Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom
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ABSTRACT |
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Na+/H+ exchanger
(NHE) proteins perform a variety of functions in the kidney and are
differentially distributed among nephron segments. The purpose of this
study was to identify NHE isoforms in murine M-1 cells as a model of
cortical collecting duct principal cells. It was found that mRNAs
corresponding to NHE1, NHE2, and NHE4 are expressed in M-1 cells.
NHE-dependent regulation of intracellular pH (pHi) was
investigated in the absence of extracellular HCO
mouse; cortical collecting duct principal cells; amiloride; adenosine 5'-triphosphate intracellular pH; sodium/hydrogen exchanger
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INTRODUCTION |
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THE RENAL CORTICAL COLLECTING duct (CCD) in mammals plays an important role in regulating Na+ absorption and H+ secretion, thus regulating the NaCl content and pH of urine.
Molecular cloning studies have identified several forms of the mammalian Na+/H+ exchangers (NHEs) 1-5 (29). NHE1-4 have been identified in the kidney. There is evidence for NHE activity in the proximal tubule (12), loop of Henle (9), distal tubule (27), and the cortical and medullary segments of the collecting duct system (5, 10, 28).
NHE1 is expressed ubiquitously among mammalian cells and is localized in the nephron to the basolateral membrane of epithelial cells (2). NHE1 is sensitive to inhibition by amiloride, a nonspecific inhibitor of Na+/H+ exchange, and is involved in controlling intracellular pH (pHi) and in regulating cell volume. NHE2 is also amiloride sensitive, and its cell membrane location may be tissue specific. In intestinal epithelial cells, NHE2 is expressed in brush-border membranes (11). In a cell line from mouse inner medullary collecting duct, NHE2 was localized in the basolateral membrane (22); however, in the pig kidney proximal tubule cell line LLC-PK1, NHE2 was reported to be present in the brush-border membranes (14). NHE3 is resistant to amiloride inhibition and is expressed in brush-border membranes of rat kidney proximal tubule (1) and intestine (11). Expression of NHE4, an amiloride-insensitive isoform, has not been fully explored, but previous work has shown NHE4 to be localized in the basolateral domain of ascending limbs of Henle's loop, distal tubules, and early proximal tubules (6).
The murine kidney cell line M-1 was derived from dissected CCDs of a
mouse transgenic for the early region of the SV40 virus (23). This cell line displays general transport and
electrophysiological characteristics of CCD principal cells (13,
16, 17, 23). The mouse is the present animal of choice for the
generation of transgenic or targeted gene knockout models. However,
compared with other species such as rat or rabbit for many
physiological systems, comparatively little functional data exist. To
date, little is known regarding either the function or the identity of
H+ and HCO
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METHODS |
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Cell culture. The M-1 cells in this study were a gift from Dr. C. Korbmacher (University Laboratory of Physiology, Oxford, UK). Cells were used between passages 27 and 35, cultured on either plastic or permeable filter supports (Snap-wells; Corning-CoStar, High Wycombe, Bucks, UK) and grown to confluency at 37°C in a humidified 95% air-5% CO2 atmosphere. The cultures were maintained in serum-free media (PC-1; Biowhittaker, Wokingham, UK) supplemented with 2 mM glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin (Sigma, Poole, UK). Media was changed every 2 days, and cells were passaged every 3 days by trypsinization.
Reverse transcription-PCR determination of NHE mRNA expression.
