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Departments of 1Internal Medicine and 2Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut
Submitted 22 June 2004 ; accepted in final form 26 October 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-glutamyltranspeptidase and megalin indicated expression of NHE8 on both the microvillar surface membrane and the coated-pit region of proximal tubule cells, suggesting that NHE8 may be subject to endocytic retrieval and recycling. Although colocalizing in the proximal tubule with NHE3, no significant alteration in NHE8 protein expression was evident in NHE3-null mice. We conclude that NHE8 is expressed on the apical brush-border membrane of proximal tubule cells, where it may play a role in mediating or regulating ion transport in this nephron segment. sodium/hydrogen exchange; proximal tubule
Our primary interest has been in elucidating the role that NHEs play in the kidney with regard to proximal tubule sodium bicarbonate reabsorption. Although NHE3 mediates the bulk of proximal tubular sodium bicarbonate reabsorption, a significant component is mediated by alternative mechanisms that have been incompletely defined (3, 24). In an effort to identify proteins that may contribute to acid-base and/or electrolyte transport in the proximal tubule, we identified and cloned NHE8 (6). Using in situ hybridization techniques, we demonstrated that NHE8 is indeed expressed in proximal tubule cells (6) and thus is an excellent candidate for mediating cation transport in this region. Using NHE8-specific antibodies, we showed by immunoblotting experiments that NHE8 cofractionates with brush-border membranes (6). Unfortunately, the precise membrane localization could not be definitely established by immunohistochemical staining due to an inability of these NHE8 antibodies to work for this modality. Determining the membrane localization of NHE8 is important because phylogenetic analysis has been interpreted to suggest that NHE8 might be an intracellular NHE isoform (12).
To understand the role that NHE8 plays in renal function, it is essential to unequivocally define its sites of expression. Thus the goal of the present study was to generate new NHE8-specific antibodies to immunolocalize NHE8 protein in the kidney. Our results demonstrate that NHE8 is expressed in the kidney on the apical brush-border membrane of proximal tubules, from S1 to S3, with no detectable staining in other tubule segments.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Preparation of immunogen, rabbit immunization, and antibody purification. The peptide sequence (NHE8.472.pep) consisted of amino acids 472–491 of mouse NHE8 with the addition of a NH2-terminal cysteine. Peptide synthesis, KLH conjugation, and rabbit immunization were performed at Pocono Rabbit Farm and Laboratory according to their standard protocol (http://www.prfal.com/).
Sera from each of the three immunized rabbits were purified over an affinity column in which NHE8.472.pep was immobilized. The affinity column was prepared by covalently cross-linking NHE8.472.pep, via the sulfhydryl residue on the NH2 cysteine, to a Sulfolink coupling gel (Pierce) according to the manufacturer's recommendations. After purification, each of the polyclonal antibodies (named Rab63, Rab64, Rab65) was stored in 50% glycerol/PBS at –20°C.
Monoclonal Antibody Generation
Preparation of NHE8 fusion proteins. The fusion protein maltose-binding protein (MBP).NHE8.t89 was prepared as described previously (6). The fusion protein gluathione S-transferase (GST).NHE8.t89 was prepared using the same component of the human NHE8 sequence that was used for generating the MBP.NHE8.t89 protein. Specifically, the COOH 267 nucleotides of human NHE8, corresponding to the terminal 89 amino acids, were subcloned into the pGex6p-1 (Pharmacia Biotech) vector. When protein expression was induced in Escherichia coli, the resultant GST.NHE8.t89 fusion protein formed insoluble intracellular inclusions. However, the modified protocol utilizing 1.5% sarkosyl followed by affinity purification over a GST column, as described in the troubleshooting section of the manufacturer's protocol, successfully enabled sufficient fusion protein purification.
