Departments of 1 Cellular and Molecular Physiology, and 2 Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8029; and 3 Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524
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ABSTRACT |
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The absorption of NaCl in the
proximal tubule is markedly stimulated by formate. This stimulation of
NaCl transport is consistent with a cell model involving
Cl-formate exchange in parallel with pH-coupled formate
recycling due to nonionic diffusion of formic acid or
H+-formate cotransport. The formate recycling process
requires H+ secretion. Although
Na+-H+ exchanger isoform NHE3 accounts for the
largest component of H+ secretion in the proximal tubule,
40-50% of the rates of HCO
oxalate; anion exchange; Na+-H+ exchanger; microperfusion
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INTRODUCTION |
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THE ABSORPTION OF
NaCl in the proximal tubule is markedly stimulated by formate and
oxalate (13, 20, 22, 24, 25, 27). This stimulation of NaCl
transport is consistent with a cell model involving
Cl-formate and Cl
-oxalate exchange
processes in parallel with recycling of formate and oxalate across the
apical membrane (3). In the case of formate-stimulated
NaCl absorption, it has been proposed that there is pH-coupled formate
recycling due to nonionic diffusion of formic acid or
H+-formate cotransport (10, 19). Such a
process in turn requires H+ secretion in parallel with
Cl
-formate exchange. In the proximal tubule,
Na+-H+ exchanger isoform NHE3 plays a major
role in H+ secretion, but 40-50% of the rates of
HCO
The purpose of the present investigation is to use NHE3 null mice to
directly test the role of apical membrane NHE3 in mediating NaCl
absorption stimulated by formate. We demonstrate that formate stimulates NaCl absorption in the mouse proximal tubule microperfused in vivo, as previously found in the rat (24, 25, 27).
Moreover, we find that the component of NaCl absorption stimulated by
formate is absent in NHE3 null mice. In contrast, stimulation of NaCl absorption by oxalate is preserved in NHE3 null mice, consistent with
the previous proposal that oxalate-stimulated NaCl absorption is
independent of Na+-H+ exchange, and is mediated
by Na+-sulfate cotransport in parallel with
Cl-oxalate exchange and sulfate-oxalate exchange
(3, 12, 24).
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MATERIALS AND METHODS |
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Animals and surgical preparation.
Knockout mice deficient in NHE3 were generated by targeted gene
disruption (21). Genotype analysis of tail DNA was
performed by PCR, using primers derived from the 5' and 3' ends of exon 6 and the 5' end of the neomycin resistance gene. When used in the same
reaction, the three primers amplify a 199-bp product from the wild-type
gene and a 113-bp product from the mutant gene. Homozygous wild-type
(NHE+/+) and null (NHE3/
) mice resulting
from breeding of heterozygotes were maintained on a regular diet and
tap water until the day of the experiment. Ages of mutant animals were
matched with their wild-type controls.
Microperfusion of proximal tubules in situ. The details of the methods for surgical preparation and microperfusion of mouse proximal tubules in vivo were described previously (21, 26) and were similar to those used in the rat (24). Briefly, proximal convoluted tubules with 3-5 loops on the kidney surface were perfused at a rate of 15 nl/min with a perfusion solution containing 20 µCi/ml of low-sodium [3H]methoxy-inulin for measuring volume absorption. Tubule fluid collections were made downstream with another micropipette. One collection was made in each perfused tubule, and two to four collections were taken in the experimental kidney of each animal. The perfused segments were marked with Sudan Black heavy mineral oil, and their lengths were determined after filling with high-viscosity microfil (Canton Bio-Medical Products, Boulder, Colorado) and dissection of the silicone rubber casts.
Measurement of rates of Cl and fluid absorption.
The rates of net Cl
(JCl) and
fluid (Jv) absorption were calculated based on
changes in the concentrations of [3H]inulin and
Cl
as described previously (23, 24).