All reagents were obtained from Promega (Southampton, UK) unless
otherwise stated. Total RNA was extracted from M-1 cells and mouse
kidney by using TriZol reagent (GIBCO BRL, Paisley, UK) according to
the manufacturer's instructions and then treated with DNAse I to
remove genomic DNA. Reverse transcription reactions used 2 µg of
total RNA, 2 µl of 25 µM JW1 primer [an
oligo(dT)(ll)], and molecular biology-grade water
up to a final volume of 12.9 µl. Samples were heated at 90°C for 2 min to denature RNA secondary structures. RT buffer (4 µl),
deoxynucleotide phosphate mix (1.6 µl), and Moloney murine leukemia
virus-RT (1.5 µl) were added to each sample and allowed to anneal on
ice. Finally, samples were heated for 1 h at 35°C and then for 5 min at 95°C to denature the RT. PCR was performed with primers on the
basis of published sequences for rat NHE isoforms
(1-5) located near the 3'-end of the coding region,
where there is low-sequence homology among the four isoforms (Table
1) (4). The PCR reaction
contained 50 mM KCl, 10 mM Tris · HCl (pH 9.0 at 25°C), 0.1%
Triton-X 100, 200 mM deoxynucleotide phosphate mix, 3.5 mM
MgCl2, 1.25 U Taq polymerase, 200 nM of each
primer (NHE1, NHE2, NHE3, NHE4, or the "housekeeping gene"
glyceraldehyde-3-phosphate dehydrogenase), plus the target DNA. Samples
were heated to 94°C for 4 min and then subjected to 35 cycles of
denaturation (94°C for 1 min), annealing (60°C for 1 min), and
extension (72°C for 1.5 min). A final extension phase of 72°C for
10 min was included for all samples. PCR products were separated by
electrophoresis on a 2% agarose gel and visualized by ethidium bromide
staining under ultraviolet (302-nm) light. Each determination was
performed with and without RT, and each RT-PCR was performed at least
three times on RNA from three separate extractions.
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Measurement of pHi. pHi was determined by using the pH-sensitive fluorescent dye [2',7'-bis-(2-carboxyethyl)-5(6')-carboxyfluorescein] acetoxymethyl ester (Molecular Probes, Leiden, The Netherlands). Cells were grown to confluency on filter supports and placed in a modified Ussing chamber that allowed independent superfusion of apical and basolateral compartments with a solution containing (in mM) 137 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 1.6 Na2HPO4, 0.4 NaH2PO4, and 5 glucose (pH 7.4). In some experiments, N-methyl-D-glucamine was isosmotically substituted for Na+. The chamber was mounted on the stage of an inverted microscope (Olympus IMT-2) and viewed with a ×10 objective. Autofluorescence (background level before the addition of dye) was determined at the beginning of each experiment and was subtracted from all subsequent fluorescence values. Cells were loaded in the presence of 10 µM 2',7'-bis-(2-carboxyethyl)-5(6')-carboxyfluorescein, acetoxymethyl ester in HEPES buffer for 30 min at room temperature. Bath superfusion was then commenced, and the bath solution temperature was raised to 37 ± 0.5°C and maintained thereafter. After 30 min, cells were illuminated with alternate excitation wavelengths of 440 and 490 nm and data acquisition commenced. The level of fluorescence was typically 50-200 times the background level. Acquisition and display of the data were controlled by the Felix 1.2 PC software (Photon Technologies).
Calibration of the 440/490 emission ratio was performed by the nigericin method described by Thomas et al. (25). Briefly, at the end of each experiment, the bath was perfused with a high-K+ solution at pH 7.4 [(in mM) 100 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES]. Nigericin was added at a final concentration of 7 × 10Statistics. Values are reported as means ± SE, and n is the number of experiments performed. To compare means, Student's unpaired or paired t-tests were used as appropriate. For all comparisons, a value of P < 0.05 was considered to be significant. Figures of gels are representative examples.
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RESULTS |
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NHE mRNA expression in mouse kidney and M-1 cells.
mRNA was amplified by PCR by using primers designed previously
(4) to recognize the COOH terminus of NHE isoforms. This region has low-sequence homology between isoforms but is highly conserved between species (4). As expected, analysis of
the PCR products from mouse kidney revealed cDNA products of
appropriate sizes (Fig. 1A):
422 bp for NHE1, 310 bp for NHE2, 321 bp for NHE3, and 501 bp
for NHE4. In M-1 cells, products of 422 bp for NHE1, 310 bp for NHE2,
and 501 bp for NHE4 were observed. However, no product was observed for
NHE3 (Fig. 1B). Each reaction was performed with and without
RT. In the absence of reverse transcription, no products were observed
(Fig. 1, A and B), indicating that genomic DNA
did not contaminate the samples. The efficiency of the RNA extraction and RT reactions was confirmed for all samples by
amplification of a 597-bp product for glyceraldehyde-3-phosphate
dehydrogenase (Fig. 1, A and B).
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Functional localization of
Na+/H+
exchange in M-1 cells.