Immunization of mice and production of hybridomas. Immunizations with MBP.NHE8.t89 were conducted in 8-wk-old Balb/c mice by the immunization services at Yale University according to standard protocols (http://info.med.yale.edu/yarc/vcs/immuniza.htm#1). A mouse whose serum showed specific reactivity by Western blot to NHE8, as defined by the presence of an NHE8 signal in NHE8-transfected COS-7 cells but not untransfected controls, was selected for the fusion. Spleen cells were fused to Ag8 mouse myeloma cells according to established protocols (2, 9).
Hybridoma selection and monoclonal antibody purification.
Hybridomas were selected for the ability of their supernatant to label NHE8 by ELISA, Western blotting, as well as immunofluorescence microscopy. First, 
1,700 resultant hybridoma supernatants were screened by ELISA against GST.NHE8.t89. By using GST.NHE8.t89 instead of the MBP.NHE8.t89 immunogen, antibodies specific for the NHE8 component of the immunogen could be identified. The 105 supernatants that yielded the strongest signals were then tested for the ability to specifically label mouse NHE8 by Western blot analysis. As with prefusion screening, specificity was determined by the ability of the hybridoma supernatant to immunolabel Western blots prepared from NHE8-transfected COS-7 cells but not Western blots prepared from untransfected COS-7 control cells. The 29 resultant clones were then screened for the ability to specifically label NHE8 by immunofluorescence. For this assay, coverslips were prepared with either PLP-fixed NHE8-transfected COS-7 cells or with PLP-fixed untransfected COS-7 cells. The eight surviving hybridomas that demonstrated specific NHE8 labeling by immunofluorescence were cloned and subcloned by limiting dilution.
Eight monoclonal antibodies (mAb; 4A9, 7A11, 9C9, 13B12, 13D2, 13E4, 15G5, 17F6) were purified from the corresponding hybridoma supernatants by affinity chromatography using protein G-Sepharose 4B (Amersham) according to the manufacturer's protocols. Each purified mAb was stored in 50% glycerol/PBS at –20°C.
ELISA. Hybridoma supernatants were initially screened for NHE8 reactivity by ELISA as described above. In brief, GST.NHE8.t89 fusion protein, solubilized in PBS at 10 µg/ml, was used to coat ELISA plates in an overnight incubation at 4°C. The plates were then washed four times (0.1% Triton X-100/PBS) after which the primary antibody (50 µl hybridoma supernatant diluted in 100 µl of 0.1% Triton X-100, 0.1%BSA/PBS) was applied to the corresponding well and incubated for 1 h at 25°C. Plates were then washed four times. The secondary antibody [horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G, Zymed] was then added to each well and incubated for 1 h at 25°C. After the final set of four washes, a 0.1% HRP substrate was added to each well to identify the wells in which bound antibody complexes were present. The optical density in each well, determined at a wavelength of 490 nm, was used as a measurement of antibody binding.
Expression of NHE isoforms in COS-7 cells. COS-7 cells were transiently transfected with cDNAs for rat NHE3 (kind gift from John Orlowski) (14) or mouse NHE8 (pCDNA/NHE8) (6) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. COS-7 cells were grown in DMEM/10% fetal calf serum at 37°C in 5% CO2-95% air. Following 24-h incubation, cells were assayed either by Western blotting or by immunofluorescence microscopy.
Cell-surface biotinylation. COS-7 cells were plated at 95% confluence in one well of a six-well tissue culture dish, transiently transfected with NHE8 cDNA, and used for biotinylation experiments 24 h after transfection. Surface biotinylation was performed using NHS-S-S-Biotin (Pierce) in biotinylation buffer (10 M triethanolamine, pH 7.5, 150 mM NaCl, 2 mM CaCl2) according to previously described protocols (5). The cells were then solubilized in solubilization buffer (1 ml TBS, 1% Triton X-100, 1 h, 4°C), and the lysate was clarified by centrifugation (20,000 g, 10 min, 4°C). Fifty microliters of washed streptavidin-agarose beads (Pierce) were then added and allowed to incubate with the cleared lysate for 1 h at 4°C. Biotinylated proteins were then pulled down with the streptavidin beads (2,000 g, 1 min). After four washes (each wash with 1 ml of solubilization buffer), bound proteins were eluted from the beads with Laemmli sample buffer containing 100 mM DTT and subjected to SDS-PAGE.