JCl and Jv were expressed per millimeter tubule length. The composition of the perfusion solution
was the same as used previously in the rat (24) (in mM):
sodium chloride 140, sodium bicarbonate 5.0, potassium chloride 4.0, calcium chloride 2.0, magnesium sulfate 1.0, dibasic sodium phosphate
1.0, and monobasic sodium phosphate 1.0, pH 6.7. Concentrations of
formate and oxalate indicated in the tables and figures were added as
sodium salts.
Statistics. Data are presented as means ± SE. Experimental groups were compared with a control group by use of Dunnett's test. Differences were considered significant if P < 0.05.
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RESULTS |
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In the first series of experiments, we evaluated whether formate
and oxalate stimulate NaCl transport in the proximal tubule of the
mouse as previously observed in the rat (24, 27). To this
end, proximal tubules were microperfused in situ with a relatively high
Cl, low HCO
reabsorption, JCl, was also measured directly.
Shown in Table 1 and Fig.
1, the addition of either 500 µM
formate or 5 µM oxalate to the luminal perfusion solution resulted in
significant stimulation of Jv and
JCl. These findings are consistent with the
contributions of luminal membrane Cl-formate and
Cl
-oxalate exchange processes to transcellular NaCl
absorption as previously demonstrated in the rabbit and rat proximal
tubule (13, 20, 22, 24, 25, 27). A key finding in the
previous studies in the rat was that stimulation of
Jv and JCl by formate but
not by oxalate was abolished by luminal application of the Na+-H+ exchange inhibitor
ethylisopropylamiloride (EIPA) (24). This finding strongly
suggested a central role for apical membrane Na+-H+ exchange in mediating formate-stimulated
NaCl absorption.
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Given that NHE3 is the principal Na+-H+ exchanger isoform in the brush border membrane of proximal tubule cells (2, 4, 14, 21, 29), it would seem most likely that inhibition of this isoform was responsible for the observed inhibition of formate-stimulated NaCl absorption by EIPA. However, an EIPA-sensitive component of apical membrane acid extrusion from proximal tubule cells was recently observed in isolated tubules from NHE3 null mice (7), suggesting that an Na+-H+ exchanger isoform other than NHE3 might be functional at the brush border membrane.
Accordingly, to directly address the specific role of NHE3 in
formate-dependent NaCl transport, we assessed the ability of formate to
stimulate NaCl transport in the proximal tubules of NHE3 null mice.
These results are shown in Table 2 and
Fig. 2. The baseline
Jv and JCl measured in
the absence of formate and oxalate were lower in the NHE3 null mice
compared with the wild-type controls (see Table 1 and Fig. 1). Formate
failed to increase Jv or
JCl significantly in the proximal tubules of
NHE3 null mice. In contrast, significant stimulation of
Jv and JCl by oxalate was
still present. The latter finding underscores the specific role of NHE3
in facilitating formate stimulation of NaCl transport in the proximal
tubule.
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DISCUSSION |
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A Cl-formate exchange activity was originally
identified in studies of isolated brush border membrane vesicles
(9, 10). For this anion exchange process to support
significant absorptive Cl
flux, a mechanism is required
to recycle formate back from the lumen into the cell in view of the low
plasma concentrations of formate (13, 24). At least two
potential mechanisms for formate recycling coupled to H+
translocation were identified in studies of brush border membrane vesicles, nonionic diffusion of formic acid, and H+-formate
cotransport (or OH
-formate exchange) (10,
19). These mechanisms in turn require continuous extrusion of
H+ across the apical membrane. In the proximal tubule, the
major pathway for cellular acid extrusion is
Na+-H+ exchange (1, 11).