The mean pHi in M-1 cells was 7.20 ± 0.11 (n = 23) when both surfaces were superfused with
control solution at pH 7.4. After a 20 mM NH4Cl pulse, the
mean reduction in pHi (pHi) was 0.27 ± 0.08 pH units (P < 0.05, n = 17). The
mean rate of recovery after acidification was 1.3 ± 0.28 × 10
3 pH/s (Fig. 2;
n = 8). Application of amiloride, a nonselective inhibitor of Na+/H+ exchange, to the apical
side reduced the rate of recovery from the acid load to 2.65 ± 0.37 × 10
4 pH/s (Fig.
3A; P < 0.001, n = 4). The rate of recovery increased toward
control values on removal of amiloride. When amiloride was applied to
the basolateral side alone, the rate of recovery from the acid load was
reduced to 1.49 ± 0.8 × 10
4 pH/s (Fig.
3B; P < 0.001, n = 5).
Addition of amiloride to both sides of the monolayers completely
abolished pH recovery from an acid load, and pHi continued
to fall at a rate of 5.6 ± 1.9 × 10
5 pH/s
(Fig. 3C; P < 0.001, n = 5)
until amiloride was removed, whereupon there was a recovery to control
levels. Substitution of extracellular Na+ with the
membrane-impermeant cation N-methyl-D-glucamine
after the acid load also resulted in continued intracellular
acidification (3.2 ± 1.6 × 10
5 pH/s,
P < 0.001, n = 5), which confirms the
dependency of the recovery process on Na+/H+
exchange.
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Extracellular nucleotides produce intracellular acidification in
M-1 cells.
Transport of both Na+ and Cl is altered in
mouse M-1 cells by extracellular nucleotides (8).
Therefore, we tested whether activation of purinergic receptors might
lead to alterations in pHi. Figure
4 shows the effects of two P2 agonists on
pHi in M-1 cells. Addition of 1 mM ATP (a mixed P2X and P2Y
agonist) to the apical superfusate caused a reversible intracellular
acidification (Fig. 4A;
pHi = 0.24 ± 0.09, n = 8, P < 0.05). Application
of 0.1 mM of 2' and 3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), a
selective P2X7 (P2Z) agonist, to the apical superfusate
also resulted in a small but statistically significant acidification
(Fig. 4B;
pHi = 0.058 ± 0.02, n = 8, P < 0.05). A reduction of
pHi was observed only on activation of receptors in the
apical membrane. In contrast, application of either ATP or Bz-ATP to
the basolateral superfusate was without effect on pHi (data
not shown). The response was not mediated by means of the
P2Y2 receptor subtype, because the P2Y2 agonist
UTP was also without effect on pHi, irrespective of the
side of application (data not shown).
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DISCUSSION |
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The overall purpose of this study was to determine the functional location of Na+/H+ exchange and to identify particular NHE isoforms that are expressed in M-1 cells as a model of murine CCD principal cells. Compared with the more extensively characterized experimental models of rabbit and rat CCD, there is little information available regarding the functional properties of this nephron segment of mouse kidney. The M-1 cell line displays general transport properties characteristic of principal cells of the CCD (23). M-1 cells express subunits of the amiloride-sensitive Na+ channel (16), and monolayers display moderate sensitivity to both glucocorticoids and mineralocorticoids (17).
By using RT-PCR, we have determined that M-1 cells express mRNAs for NHE1, NHE2, and NHE4. These results are supported by studies of the rabbit CCD cell line RC.SV3, which expresses an identical NHE profile (10). It is clear that there are highly specific differences in NHE expression between segments of the nephron. Cortical and medullary thick ascending limbs (TALs) of rat kidney have been reported to express both NHE1 and NHE3 but not NHE2 or NHE4 (4). Cells of the macula densa of rabbit kidney express both NHE2 and NHE4 in the apical and basolateral membranes, respectively (19). This expression pattern is similar to that reported for mouse inner medullary collecting ducts (24). There is considerable controversy regarding NHE2 expression in the kidney. One report (3) failed to detect either NHE2 mRNA or protein in the kidney, whereas others (7) have reported that NHE2 is expressed in the apical membranes of cortical and medullary TALs but not in the collecting duct of rat kidney. The present results are consistent with the absence of NHE3 from mouse CCD, because no mRNA was demonstrated by RT-PCR, although the primers used in the study were able to detect NHE3 mRNA from whole mouse kidney. NHE3 is expressed in the apical membrane of the proximal convoluted tubule, where it appears to mediate a significant fraction of Na+ and water reabsorption (26). In the TAL, NHE3 is expressed in the apical membranes of both cortical and medullary segments; however, this isoform does not appear to contribute to salt reabsorption in the TAL under normal conditions (26), but its activity is increased by chronic metabolic acidosis (15). A question arising from the present study is whether the profile of NHE mRNA expression that we have observed reflects functional protein expression. This is not always known to be the case. As reported previously (20), NHE4 protein is undetectable in brain tissue despite detectable levels of mRNA expression. Such discrepancies may be the result of various mechanisms of posttranscriptional regulation of gene expression. Successful identification of specific NHE proteins in M-1 cells has so far evaded us. We have made exhaustive attempts (Hill C and White SJ, unpublished observations), using a variety of available antibodies raised against NHE isoforms, to determine expression and localization of these proteins by immunoblotting and immunocytochemistry. However, to date, these studies have proven inconclusive because of the lack of specificity of such antibodies.
