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Am J Physiol Cell Physiol 273: C1496-C1505, 1997;
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Vol. 273, Issue 5, C1496-C1505, November 1997

Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney

Crescence Bookstein1, Yue Xie1, Karen Rabenau1, Mark W. Musch1, Rebbecca L. McSwine1, Mrinalini C. Rao2, and Eugene B. Chang1

1 Section of Gastroenterology, Department of Medicine, University of Chicago, Chicago 60637; and 2 Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We present evidence that tissue distribution of two highly conserved Na+/H+ exchanger isoforms, NHE2 and NHE4, differs significantly from previously published reports. Riboprobes unique to each of these antiporters, from 5' (noncoding and coding) and 3' coding regions, were used to analyze mRNA from adult rat kidney and intestine by ribonuclease protection assay and in situ hybridization. In contrast to earlier work that concluded that both NHE2 and NHE4 were expressed throughout the intestine and in the kidney, our data show that there is no NHE2 message in the kidney and NHE4 is not expressed in small or large intestine. Analyses of intestinal epithelial and kidney membrane proteins by an NHE2-specific antibody identified a doublet at <90 kDa in intestine but not in kidney. NHE2 is highly expressed in the Na+-absorptive epithelium of jejunum, ileum, and ascending and descending colon. NHE4 mRNA message is found in the inner medulla of the kidney as previously reported (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao, and E. B. Chang. J. Biol. Chem. 269: 29704-29709, 1994) and not in the intestine. From these data, we speculate that neither NHE2 nor NHE4 has a role in renal Na+ absorption. NHE2 is likely involved in gut Na+ absorption, whereas NHE4 may have a specialized role in cell volume rectification of inner medullary collecting duct cells. Knowledge of the correct tissue and cell-specific distribution of these two antiporters should help significantly in understanding their physiological roles.

sodium/proton exchanger; ion transport

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

SODIUM/PROTON EXCHANGER activity, the electroneutral influx of one Na+ accompanied by efflux of one H+, appears in nearly every cell from bacteria to mammals. Cloning of the ubiquitously expressed regulator of intracellular pH (pHi) and cell volume, the Na+/H+ exchanger NHE1 (18), led to identification of a family of NHE isoforms (5, 11, 15, 21, 23). All have similar structural organization, with 10 or 12 transmembrane domains and a large cytosolic carboxy domain. The greatest conservation of amino acid residues occurs within the membranous region, which is believed to be all that is required for Na+/H+ exchange activity (24). The soluble carboxy-terminal domain, with the most divergent sequences, consists of regulatory elements. Of the five rat NHE isoforms cloned thus far, little is known about the two that are most closely related: NHE2 and NHE4.

This study was undertaken in response to discrepancies we encountered in determining expression patterns of NHE4 mRNA. Initial studies describing the tissue distribution of NHE2 and NHE4 in rat were largely restricted to Northern blot analyses using full- or nearly full-length cDNA probes (5, 15, 25). Our preliminary efforts to determine by in situ hybridization the patterns of expression of NHE4 mRNA in specific cell types and under different conditions in vivo, using a small isoform-specific 5'-end probe, led to the suspicion that our interpretation of the Northern data might be inaccurate. We detected NHE4 mRNA in stomach epithelium, kidney inner medulla, and hippocampal cavi amnoni fields 1-4; however, contrary to published studies, we could detect no expression in intestine. This observation led us to a systematic and rigorous examination of the expression patterns of both NHE2 and NHE4, the results of which are presented here. We have used two highly specific approaches: ribonuclease protection assay (RPA) and in situ hybridization, each employing unique riboprobes to unambiguous regions of NHE2 and NHE4 to define the tissue-specific expression of their mRNA. In addition, we have examined the expression of NHE2 protein by an isoform-specific antibody. We present evidence, contrary to previous reports, that the rat isoform NHE4 is not expressed in rat intestine and that NHE2 is not expressed in rat kidney.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Animals. Sprague-Dawley rats, 180-200 g, were obtained from Harlan Animal Supply. Animals were killed by injection with 0.5 ml ketamine and 0.3 ml Nembutal. Tissues were removed and processed immediately or frozen in liquid nitrogen. Epithelial cells from stomach and intestine were removed by cutting lengthwise and gently scraping with a microscope slide.

Cells. NHE-deficient Chinese hamster lung fibroblasts (PS120 cell line) were obtained from J. Pouyssegur (17). Rat full-length NHE2 and NHE4 cDNA were obtained from G. E. Shull (15, 25). Both cDNAs were subcloned into the expression vector pCB6+ (courtesy of J. Stinski) on which the cytomegalovirus (CMV) promoter drives expression of the inserted DNA and carries the eucaryotic resistance gene for gentamycin/neomycin resistance. Transfection and verification of the PS120/NHE4 clones by Northern and Western analyses have been described elsewhere (3). Xba I and Kpn I restriction sites were added to the NHE2 cDNA at base 51 to facilitate subcloning. The pCN2 construct (pCB6 + NHE2 cDNA) carried the full-length cDNA from Kpn I (base 52) to 3689 and was transfected into PS120 cells by Lipofectin (Life Technologies) and selected by resistance to the G418 antibiotic (Life Technologies) and repeated acid loads by the method of Franchi et al. (6). Clonal populations were then analyzed for NHE2 mRNA with the use of isoform-specific probes and with Western blot using NHE2-specific antibodies.