Kidney membrane preparation. Sprague-Dawley rats were anesthetized by intraperitioneal injection of pentobarbital sodium. The kidneys were removed, and the dissected cortex was homogenized. This homogenate was subjected to a low-speed spin (2,400 g, 4°C, 10 min), and the resultant supernatant was then subjected to a high-speed spin (30,000 g, 60 min, 4°C). The pellet from this final spin was resuspended in TBS with protease inhibitors (40 µg/ml PMSF, 0.5 µg/ml leupeptin, and 0.7 µg/ml pepstatin) and stored at –20°C. Protein concentration was determined by Lowry assay.
Mouse kidney membranes were prepared in a similar fashion except that both cortex and medulla were included in the homogenate. The mice used included three NHE3-null mice (18) on a Black Swiss background (25), as well as three age-matched Black Swiss wild-type controls.
SDS-PAGE and immunoblotting. Cells or membranes were solubilized in Laemmli sample buffer, and proteins were separated by SDS-PAGE using 7.5% gels. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon) from polyacrylamide gels (350 mA for 1 h at 4°C using the Bio-Rad MINIPROTEAN3 electrophoresis system). Immunoblotting was performed as follows. Polyvinylidene difluoride membranes were incubated first in Blotto (5% nonfat dry milk, 0.1% Tween 20 in PBS, pH 7.4) for 1 h at 25°C to block nonspecific protein binding, followed by an overnight incubation in the primary antibody diluted in Blotto. After four washes with Blotto, the membrane was incubated with the appropriate HRP-labeled secondary antibody, diluted in Blotto, for 1 h at 25°C. After four washes with Blotto and two washes with PBS, bound antibody was detected with the enhanced chemiluminescence system (Amersham Biosciences).
Primary antibodies used include the following. Anti-NHE8 monoclonal antibody 7A11 (1 mg/ml purified stock) was used on immunoblots of kidney membranes (1:1,000 dilution) or on transfected cells (1:2,000 dilution). Polyclonal anti-NHE8 antibody Rab65 (1 mg/ml purified stock) was used on immunoblots of kidney membranes (1:2,000 dilution) or transfected cells (1:16,000 dilution). Monoclonal anti-NHE3 antibody 3H3 (a kind gift from Dr. Daniel Biemesderfer, Yale University) was used on immunoblots of kidney membranes (1:1,000 dilution) or transfected cells (1:1,000 dilution).
Blocking studies were performed as described previously (8). In particular, the primary antibody was incubated with the blocking peptide (3 µg blocking peptide/µl of antibody) in an overnight incubation (4°C in TBS buffer). This mixture was then used at the appropriate dilution for subsequent immunoblotting or immunofluorescence experiments.
Tissue and cell preparation for immunohistocytochemistry.
Adult Sprague-Dawley rats were anesthetized with pentobarbital sodium. The kidneys were cleared (PBS) and then fixed (modified high-osmolar PLP fixative: 2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate, 750 mM sucrose in phosphate buffer, pH 7.4) via distal aortic perfusion of renal arteries (19, 21). This fixative was modified from the standard PLP fixative in that it included 750 mM sucrose. The sucrose increased effective osmolarity of the perfusate from 300 to 1,050 mosmol/kgH2O, thereby allowing better architectural preservation of the hypertonic medullary interstitium. Kidneys were removed, cut into 1- to 2-mm-thick blocks from representative cortical, medullary, and papillary sections, and postfixed in the same fixative for 4 h. In preparation for embedding, blocks were washed with TBS, dehydrated in a series of ethanol incubations, washed with propylene oxide, and finally embedded in EMbed 812 (Electron Microscopy Sciences, Fort Washington, PA) in an overnight incubation at 60°C (20).
