1 Departments of 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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NHE3 is the predominant isoform responsible for apical membrane
Na+/H+
exchange in the proximal tubule. Deletion of NHE3 by gene targeting results in an NHE3/
mouse with greatly reduced proximal tubule
HCO
3 absorption compared with
NHE3+/+ animals (P. J. Schultheis, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani,
L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282-285, 1998).
The purpose of the present study was to evaluate the role of other
acidification mechanisms in mediating the remaining component of
proximal tubule HCO
3 reabsorption in
NHE3
/
mice. Proximal
tubule transport was studied by in situ microperfusion. Net rates of
HCO
3 (JHCO3) and fluid
absorption (Jv)
were reduced by 54 and 63%, respectively, in NHE3 null mice compared
with controls. Addition of 100 µM ethylisopropylamiloride (EIPA) to
the luminal perfusate caused significant inhibition of
JHCO3 and
Jv in
NHE3+/+ mice but failed to inhibit
JHCO3 or
Jv in
NHE3
/
mice,
indicating lack of activity of NHE2 or other EIPA-sensitive NHE
isoforms in the null mice. Addition of 1 µM bafilomycin
caused a similar absolute decrement in
JHCO3 in
wild-type and NHE3 null mice, indicating equivalent rates of
HCO
3 absorption mediated by
H+-ATPase. Addition of 10 µM
Sch-28080 did not reduce
JHCO3 in either wild-type or NHE3 null mice, indicating lack of detectable
H+-K+-ATPase
activity in the proximal tubule. We conclude that, in the absence of
NHE3, neither NHE2 nor any other EIPA-sensitive NHE isoform contributes
to mediating HCO
3 reabsorption in the
proximal tubule. A significant component of
HCO
3 reabsorption in the proximal
tubule is mediated by bafilomycin-sensitive H+-ATPase, but its activity
is not significantly upregulated in NHE3 null mice.
sodium/proton exchange; proton-adenosinetriphosphatase; proton-potassium-adenosinetriphosphatase; acidification
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INTRODUCTION |
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A MAJOR FRACTION of proximal tubule
HCO3 reabsorption is mediated by
apical membrane
Na+/H+
exchange (1, 4). Molecular cloning studies have shown that at least
four
Na+/H+
exchanger (NHE) isoforms are expressed in the mammalian kidney (23, 26,
30, 31). Immunocytochemical studies using isoform-specific antibodies
have indicated that, whereas NHE1 and NHE4 have a basolateral distribution (6, 9, 24), NHE2 and NHE3 are located along the apical
membranes of various nephron segments, including the proximal tubule
(3, 5, 7, 10, 28, 29, 39, 40). Analysis of inhibitor sensitivity has
suggested that virtually all of the measured
Na+/H+
exchange activity in isolated renal cortical brush-border membrane vesicles is mediated by NHE3 (38).
Direct evidence for the functional importance of NHE3 is that proximal
tubule HCO3 reabsorption is reduced by
~60% in NHE3 null mice (27). However, the mechanisms accounting for
the remaining component of HCO
3
reabsorption in the proximal tubules of NHE3 null mice are not known.
Possible mechanisms include participation of another apical NHE
isoform, such as NHE2, or of primary active
H+ transport pathways, such as
H+-ATPase and
H+-K+-ATPase.
The aims of the present study are to evaluate the relative contributions of these possible mechanisms to mediating
HCO
3 reabsorption in the proximal
tubules of NHE3 null mice.
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METHODS |
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Animals and surgical preparation.
Knockout mice deficient in NHE3 were generated by targeted gene
disruption (27). Genotype analysis of tail DNA was performed by PCR.
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. The mice
were anesthetized by intraperitoneal injection of 100 mg/kg body wt
Inactin [5-ethyl-5-(L-methylpropyl)-2-thiobarbituric acid; BYK-Gulden, Konstanz, Germany] and were placed on a
thermostatically controlled surgical table to maintain body temperature
at 37°C. After tracheotomy, the left jugular vein was exposed and
cannulated with a PE-10 catheter for intravenous infusion. A carotid
artery was also catheterized with PE-10 tubing for arterial blood
collection for blood gas analysis and for measurement of mean arterial
pressure. Blood gas analysis was performed on freshly drawn blood by
use of a Corning Blood Gas Analyzer.