Development of antibodies to NHE2. A glutathione-S-transferase (GST) protein fusion to NHE2 cDNA (GST-NHE2) corresponding to amino acids 260-280 was constructed via polymerase chain reaction (PCR) and subcloning into pGEX-KT. This region was chosen as one of three with no homology to other cloned NHEs; notably, it shows no homology with closely related NHE4. An in-frame, correctly copied sequence was confirmed by Sanger dideoxy sequencing. This protein was induced and purified according to protocol of Pharmacia, with modifications to maximize yield of intact protein. Purified fusion protein was then used to inoculate rabbits. Pre- and postimmune sera were tested on PS120, PS120/NHE1, PS120/NHE2, PS120/NHE3, and PS120/NHE4 membranes. Postimmune sera cross-reacted with two <90-kDa bands in the PS120/NHE2 cells only.

RNA isolation. Epithelial scrapings from small and large intestine and stomach were immediately suspended in GTC lysis buffer [4 M guanidinium thiocyanate in 100 mM tris(hydroxymethyl)aminomethane (Tris)-chloride (pH 7.5), 1% vol/vol beta -mercaptoethanol, 0.5% wt/vol sodium lauryl sarcosinate, and 0.07% vol/vol Anti-Foam A]. Whole organ samples from kidney, brain, and liver were quickly frozen in liquid nitrogen, pulverized, and suspended in GTC lysis buffer. Cells were disrupted at high speed by Ultra-Turax for 60 s and immediately frozen in a dry ice-isopropanol bath. Total RNA was purified by centrifugation through a CsCl cushion (7).

Northern blot analysis. Polyadenylated mRNA was recovered on immobilized oligo(dT) (14). The flow-through fraction, nonpolyadenylated and ribosomal RNA, was included on the gel to serve as control for hybridization specificity. RNA markers (Novagen, Madison, WI) served as molecular weight size standards. The RNA was transferred to Hybond-N (Amersham, Arlington Heights, IL) by capillary action in 20× SSC (3 M NaCl and 300 mM sodium citrate, pH 7); the RNA cross-linked to the membrane by ultraviolet light (Stratalinker, Stratagene, La Jolla, CA). cDNA probes (as gel purified inserts) were labeled by incorporation of [alpha -32P]dCTP by random decamer primers (Ambion, Austin, TX). After unincorporated nucleotides were removed (Pharmacia nick column, Pharmacia LKB Biotech, Piscataway, NJ), 105-107 counts/min (cpm) of probe per milliliter of hybridization buffer were added to the prehybridized membrane. The blots were prehybridized (5 min to overnight) and hybridized for 16-20 h at 55°C in 10 mM EDTA, 200 mM NaH2PO4 (pH 7), 7% wt/vol sodium dodecyl sulfate (SDS), 1% wt/vol bovine serum albumin (BSA), and 15% vol/vol deionized formamide (50 µl/cm2 membrane). Wash times, at 65°C, were as follows: 15 min in 2× SSC (twice), 30 min in 2× SSC + 0.1% SDS, and 10 min in 0.1× SSPE (18 mM NaCl, 1 mM NaPO4, pH 7.7, and 0.1 mM EDTA) + 0.5% SDS. The final wash time was adjusted as necessary to reduce background. Equal loading was determined by spectrophotometric quantitation of total RNA and monitored by ethidium staining of the gel before transfer (via residual ribosomal bands). Integrity of the mRNA was verified by hybridization with glyceraldehyde phosphate dehydrogenase cDNA. If they were to be reprobed, blots were stripped by incubating in boiling water until all trace of radioactivity was removed, as determined by a hand-held Geiger counter. Removal of probe was verified by 2-day exposure to film.

Preparation of riboprobes for RPA. The RNA probes (Table 1) were prepared by incorporating [32P]UTP (800 Ci/mmol; NEN) in transcripts generated by T7 or SP6 RNA polymerase (according to instructions) with an Ambion MAXIscript kit from linearized, buffer-saturated phenol-chloroform-extracted and ethanol-precipitated CsCl-purified plasmid DNA. The DNA templates were digested by ribonuclease (RNase)-free deoxyribonuclease I (Ambion), and full-length riboprobes were gel purified from 5% polyacrylamide before hybridization. Hybridization reactions included aliquots of probe and tRNA; one-half was RNase A and T1 digested, the other one-half was not. Both undigested hybridized probe and digested hybridized probe reactions were always included with the experimental reactions in the RPA gels as controls for many aspects of these experiments. Also included as controls of specificity were NHE-negative PS120 fibroblasts (17) that had been transfected with NHE2 or NHE4 cDNA, under the control of a CMV promoter.

                              
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  		Table 1.   Construction of unique cRNA probes

RPA. For each experimental condition, total RNA was coprecipitated with 1-5 × 105 cpm of the 32P-labeled, isoform-specific riboprobe in a total volume of 20 µl Ambion hybridization buffer [80% deionized formamide, 100 mM sodium citrate (pH 6.4), 300 mM sodium acetate (pH 6.4), and 1 mM EDTA]. Sample RNA and riboprobe were heat denatured at 90°C for 4 min, briefly centrifuged, and allowed to anneal for 18-20 h at 45°C. All remaining single-stranded RNA was then digested with the addition of RNase A and RNase T1 in buffer provided with the Ambion RPA II kit. Protected fragments were visualized by autoradiography after separation on a 5% polyacrylamide-8 M urea gel. Controls were included to demonstrate complete digestion of unhybridized probe. RNA probes for rat beta -actin (Ambion) were included in each sample to control for uniform loading as well as RNA integrity.