In preparation for immunofluorescence experiments, 1- to 2-µm-thick sections were cut from the EMbed-embedded block using a Reichert Ultracut E ultramicrotome, mounted on Superfrost Plus glass slides (Electron Microscopy Sciences), and etched for 5 min in a solution containing 2 g KOH, 10 ml 100% methanol, and 5 ml propylene oxide.
For immunofluorescence experiments in transfected cells, COS-7 cells grown on glass coverslips were simply incubated in a standard PLP fixative (2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate, in phosphate buffer, pH 7.4) for 1 h and then stored in TBS.
Indirect immunofluorescence microscopy. Tissue sections were washed with TBS, followed by 500 mM ammonium chloride, followed again by TBS. Sections were then incubated with a blocking solution (TBS, 0.1% BSA, 10% goat serum) for 15 min and then incubated with the primary antibody (diluted in blocking solution) for 1 h. After being washed with a high-salt wash solution (TBS containing 2.5% NaCl instead of the typical 0.9% NaCl, 0.1% BSA), the sections were incubated with the appropriate FITC or rhodomine-labeled secondary antibody for 1 h. After additional high-salt washes followed by a final TBS wash, sections were mounted in VectaShield (Vector Laboratories) and then visualized with a Zeiss Axiophot microscope (20).
The only major difference in the performance of immunofluorescence on cells instead of tissue was that, before the sections were incubated with the blocking solution, the cells were incubated in 0.1% Triton X-100 for 5 min to ensure permeabilization. Following TBS washes, cells were then incubated in blocking solution as above.
Antibody dilutions were as follows: anti-NHE8 mAb 7A11 (tissue 1:5, cells 1:100); anti-NHE8 polyclonal antibody Rab65 (tissue 1:2, cells 1:10); and anti-NHE3 mAb 2B9 [Chemicon (1), cells 1:1,000]. For double-labeling of NHE8 with a known brush-border marker, a rabbit anti--glutamyltranspeptidase antibody (1) (a kind gift from Dr. David Castle, University of Virginia) was utilized (1:1,000 dilution). For double-labeling of NHE8 with a marker for the coated pits and dense subapical tubules, a rabbit anti-megalin antibody (anti-MC-220) (27) (a kind gift from Dr. Daniel Biemesderfer) was utilized (1:500 dilution).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To demonstrate NHE8 reactivity, mAb 7A11 and polyclonal antibody Rab65 were used to probe immunoblots prepared from NHE8-transfected COS-7 cells. The presence of the expected band in NHE8-transfected COS-7 cells, but its absence in untransfected controls, confirmed NHE8 reactivity by Western blot analysis (Fig. 2A). Similarly, the antibodies were used to probe coverslips prepared with PLP-fixed NHE8-transfected COS-7 cells. A fluorescence signal in NHE8-transfected cells (Fig. 2B) but not untransfected controls (not shown) confirmed NHE8 reactivity by immunofluorescence microscopy.
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In native rat kidney membranes, both 7A11 and Rab65 labeled a protein the size of mature NHE8 (Fig. 3). The signal mediated by Rab65 was blocked by its peptide (NHE8.472.pep) but not by the MBP.NHE8.t89 fusion protein. Conversely, the signal mediated by the 7A11 antibody was blocked by the fusion protein against which it was generated (MBP.NHE8.t89) but not by the NHE8.472.pep peptide. These blocking studies demonstrate that the antibodies recognize different NHE8 epitopes. Since antibodies directed against two different epitopes of NHE8 labeled the same apparent protein on Western blot analysis, the specificity of the observed signal was confirmed.