It is clear that NHE activity is present in both the apical and the
basolateral membranes of M-1 cells. Amiloride applied to either the
apical or the basolateral membranes of M-1 cells reduced the rate at
which pHi recovered from an intracellular acid load.
Although amiloride at the concentration used in this study would also
inhibit Na+ entry via the amiloride-sensitive
Na+ channel, this would tend to increase the driving force
for apical proton extrusion. Simultaneous inhibition of
Na+/H+ exchange at both the apical and the
basolateral membranes by amiloride or by removal of extracellular
Na+ abolished recovery of pHi from an
intracellular acidification. From these results, it is concluded that,
under nominally HCO
An observation of additional interest arising from the present study is
that ATP reduced pHi when applied to the apical, but not
the basolateral, membrane. ATP (a mixed P2X and P2Y agonist) and the
specific P2Z agonist Bz-ATP both produced an intracellular acidification. The decrease in intracellular H+ activity
could not have been caused by activation of P2Y2 receptors, because the P2Y2 agonist UTP was without effect. A recent
study by Cuffe et al. (8) has shown that activation of
apical and basolateral P2Y2 receptors by ATP stimulates
Cl secretion in M-1 cells while inhibiting
amiloride-sensitive Na+ transport. The mechanism of
inhibition of Na+ transport by ATP is unknown. Although we
have not measured Na+ transport in this study, our results
are consistent with the idea that inhibition of Na+
transport by luminal ATP (8) may be in part mediated by
means of intracellular acidification. The increase in H+
activity resulting from activation of apical purinoceptors would reduce
Na+ entry across the apical membrane by inhibiting activity
of the amiloride-sensitive Na+ channel, which is highly pH
sensitive (18). Further experiments will be required to
directly test this hypothesis. We also found that, although Bz-ATP
produced an acidification, it was not as effective as ATP, suggesting
that the reduction in pHi did not result from activation of
the P2X7 receptor subtype. None of the nucleotides tested
had an effect on pHi when applied in the basolateral superfusate, even though M-1 cells express functional P2Y2
receptors in the basolateral membrane (8). The polarity of
the effect observed here strengthens the suggestion that apical and
basolateral purinoceptors are able to act independently of one another
in Na+-transporting epithelia (8).
In summary, the results from this study show that the M-1 cell line expresses NHE1, NHE2, and NHE4 mRNAs and that functional Na+/H+ exchange activity is present at both the apical and the basolateral plasma membranes. Activation of apical purinoceptors leads to a decrease in pHi, which may contribute to the mechanism by which extracellular nucleotides influence Na+ entry across the apical membrane in principal cells of the collecting duct.
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ACKNOWLEDGEMENTS |
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We thank Dr. Christoph Korbmacher (University Laboratory of Physiology, Oxford, UK) for the M-1 cells and Andrew J. Parker for general technical support.
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FOOTNOTES |
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This work was supported by the National Kidney Research Fund of the United Kingdom.
Address for reprint requests and other correspondence: S. J. White, Dept. of Biomedical Sciences, Univ. of Sheffield, Western Bank, Sheffield S10 2TN, UK (E-mail: s.j.white{at}sheffield.ac.uk).
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.
10.1152/ajprenal.00291.2000
Received 20 September 2000; accepted in final form 31 October 2001.
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