Preparation of tissue sections for in situ hybridization. Tissues were mounted in OCT freezing compound (Baxter) and frozen in liquid nitrogen immediately after harvesting. The samples were then sectioned (10 µm) and adhered to gelatin-subbed (EM Sciences) and poly-L-lysine-coated slides (Sigma). Mounted tissue was then fixed with ice-cold 4% paraformaldehyde in 1× phosphate-buffered saline (pH 7.4) for 2 min and ice-cold 70% ethanol for 10 min. Rehydration of the slides consisted of a series of 15-s incubations in ethanol at 70, 50, and 30% and finally incubation in diethyl pyrocarbonate-treated water. Equilibration in 0.1 M triethanolamine (Sigma), pH 8.0, for 15 s and a 10-min incubation in 0.1 M tetraethylammonium and 0.25% acetic anhydride allowed for blocking of positive charges. After a 15-s rinse in water and dehydration of the slides successively in 60 and 80% ethanol (15 s each) and two changes of 100% ethanol (2 min each), the slides were ready for hybridization with the probe.

Preparation of riboprobes for in situ hybridization. Sense and antisense strand riboprobes were generated from freshly linearized and gel-purified pGEM7Z plasmid (Promega) containing the NHE2 and NHE4 sequences. In a transcription reaction of 10 µl, final concentrations of [alpha -33P]UTP (NEN) did not fall below 12 µM and no cold UTP was added. After 2 h at 37°C, enzymes were heat inactivated at 65°C for 10 min and 50 µl of 1% SDS, 10 mM Tris-Cl (pH 7.4), and 1 mM EDTA were added. For in situ hybridization, the entire reaction was loaded onto a Sephadex G-50 column (Boehringer Mannheim Biochemicals) and purified. Probes larger than 400 base pairs (bp) were hydrolyzed in 60 mM Na2CO3 and 40 mM NaHCO3 (pH 10.2) for 40-60 min at 60°C, precipitated, and resuspended in 10 mM Tris-Cl (pH 7.4) and 1 mM EDTA. Probes were added to the hybridization fluid to an average final concentration of 1 × 1087 cpm/ml. Hybridization solution contained a final concentration of 50% formamide (Ambion), 2 µg/ml nuclease-free BSA (Boehringer Mannheim), 1 µg/ml tRNA (Boehringer Mannheim), 1 µg/ml salmon sperm DNA (GIBCO BRL), and 2× SSC.

In situ hybridization. Prepared slides were hybridized overnight at 55°C on a prewarmed slide warmer. Hybridization solution was added to cover the section (35 µl), and 22 × 40 mm coverslips were placed over the sections and sealed with Gallard-Schleisenger DPX mountant (BDH Supplies). After overnight hybridization, the DPX was carefully removed and the coverslips were removed by soaking in 2× SSC. Slides were then added to fresh 2× SSC and incubated for 30 min at 55°C. For digestion of single-stranded RNA, slides were incubated at 37°C in 50 µg/ml RNase A, 500 mM NaCl, 1 mM EDTA (pH 8.0), and 10 mM Tris-Cl (pH 8.0) for 1 h. Serial high-stringency washes in 2, 1, 0.5, and 0.1× SSC (30 min each, 55°C) were followed by dehydration in 50, 70, 95, and 100% ethanol. Slides were then exposed for autoradiography (Amersham beta max) to estimate strength of signal and determine subsequent incubation time in autoradiographic emulsion (Kodak NTB-2). Duplicate slides were processed, and the first set was developed 4 days to 1 wk later (based on film exposure) in Kodak D-19 developer for 4 min, water for 15 s, and Kodak fixer for 2 min, each at 14°C. Slides were then rinsed for 30 min with running distilled water, dehydrated, cleared in xylene, and then mounted with Permount (Fisher). Slides were viewed by dark field to visualize silver grains appearing over regions of probe hybridization and bright field for morphology.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Northern blot ambiguity. Because the physiological function of each exchanger is inferred in part from tissue and cellular localization, the need for accuracy of this information cannot be minimized. Figure 1 shows the results of sequential hybridization of the same membrane with NHE4 cDNA first, stripping and then hybridizing with NHE2 cDNA. Lanes 1-5 (Fig. 1) contain mRNA from jejunum (villus to crypt enterocyte fractions); lanes 6-10 contain mRNA from ileum (villus to crypt). Lanes 11 and 12 contain mRNA from proximal and distal colon scrapings. We used the random decamer-primed NHE2 cDNA Pst I fragment as the probe (Fig. 1, top), nucleotides 260-3598. This region corresponds to amino acids 26-717 and includes 970 bp of the 3' untranslated region. We also used as probe the complete NHE4 cDNA (Fig. 1, bottom). The Northern blots in Fig. 1 are representative of several different experiments. We used the same two probes on different blots in the reverse order (i.e., NHE2 cDNA probes first, then stripped and reprobed with NHE4 cDNA), or simultaneously, by dividing the mRNA from each tissue into two parallel blots and probing one-half with NHE2 cDNA and the other one-half with NHE4. In every Northern blot, we found that the two isoforms exhibited essentially identical expression patterns. Both appeared to have similar relative intensities in corresponding intestinal fractions, and both probes identified doublet bands at 4.2-4.4 kilobases (kb) and a faint secondary band at ~3 kb. These findings were in agreement with previously published reports from other laboratories (5, 15, 25).