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Having verified their specificity, these new NHE8 antibodies were used to localize NHE8 protein in native rat kidney by immunofluorescence microscopy. Cortical sections were labeled with Rab65 (Fig. 4A) or 7A11 (Fig. 4B). Both antibodies gave the same immunolabeling pattern, namely, brush-border staining of all proximal tubules. No staining of other tubular segments was detected. Since we had shown that these antibodies react with different epitopes on NHE8, the observation that both antibodies show the same brush-border staining makes it extremely unlikely that this staining results from cross-reactivity with other proteins. Moreover, as would be expected, the 7A11 signal was blocked with MBP.NHE8.t89 fusion protein, whereas the Rab65 signal was blocked with the NHE8.472.pep peptide (not shown). Since the 7A11 antibody produced the stronger immunofluorescence signal with less background, all further immunlocalization studies were performed using this antibody.
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The localization pattern of NHE8 in the rat medulla is depicted in Fig. 5. Intense NHE8 staining was evident in proximal tubules (S3) within the outer stripe of the outer medulla. The staining extends until the point where the S3 segment transitions into the thin limb. These transitions, in which half of the tubular cross section contains a brush border (which labels for NHE8) while the other half has the morphology of thin limbs (which does not label for NHE8), are evident in the deeper portions of the outer stripe (Fig. 5, B and C). Again, no other tubules labeled for NHE8 in the outer stripe. No proximal tubules are present in the inner stripe of the outer medulla, and, as would be expected, no NHE8 staining was present in this region. Additionally, no NHE8 protein was evident in the inner medulla (not shown).
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-glutamyl transpeptidase (GGT) (Fig. 6, A–D). Essentially, a complete overlapping of expression of the two proteins was evident (yellow fluorescence in Fig. 6C), thereby confirming brush-border localization of NHE8. Second, NHE8 was colocalized with anti-MC-220, an antibody raised against the cytoplasmic domain of megalin (Fig. 6, E–H). This antibody specifically labels a pool of megalin that is present in the intermicrovillar coated pits and subapical dense tubules in proximal tubule cells but does not label microvillar megalin (27). It also labels megalin in an intracellular membrane compartment (27), as evidenced by the small punctate areas of green fluorescence in Fig. 6, F and G. The bulk of the NHE8 staining was luminal to the megalin staining, indicating microvillar localization (Fig. 6, E and G). In addition, there was colocalization of NHE8 with megalin (yellow fluorescence in Fig. 6G), suggesting that some NHE8 is expressed in the coated pits and/or subapical tubules. Although deeper intracellular staining for NHE8 was not consistently detected with certainty, such negative findings do not exclude the possible existence of an intracellular store of NHE8 deeper within the cell that is below the level of detection.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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These findings correlate with results from a previous study, in which we used in situ hybridization to ascertain the localization of NHE8 in mouse kidney (6). In that study, NHE8 message was present in proximal tubules within the outer stripe of the outer medulla as well as a lower but significant expression diffusely throughout the cortex. Although the differential expression of NHE8 between the cortex and medulla appeared to be more pronounced than in the present study, the same tendency for higher expression in the deeper cortex/medulla was observed. Differences in technique (in situ hybridization of mRNA vs. antibody immunolocalization of NHE8 protein) or species (mouse vs. rat) may account for the observed variability. Most importantly, the present antibody-based immunolocalization results provide strong and direct evidence for the apical plasma membrane localization of NHE8 protein in proximal tubules.
Unfortunately, the function of NHE8 remains elusive. Given that NHE8 shares significant sequence identity with other members of the sodium/hydrogen exchanger family, one would predict that it mediates cation/hydrogen exchange. To date, however, we have been unable to detect ion transport activity mediated by NHE8 in functional expression studies using transfected cell systems. However, such negative studies do not preclude a role for NHE8 in mediating ion transport when expressed endogenously in an appropriate regulatory context. Thus the localization of NHE8 to the apical membrane of proximal tubule cells raises the possibility that it may indeed play a role in mediating or regulating ion transport in this nephron segment. If NHE8 does play a role in mediating ion transport in the proximal tubule, it is noteworthy that we did not detect a significant alteration in the expression of NHE8 in NHE3-null mice.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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