Immunocytochemical studies have identified the expression of
Na+-H+ exchanger isoform NHE3 on the brush
border membrane (2, 4, 5), and the profile of inhibitor
sensitivity of brush border Na+-H+ exchange is
characteristic of NHE3 (29). The rate of
HCO
The models in Fig. 3 illustrate
formate-dependent NaCl absorption as occurring by
Cl-formate exchange in parallel with H+
secretion by either Na+-H+ exchange or
H+-ATPase. As shown in Fig. 3A, in which
H+ secretion takes place by Na+-H+
exchange, the Na+ absorption associated with
formate-dependent Cl
transport is transcellular. In
contrast, as shown in Fig. 3B, in which H+
secretion is mediated by H+-ATPase, Cl
absorption is electrogenic, and is accompanied by paracellular Na+ absorption. In either case, formate would induce net
NaCl absorption.
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In microperfused proximal tubules of rabbit and rat, formate markedly
stimulates Jv and JCl
(13, 20, 22, 24, 25, 27), consistent with the proposed
role of Cl-formate exchange in mediating NaCl absorption
according to both of the models in Fig. 3. Moreover, the ability of
formate to stimulate proximal NaCl absorption is dependent on luminal
acidification, consistent with the necessity for H+-coupled
formate recycling (20). Despite the evidence that a significant fraction of proximal acidification is independent of NHE3
(7, 21, 26), the Na+-H+ exchange
inhibitor EIPA completely abolishes formate stimulation of
Jv and JCl
(24), suggesting a key role for NHE3 or another EIPA-sensitive process.
We now find that the ability of formate to stimulate NaCl transport is
virtually abolished in the proximal tubules of NHE3 null mice. This
observation indicates that neither another EIPA-sensitive process nor
the H+-ATPase can sustain the H+ secretion
needed for appreciable Cl absorption by
Cl
-formate exchange to occur. Given that NHE3 accounts
for only 50-60% of H+ secretion across the brush
border membrane and that there exist several alternative mechanisms for
acid extrusion, the question arises as to why there is an absolute
requirement for NHE3 activity for formate-stimulated NaCl absorption.
Indeed, in our studies, the tubules were perfused with a low
HCO
It may be noted that the baseline Jv and
JCl measured in the absence of added formate or
oxalate are lower in the NHE3 null mice compared with the wild-type
controls. One possible explanation is that a component of NaCl
absorption dependent on NHE3 activity is present under baseline
conditions due to Cl-formate exchange resulting from the
presence of formate in peritubular capillary blood. Alternatively,
Cl
-base exchange independent of formate (e.g.,
Cl
-OH
exchange) might be present, and, in
parallel with Na+-H+ exchange, contribute to
NaCl absorption (13, 22, 28). In addition, glomerular
filtration rate is chronically and markedly reduced in NHE3 null mice
(14), possibly resulting in reduced cell size, membrane
surface area, and transporter expression, the opposite of what results
from glomerular hyperfiltration (6, 8, 16-18).
Significant stimulation of Jv and
JCl by oxalate is still observed in NHE3 null
mice, reflecting the presence of the component of transcellular
Cl absorption taking place by Cl
-oxalate
exchange. These findings are consistent with the previous observation
that EIPA does not inhibit oxalate-stimulated NaCl transport
(24). Taken together, the present and previous results indicate that NHE3 has no role in mediating oxalate-stimulated NaCl
absorption. In fact, previous evidence strongly suggests that
oxalate-stimulated NaCl absorption is mediated by
Na+-sulfate cotransport in parallel with
Cl
-oxalate exchange and sulfate-oxalate exchange
(3, 12, 24).
In conclusion, we find that NHE3 has a specific role in mediating formate-stimulated NaCl absorption in the proximal tubule. In view of the fact that other secretory pathways contribute to H+ secretion in the proximal tubule, the virtually complete dependence of formate-induced NaCl absorption on NHE3 activity raises the possibility that NHE3 and the formate transporters are functionally coupled in the brush border membrane.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-17433, DK-33793, and DK-50594.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. S. Aronson, Dept. of Internal Medicine, Yale School of Medicine, 333 Cedar St., P.O. Box 208029, New Haven, CT 06520-8029
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.
Received 20 February 2001; accepted in final form 17 April 2001.
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