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  		Fig. 1.   Northern blot analysis of expression of Na+/H+ exchanger (NHE) isoforms NHE2 and NHE4 throughout rat intestine. mRNA (5 µg) was separated by electrophoresis, transferred to nylon membrane, and probed with gel-purified cDNA inserts, radioactively labeled by random primer and then column purified. Lanes 1-5 are from enterocytes isolated as sequential Weizer fractions (villus to crypt) from rat jejunum. Lanes 6-10 are from similarly fractionated ileum. Lane 11: mRNA from proximal colon scrapings. Lane 12: mRNA from distal colon. At top, probe was NHE2, nucleotides 260-3598 (stripped after hybridization with NHE4, shown at bottom, with stripping of all probe verified by autoradiography). At bottom, same blot is shown when probed with NHE4. Arrows indicate the position of a less-intense 3-kilobase (kb) band frequently seen with these 2 probes.

Because these findings suggested that cross hybridization of the two cDNAs might be occurring, we began to look more closely at the sequences of the two antiporters. These two isoforms share considerable amino acid sequence identity, probably indicative of gene duplication. Not surprisingly, the nucleotide sequences are also very similar. There are a number of identical stretches, with at least two being of sufficient length to possibly allow cross hybridization of the cDNA probes used for Northern analyses. Because we felt that the Northern data might be misleading, we decided to revisit the issue of tissue- and cell-specific expression, particularly in the intestine and the kidney.

Northern blot analysis using isoform-unique riboprobes. A more detailed analysis of the tissue distribution of these two isoforms required the design of cRNA probes that excluded any possibility of cross hybridization. For this purpose, we subcloned unique 5' sequences as well as 3' probes that included DNA encoding the singular carboxy-terminal tails of these two isoforms (as detailed in Table 1). No probe included significant sequence similarity to hybridize to any region of the other isoform. For NHE2, two small probes were made. The 2N probe includes all of the 5' cloned cDNA to the EcoR I site at base 726 and encompasses codons for the first 180 amino acids of this protein. The 2C probe covers bases 2210-2630, with oversequence encoding the unique final amino acids as well as the 3' noncoding sequence. Three probes were made for NHE4. The 4Na probe covers bases 42-636 (5' untranslated sequence and the first 56 codons of the protein). The 4Nb probe is immediately distal, from base 637 to 964, and includes the next 109 codons. The latter probe includes a region conserved at the protein level but not as highly conserved at the DNA level. At the 3' end, the 4C probe extends from base 2456 to 2755, an entirely unique sequence covering the final 53 codons. In contrast to the results of Northern analysis using full-length probes (Fig. 1), when we used isoform-specific probes on rat intestinal epithelial and kidney mRNA (Fig. 2), we were unable to demonstrate significant NHE4 mRNA expression in the intestine or NHE2 expression in the kidney. The conditions used on the latter Northern blots were identical to those used for the first, and apparently ambiguous, Northern blots. These data suggested that NHE4 was not an intestinal NHE, whereas NHE2 was abundantly expressed throughout intestinal epithelium.


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  		Fig. 2.   Northern blot analysis of NHE2 and NHE4 expression using small isoform-specific probes. Identical blots were prepared, hybridized, and washed exactly as described for Northern blots in Fig. 1. J, I, and C, lanes containing epithelial scrapings from jejunum, ileum, and colon, respectively. Km and Kc, lanes containing kidney medulla mRNA and kidney cortex mRNA, respectively. Probes were radioactively labeled by random primer (Ambion) from gel-purified cDNA. Similar specific activity was added to each blot, and autoradiographs were exposed for the same length of time (24 h). Each lane was loaded with 5 µg of mRNA. Probe used is indicated at top. Bands have identical mobility to the bands in Fig. 1. Appearance of a band in colon with 4Na probe may result from overflow from the Km lane; it was not a consistent finding. The 2C blot shows a similar type of spillover in the lane next to jejunum that actually contained no mRNA. After further examination with more sensitive detection methods, we could not confirm any NHE4 expression in the colon. The very abundant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message for each lane was exposed only long enough to show relative quantities of mRNA and does not reflect the length of exposure time for the NHE4- and NHE2-probed Northern blots.

