immunohistochemical method to detect sites of low NHE2 density. In this regard, preliminary indirect immunofluorescence studies by Yip et al. (30) showed weak staining in the brush border of proximal tubules with an anti-NHE2 antiserum. However, a preliminary in vivo microperfusion study demonstrated the absence of inhibition by luminal 100 µM HOE-694, a concentration that should have completely inhibited NHE2 activity and only marginally that of NHE3, on bicarbonate and fluid reabsorption in the rat proximal tubule, indicating that NHE3 is the principal NHE isoform mediating bicarbonate reabsorption in the rat proximal tubule (26). In addition, our results are consistent with a previous study identifying NHE2 mRNA in distal but not in proximal tubules using the method of in situ hybridization on rat kidney cortex sections (12). Because the proximal tubules, which constitute ~80% of the cortical mass, showed no measurable immunoperoxidase staining with our anti-NHE2 antibody, NHE2-stained immunoblots of cortical apical membrane preparations must reflect reactivity against NHE2 that occurs in non-proximal-tubule segments, predominantly the cortical thick ascending limbs and the distal convoluted/connecting tubules. In this regard, the ability to detect the Na-K-Cl cotransporter BSC1, a protein of apical location in thick ascending limbs (14), in immunoblots of apical membrane preparations from whole renal cortex demonstrates the presence of apical membranes of distal tubular origin in our preparation (data not shown).
Some of the NHE2 immunoreactive tubules within the cortical labyrinth
were distal convoluted tubules, as demonstrated by staining for NHE2
and the thiazide-sensitive NCC on two consecutive 4-µm sections. Some of the immunoreactive tubules within the cortical labyrinth were identified as connecting tubules based on their shape
and localization within the cortex. By immunofluorescence microscopy,
Amemiya et al. (2) have recently demonstrated that NHE3 is expressed in
apical membrane of proximal tubules, but absent in distal convoluted
tubules. However, there is significant bicarbonate reabsorption in the
early distal tubule in the rat that is inhibited by
104 M
ethylisopropylamiloride, suggesting that luminal acidification is
mediated by an apical membrane
Na+/H+
exchanger (27). Besides the thiazide-sensitive NCC,
parallel exchange of
Na+/H+
and Cl/anions has also been
shown to account for NaCl reabsorption in the distal convoluted tubules
(25) and connecting tubules (19). In these latter segments, the apical
membrane
Na+/H+
exchange activity is thus likely to be mediated by the NHE2 isoform rather than NHE3.
The presence of NHE2 in apical domain of cortical and medullary thick ascending limbs is best illustrated in consecutive serial sections labeled with anti-NHE2 antibody and antibody against the THP. Indeed, we localized NHE2 to the same tubular structures as those labeled with antibody to the THP, namely, thick ascending limbs of Henle. Anti-NHE2 antibody did not bind to any other tubule segment within the medullary rays of the cortex and outer medulla, indicating that neither straight proximal tubules, thin descending limbs, nor collecting tubules expressed NHE2. Of note, heterogeneous reactivity with the anti-NHE2 antibody was observed along the course of the thick ascending limb, highest in its cortical portion with decreasing abundance along this segment in the outer medulla. At the mRNA level, NHE2 that could not be detected by the RT-PCR reactions from microdissected medullary thick ascending limbs by Borensztein et al. (7) was nevertheless detectable using optimized RT-PCR reactions in a recent study by Sun et al. (20). These results reflect the low level of NHE2 expression in this nephron segment. Finally, we would like to point out that, in contrast to NHE2, NHE3 was abundant in the medullary portion of the thick ascending limb with decreasing intensity along the medullary rays (2). If NHE3 is likely to be involved in bicarbonate reabsorption in this nephron segment, the physiological significance of the presence of another NHE isoform (NHE2) in thick ascending limb luminal membranes remains to be established.
In addition to the thick ascending limb, we also found that some thin limbs of long loops of Henle displayed strong labeling intensities for NHE2. Since thin descending limbs of Henle's loop in the inner stripe of the outer medulla were unstained, stained thin limbs in the inner medulla might represent ascending rather than descending thin limbs of long loops. To our knowledge, direct measurement of Na+/H+ exchange has not been performed in this segment.
In summary, we find that rat renal NHE2 is an apical membrane protein with an apparent molecular mass of 85 kDa that is present in thin limbs of long loops and distal tubules, strongest in the distal convoluted tubules and the cortical portions of thick ascending limbs, and weakest in their medullary portions. It does not appear that NHE2 protein is expressed in the rat proximal convoluted tubule.
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ACKNOWLEDGEMENTS |
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Antibodies to NHE1, NaPi-2, BSC-1, and thiazide-sensitive NaCl cotransporter were generous gifts from Drs. S. Grinstein (University of Toronto, Canada), J. Biber (University of Zürich-Irchel, Switzerland), S. C. Hebert (Vanderbilt University, Nashville), and D. Ellison (University of Colorado Health Sciences Center, Denver), respectively. We thank Pierre Kitmacher and Michel Paing for photographs.
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FOOTNOTES |
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This research was supported by grants from the Institut National de la Santé et de la Recherche Médicale and the Université Paris 6 and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-19407. R. Chambrey was supported by grants from the Société de Secours des Amis des Sciences and the Fondation pour la Recherche Médicale.
The present study was presented in part at the 1997 Annual Meeting of the American Society of Nephrology.
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: R. Chambrey, Unité INSERM 356, Institut de Recherche des Cordeliers, 15, rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France (e-mail: chambrey{at}ccr.jussieu.fr).
Received 12 January 1998; accepted in final form 29 May 1998.
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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