RPA. To more thoroughly examine the patterns of tissue-specific expression for each isoform, we utilized two potentially more sensitive methods: RPA and in situ hybridization of tissue sections. The advantages of RPA over Northern analysis to analyze expression of the different isoforms include the following reasons: 1) the probes can be designed with absolute lack of ambiguity, 2) low-abundance transcripts are readily detected from relatively small tissue samples, 3) the sequence of the target is known, and we can predict the exact size of the RNase-protected hybrid, and 4) any differences in sequence internal to the probe are digested, yielding smaller fragments. This assay employs RNase A, which digests all single-stranded RNA 3' to cytosine and uridine residues, and T1, which cuts 3' to guanosine. The combination effectively and efficiently digests all single-stranded, nonhybridized RNA to single nucleotides or very small fragments. Tissues were separated, and total RNA was isolated and purified as described in METHODS. Probes were gel purified before hybridization, and samples were included on each gel with and without RNase digestion as controls for the reaction conditions, as well as comparative size markers. Rat beta -actin probe (which protects a doublet) was included in each reaction to monitor the integrity of the RNA. As a control for nonspecific hybridization, the sense strand for each probe was hybridized and digested exactly as described for the antisense probes. No double-stranded product was protected by the sense strand of any of the probes used (not shown). Figure 3 shows the results of RPA on rat tissues and transfected fibroblasts with the antisense probes as described. With the use of the 2C probe (Fig. 3A), the predicted size fragment (based on cDNA sequence) appeared in RNA from stomach, duodenum, jejunum, ileum, and colon. In contrast, the corresponding 4C probe (Fig. 3B) protected its complement mRNA transcript only in stomach and the PS120/NHE4 transfectants. No transcript was detected in duodenum, jejunum, ileum, or colon. The 5'-end 2N probe (Fig. 3C) protected mRNA in the same tissues as the 2C probe (stomach, duodenum, jejunum, ileum, and colon), but no transcript was detected in RNA from kidney. In contrast, the 4Nb probe (Fig. 3D) protected RNA fragments in stomach, kidney, and brain. As with the 4C probe, protected fragments were not recovered from jejunum, ileum, or colon. This was true for all of the NHE4 probes at up to 100 µg of RNA per lane (data not shown).


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  		Fig. 3.   Ribonuclease protection assays. A: probe 2C. RNA from epithelial scrapings of stomach (S), duodenum (D), jejunum (J), ileum (I) (20 µg of RNA for each), and colon (C; 40 µg) were used. B: probe 4C. RNA from transfected PS120 fibroblasts (PS/NHE4, 2.5 µg RNA), duodenum and stomach (20 µg for each), and jejunum, ileum, and colon (40 µg for each) were used. C: probe 2N. RNA from stomach, duodenum, jejunum, ileum (20 µg for each), and colon (40 µg) were used. D: probe 4Nb. RNA from stomach and duodenum (20 µg for each), jejunum, ileum, and colon (40 µg for each), and whole kidney (K) and brain (B) (40 µg for each) were used. Bands in kidney and brain, which appear to run higher because of the "smile" in the gel, are the same size as the stomach fragment. In A-D, lower doublet bands (126-130 base pairs) are rat beta -actin-protected hybrids, included as a control to show the presence of RNA in lanes in which NHE messages were absent. Relative amounts of beta -actin are not strictly comparable from tissue to tissue; A-D are also not comparable, since each was exposed for different lengths of time.

In situ localization of NHE2 and NHE4 mRNA. Further analyses of the distribution of mRNA species were made via in situ hybridization of tissue sections. Whenever the double-stranded probe-mRNA hybrid survived single-stranded RNA digestion, the radioactive nucleotides incorporated in the cRNA probe reacted with a photographic emulsion, causing the precipitation of silver grains (which by dark-field microscopy appear as bright spots on a black background). These images identify those cells that are actively transcribing the target mRNA. Representative images taken of NHE2-probed intestinal tissue sections are shown in Fig. 4. Dark-field images of jejunum (Fig. 4A) and ileum (Fig. 4C) show the signal concentrated in the villous cells. Relatively little signal appears in the crypt regions. (The corresponding bright-field, unstained image of each tissue is shown in Fig. 4, B and D.) The 2N probe signal in proximal and distal colon (Fig. 4, E and G) was similarly concentrated in the absorptive epithelial cells. Intestinal tissue sections from the same animals were probed with NHE4 cRNA, and one representative image (proximal colon probed with 4C) is shown in Fig. 4, I and J. As in the analogous RPA, no NHE4-specific signal was detected. No NHE4 message, with the use of any of its three probes, was observed in small or large intestine by in situ hybridization.


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  		Fig. 4.   In situ hybridization of rat intestinal tissue with NHE2 and NHE4 cRNA probes. A, C, E, G, and I: dark-field images of 10-µm-thick sections. Radioactive signal of the antisense cRNA-protected RNA hybrids is seen as white against the black background (×100 magnification, Zeiss Axiophot). B, D, F, H, and J: corresponding images under bright field (same magnification, same image field). Tissues were not stained. S, serosa; L, lumen. A and B: jejunum, probe 2N. C and D: ileum, probe 2N. E and F: proximal colon, probe 2N. G and H: distal colon, probe 2N. I and J: proximal colon, probe 4C.

Localization of NHE2 and NHE4 mRNA in kidney. Using much greater quantities of kidney RNA, we reanalyzed this tissue for NHE2 and NHE4 expression by RPA (Fig. 5). With up to 100 µg of RNA, we could detect no message in kidney with the 2N probe. In comparison, NHE2 message was abundant in 20 µg of jejunum RNA. In contrast, both 4Na and 4Nb probes (Fig. 5, C and D) detected NHE4 transcript with 50 µg of kidney RNA (probe 4C also detected the NHE4 transcript, not shown). After extended exposure of the RPA autoradiograph shown in Fig. 5B, a very faint band corresponding to riboprobe 2C appeared in the kidney cortex lane. This was not confirmed by Northern analysis (Fig. 2), in situ hybridization of whole kidney (Fig. 6), or Western analysis (Fig. 7). However, we cannot distinguish among adrenal gland contamination or spillover from colon at these concentrations or extremely low expression of a transcript with sequence identical to 2C but not containing the corresponding 5' region included in probe 2N.


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  		Fig. 5.   Ribonuclease protection assays of rat kidney RNA. A: probe 2N. RNA from jejunum (20 µg) and kidney (50 and 100 µg) were used. B: probe 2C, 7-day exposure. RNA from PS/NHE4 (2.5 µg), stomach (20 and 50 µg), duodenum (50 and 20 µg), jejunum (20 and 50 µg), ileum (20 and 50 µg), colon (20 and 50 µg), and kidney cortex (50 µg) were used. C: probe 4Na. RNA from stomach and duodenum (20 µg each) and whole kidney (50 and 100 µg) were used. D: probe 4Nb. RNA from PS/NHE4 (2.5 µg), stomach (20 µg), and whole kidney (10, 50, and 100 µg) were used. [Note the faint NHE2 mRNA band in kidney (B) with 7-day exposure compared with relative abundance of NHE2 message in intestinal epithelial RNA and in kidney with NHE4 probes in C and D (both 24-h exposures).]


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  		Fig. 6.   Autoradiographs of rat kidney in situ hybridizations. Sequential sections of rat kidney, prepared as described in METHODS, were probed with antisense or sense cRNA probes. A: 4C antisense probe. Tubules expressing NHE4 mRNA are marked by dark black lines in collecting tubules in the medulla and scattered patches along the outer rim of the cortex. B: 4C sense strand, which shows no specific signal. C: 2N antisense probe. D: 2N sense probe. E: 2C antisense probe. F: 2C sense probe. Tissues were not counterstained.


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  		Fig. 7.   Western blot analysis of NHE2 protein. A: each lane contains ~40 µg of membrane proteins; from left, PS120 cells, rat ileum brush border (Il-BB), 4 lanes of membranes primarily from kidney cortex (BL, basolateral). Positions of the molecular mass markers are indicated at right. Arrow, position of the doublet at <90 kDa. B: PS120, PS/NHE1, and PS/NHE3 transfectants, ileum, and PS/NHE2 membranes (~40 µg each). C: competition by antigen. 2M5 immune serum was preincubated with 0, 75, and 150 µg/ml NHE2 2M5-glutathione-S-transferase fusion protein. Arrow, NHE2 doublet. (Two lower bands in the ileal membrane preparation also disappeared with the fusion protein competition and may represent proteolytic breakdown products.)

Analysis of NHE2 and NHE4 in kidney by in situ hybridization. If NHE2 kidney expression were limited to a subset of specialized tubules, we could expect to be at the limits of sensitivity with RPA, as we had found with NHE4 (3). We had previously demonstrated by in situ hybridization that NHE4 expression in the kidney was primarily in the inner medullary collecting tubules, representing a tiny fraction of the total kidney RNA. To clarify this issue by in situ hybridization, we used NHE2 and NHE4 probes on sequential slices of the same kidney (Fig. 6). As shown in Fig. 6A, the 4C antisense probe detected NHE4 mRNA in the inner medulla, consistent with our previous findings using the 4Na probe. The sense strand 4C (Fig. 6B) showed no specific signal. Neither antisense NHE2 probe 2N (Fig. 6C) nor 2C (Fig. 6E) detected NHE2 mRNA in any part of the rat kidney.

NHE2 protein in intestine and kidney. We next analyzed membrane proteins by Western blot to more thoroughly investigate the pattern of NHE2 expression. An antibody (2M5) was made to a GST-NHE2 fusion protein (amino acids 260-280, between membrane domains 5b and 6). This antibody cross-reacts to a doublet below 90 kDa in brush-border membranes from rat intestinal epithelial scrapings (Fig. 7, ileum brush border) and to PS120 cells transfected with rat NHE2 cDNA (Fig. 7B). These bands do not appear in PS120 membranes or in brush-border or basolateral membranes from kidney cortex. As shown in Fig. 7B, 2M5 antibody does not cross-react with membrane proteins in PS120, PS120/NHE1, or PS120/NHE3 transfectants (or PS120/NHE4 cells, not shown). Preimmune serum was negative for the specific bands shown (data not shown). Specificity of the 2M5 serum to these two brush-border proteins was further confirmed by preincubation with the fusion protein antigen. Cross-reaction to these two proteins in ileum brush-border membranes was competitively inhibited with increasing concentrations of the GST-NHE2 fusion protein (Fig. 7C). Using RPA, in situ hybridization, and Western analysis, we found NHE2 mRNA and protein in epithelial cells of the small and large intestine and none in the kidney. In contrast, NHE4 mRNA was found only in the kidney and stomach, with none being detected in the duodenum or intestine.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

NHE2 and NHE4 proteins are highly conserved. The amino acid sequences of NHE2 and NHE4 are 57% identical overall, with 63% identity in the membranous domains and 46% in the cytoplasmic domains. Expression studies of the cloned cDNAs in NHE-deficient Chinese hamster fibroblasts have demonstrated amiloride-sensitive exchange activity for NHE2 in response to increasing intracellular H+ concentration ([H+]i) and pharmacological kinetics that distinguish it from the other isoforms (9, 26). We have been able to demonstrate [H+]i-sensitive NHE4 activity, in transfected fibroblasts, only during hyperosmolar-induced cell shrinkage, and, under those conditions, its pharmacological and kinetic characteristics are distinctive (2, 3).

On the basis of chromosomal mapping, NHE2 and NHE4 are products of separate genes located on human chromosome 2 and rat chromosome 9 (20) and may represent recent gene duplication events. Nucleotide sequences of these two isoforms are very similar, with two highly conserved regions of 83% and 95% identity. We present evidence here that the full-length cDNA probes may cross hybridize. This is problematic and impacts on the validity of previous reports describing tissue localization as determined by Northern analysis, and, as a consequence, physiological roles ascribed to NHE2 and NHE4 may have been premature. On the basis of these previous studies, NHE2 was believed to be a major isoform of both kidney and gut (5, 15, 23, 25). Understanding the physiological role of cell volume-regulated NHE4 has also been hampered by the assumption that NHE4 is expressed in both kidney and gut. In view of the significant cross hybridization of these probes even under conditions of high stringency, we decided to analyze this issue in more depth. Accurate tissue and cellular localization is essential for understanding the physiological role of these two closely related NHE isoforms.

Previous analyses of mRNA from various rat tissues by Northern blot (5, 15, 25) produced an interpretation of NHE2 and NHE4 tissue-specific expression that conflicts with the data presented here. In those reports, the NHE2 message was found in greatest abundance in small intestine, colon, and stomach, with lesser amounts in skeletal muscle, kidney, brain, testis, uterus, heart, and lung. The NHE2 isoform cloned by Collins et al. (5) from rat [different in sequence from the NHE2 cloned by Wang et al. (25)] appeared to be most abundant in uterus, liver, and stomach, with lesser amounts in jejunum, large intestine, and ileum (although no standardizing transcript was included to normalize). The rat NHE4 signal (15) was strongest in stomach, with significantly lower intensity in small and large intestine, and, after a 120-h exposure, signal was detected from uterus, brain, kidney, and skeletal muscle. No NHE4 message was detected in testis, spleen, liver, lung, or heart by Northern blot. Similar studies by our laboratory, using similar nearly full-length cDNA as probes, led us to the same conclusions. An ~4.2-kb band (possibly a doublet) and a fainter 3-kb band appeared with nearly identical distribution pattern when either the NHE2 or NHE4 cDNA full-length probe was used. However, when we used smaller, isoform-specific cDNA or cRNA probes, the pattern of expression changed. In fact, it was our inability to find NHE4 mRNA in small or large intestine by in situ hybridization that led to the study presented here.

We believe that the extended regions of identical sequence shared by these two isoforms, regions that were used as probes for Northern analyses, led to cross hybridization and consequent misinterpretation of their tissue distribution. Furthermore, it is our conclusion that the relative abundance as determined by Northern analysis is misleading. Even if expression is fairly abundant in only one type of cell within an organ, that mRNA species may represent only a small fraction of the total that was harvested from the whole tissue. For instance, by Northern analysis of total kidney mRNA, the NHE4 signal is rare, whereas, by in situ hybridization, it is clear that it is abundantly expressed in the relatively small population of cells that line the inner medullary collecting tubules. Similarly, by Northern analysis of small and large intestine with nearly full-length probes, NHE4 appeared to be expressed in the same abundance as NHE2, whereas by in situ hybridization and RPA it is clearly not expressed at all in these tissues. Northern analysis, valuable as a first indicator of mRNA distribution, cannot be as definitive for cell-specific expression as carefully controlled in situ hybridization. In fact, the Northern blot may produce a misleading and inaccurate impression, as we demonstrate here.

We present evidence here that NHE2 is not expressed to any significant extent in the rat kidney, a conclusion that conflicts with the data of other groups (1, 4, 19, 23, 25). Tse et al. (23) analyzed the tissue distribution of the rabbit NHE2 by Northern analysis and RPA using a 223-base riboprobe (nucleotides 1773-1995) corresponding to amino acids 592-665 (mid-carboxy domain). With this probe, they detected three different size transcripts in ileum and kidney as well as a protected 223-bp fragment in these (and other) tissues by RPA. Using full or nearly full-length cDNA probes, we also detected multiple different-sized transcripts in rat kidney and intestine by Northern analysis. We have also used two isoform-specific cRNA riboprobes, one from the extreme 5' end of the cDNA, which included amino acids 1-180, and the other from the 3' end of the transcript, which included amino acids 676-808. The regions of dissimilarity between these two NHE isoforms, which are useful as discriminating probes, are distributed throughout each protein and include the extreme amino terminus, short sequences in the membrane domains, and the extreme carboxy terminus. Using these two probes in RPA and in situ hybridization, we were able to confirm the expression of NHE2 in the intestine but not in the kidney.

We also developed an antibody (2M5) to a GST-NHE2 fusion protein (amino acids 260-280, between membrane domains 5b and 6) that confirms NHE2 protein expression (as two bands of <90 kDa) in intestinal epithelial cells but not in the kidney. Tse et al. (22) also described the development of an antibody made against a fusion protein of GST and the last 87 amino acids of NHE2. By Western blotting, this antibody recognized two specific proteins of 75 and 85 kDa in PS120/NHE2 membranes as well as two nonspecific proteins of 97 and 70 kDa. Using the latter antibody, Hoogerwerf et al. (8) found two bands of 95 (or 97) kDa and 85 kDa in brush-border membrane preparations from rabbit, rat, and human small intestine and rat kidney cortex. Although they could not definitively state whether the larger protein represented NHE2 or nonspecific binding, they concluded that the 85-kDa band was NHE2. Both the antibodies used by Hoogerwerf et al. (8) and our antibodies were made against regions of NHE2 that are unique and bear no homology with other cloned NHE isoforms, especially with the closely related NHE4. In addition, both antibodies recognize similar nonspecific lower bands in the intestinal membrane preparations and the PS120 fibroblasts. However, our NHE2 antibody neither cross-reacts with the 97-kDa band that Hoogerwerf et al. (8) reported nor cross-reacts with membrane proteins in kidney. In addition, one of our riboprobes (2C) includes the same region of cDNA as used for the Hoogerwerf et al. (8) antigen fusion. With our 2C riboprobe, we detected only a very weak signal by RPA when 100 µg of kidney cortex RNA were used and detected no NHE2 message in kidney by in situ hybridization. Borensztein et al. (4), using PCR primers at nucleotides 2254 and 2546, a region within our 2C probe, also detected NHE2 in whole kidney homogenate. PCR is an extremely sensitive method of detection, which, along with the RPA results, suggests that there may be some expression. However, the corresponding NHE2 in situ analyses of whole kidney, with both 2N and 2C antisense riboprobes, gave results that were negative for positive signal and identical to the sense strand-probed kidney. The same probes detected abundant NHE2 message in intestinal epithelium by both RPA and in situ hybridization. Each method of analysis (Northern, Western, RPA, reverse transcription-PCR, and in situ hybridization) uses different physical parameters and has different levels of sensitivity, which, at least in this case, may complicate interpretation, particularly when the results conflict. Antibodies made to regions of a protein isolated from one species frequently will cross-react to the protein from a different species, even though there may be amino acid substitutions. As long as the epitope that the antibody recognizes remains, cross-reaction will occur. In contrast, if even one base of a codon differs, a riboprobe will no longer protect a full-length fragment.

The disparity may be explained if we hypothesize the existence of another NHE isoform (not NHE4) closely related to NHE2, either the product of a separate gene or an alternatively spliced variation of the cloned NHE2. To support this hypothesis, we return to the original Northern analyses done by several laboratories to define tissue distribution of NHE2 and NHE4. Two or more different size transcripts were frequently detected in mRNA from both intestine and kidney, using full-length cDNA probes or small riboprobes. Alternatively spliced variants of other proteins (10, 12, 13, 16), even in the same tissue, have been reported. The renal Na+-K+-Cl- cotransporter clones included different but homologous variants, identical over all regions of overlap except for 96 bp (16). Collins et al. (5) reported a rat ileal NHE2 of a different sequence than the NHE2 isoforms cloned by Tse et al. (23) or Wang et al. (25). Although our riboprobes were designed to rule out overlapping expression of NHE2 and NHE4, a much more detailed study would be required to rule out variants of NHE2 being expressed in the kidney.

The NHEs cloned to date appear to constitute a family of highly conserved transporters, each of which has modifications adapted to the specialized requirements of its environment. Our previous studies presented evidence that NHE4 is unique, distinct from other NHE isoforms in its regulation. Unlike the other cloned isoforms, it cannot be activated by lowering the pHi, except under acute hyperosmolarity. Cloned from a rat stomach cDNA library by its similarity to NHE1, the NHE4 is structurally organized similarly to every other NHE but most closely related in DNA and amino acid sequence to NHE2. Unlike the ubiquitously expressed NHE1, the unusual tissue distribution pattern we observe for NHE4 [cavi amnoni fields of the hippocampus (2), stomach epithelium, and renal inner medullary collecting tubules (3)] would argue that this isoform has a highly specialized role that probably requires regulatory proteins specific to these tissues for activation. Rat NHE2, cloned from a stomach cDNA library by Wang et al. (25) or from rat ileal cDNA by Collins et al. (5), is 56-57% identical in amino acid sequence to but functionally quite distinct from NHE4 (26). When expressed in NHE-negative fibroblasts, NHE2 can be activated in the presence of a pH gradient under isosmolar conditions; NHE4 cannot.

The distinctions between these two isoforms go beyond activation parameters in transfected fibroblasts. On the basis of evidence presented here, rat NHE2 is abundantly expressed in the epithelial cells of ascending and descending colon, ileum, jejunum, and stomach. In contrast, we can detect no NHE4 message in small or large intestine, although it is abundant in inner medullary collecting tubules in the kidney. With the use of the same isoform-specific assays, we are unable to detect significant NHE2 expression in rat kidney.

This study is important because it attempts to clarify the tissue-specific expression patterns that distinguish these two isoforms from each other. Our hypotheses about their respective functions should now include the recognition that their distribution is more limited and specific than we had supposed. We can use their similarities as a base line to suggest comparable roles and use their differences to predict cell-specific dissimilarities in function and regulation. Our data would also suggest that a single method of analysis may present misleading interpretations in the case of closely related genes. In conclusion, we believe that we have presented convincing evidence that NHE2 is abundantly expressed in epithelial cells throughout the small and large intestine but not in the rat kidney. In contrast, NHE4 message is found in renal inner medullary collecting tubules but is not expressed in normal adult rat intestine.

    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grant DK-38510, the Digestive Disease Core Grant DK-42086, and the GastroIntestinal Research Foundation.

    FOOTNOTES

Address for reprint requests: C. Bookstein, Univ. of Chicago, Dept. of Medicine mc 6084, 5841 S. Maryland Ave., Chicago, IL 60637.

Received 30 July 1996; accepted in final form 11 July 1997.

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Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 273(5):C1496-C1505
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society


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