Microperfusion of proximal tubules in
situ. After surgical preparation, saline solution
(0.9%) was infused at a rate of 0.15 ml/h ( of the infusion
rate used in rat). The left kidney was exposed by lateral abdominal
incision, carefully isolated, and immobilized in a special kidney cup
filled with light mineral oil (37°C). The kidney surface was
illuminated by a fiber optical light. The details of the method for
microperfusion of proximal tubules in vivo were described previously
for the rat (35). Briefly, a proximal convoluted tubule with three to five loops on the kidney surface was selected and perfused at a rate of
15 nl/min with a proximal oil block. The perfusion solution contained
20 µCi/ml of low-sodium
[methoxy-3H]inulin
for measuring volume absorption and 0.1% FD & C green dye
for identification of the perfused loops. Tubule fluid collections were
made downstream with another micropipette with distal oil block. One
collection was made in each perfused tubule, and two to four
collections were taken in each kidney. The perfused tubules were marked
after collection with sudan black heavy mineral oil. To determine the
length of the perfused segment, tubules were filled with high-viscosity
microfil (Canton Bio-Medical Products, Boulder, CO), the kidney was
partially digested in 20% NaOH, and silicone rubber casts of the
tubule segments were dissected.
Measurement of net
HCO3 and fluid
absorption. The rates of net
HCO
3 (JHCO3) and
fluid (Jv)
absorption were calculated based on changes in the concentrations of
[3H]inulin and total
CO2 as described previously (35).
The total CO2 concentrations in
both initial and collected fluids were measured by a microcalorimetric
(Picapnotherm) method (35).
JHCO3 and Jv were expressed
per millimeter tubule length. The composition of the perfusion solution
was the same as used previously in the rat (36) (in mM): 115 NaCl, 25 NaHCO3, 4 KCl, 1 CaCl2, 5 sodium acetate, 2.5 Na2HPO4,
0.5 NaH2PO4,
5 L-alanine, and 5 glucose. Solutions were bubbled at room
temperature with a 5% CO2-95%
O2 gas mixture before use. The pH
was titrated to 7.4 with NaOH or HCl as required.
Statistics. Data are presented as
means ± SE. Student's t-test was
used when a single experimental group was compared with a control group
(Table 1). Several experimental groups were
compared with a control group (Table 2) by
use of Dunnett's test. Differences were considered significant at
P < 0.05.
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RESULTS |
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Blood gas analysis, as shown in Table 1, indicated that NHE3 null mice
have a mild to moderate metabolic acidosis with reduction in arterial
blood HCO3 concentration from 25.7 to
20.4 mM and reduction of pH from 7.34 to 7.18. A moderate respiratory acidosis was also noted in both sets of animals
(PCO2 50-55), most likely
secondary to anesthesia. The mild metabolic acidosis in NHE3 null mice
confirms previous results (27).
As indicated in Table 2 and Fig. 1,
JHCO3 and
Jv were reduced
by 54 and 63%, respectively, in NHE3 null mice compared with controls.
These findings confirm previous results indicating a major role of NHE3
in mediating proximal tubule HCO3 and
fluid absorption (27). Clearly, a significant fraction of JHCO3 persists in
the NHE3 null mice, indicating contributions from alternative
acidification processes. We therefore used inhibitors to investigate
the mechanisms mediating the remaining
JHCO3 and Jv.
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To investigate whether any other NHE isoform such as NHE2 contributes
to the remaining component of
JHCO3 in NHE3
knockout mice, we examined the effects of the
Na+/H+
exchange inhibitor ethylisopropylamiloride (EIPA). This inhibitor was
added to the lumen perfusate at a concentration of 100 µM. As shown
in Table 2 and Fig. 1, EIPA significantly decreased JHCO3 and
Jv by 40 and
47%, respectively, in wild-type mice. These results are similar to
previous studies in rat proximal tubule (25, 35). In contrast, EIPA did
not reduce either JHCO3 or Jv in NHE3
knockout mice. These results indicate that other NHE isoforms such as
NHE2 do not play a role in mediating HCO3 absorption in the absence of NHE3. It may be noted that EIPA did not
reduce JHCO3 and
Jv in the
wild-type mice to the levels observed in the knockout mice. This can be
explained by the known resistance of NHE3 to amiloride analogs in the
presence of physiological Na+
concentrations (12, 18, 25, 35), resulting in incomplete inhibition of
NHE3 activity in the wild-type mice.
To evaluate the contribution of the
H+-ATPase to mediating
HCO3 absorption in the proximal tubules of
wild-type and NHE3 null mice, we studied the effect of 1 µM
bafilomycin A1 (17, 32, 34). The results in Table 2 and Fig. 1 show that bafilomycin decreased
JHCO3 in
wild-type mice by 22%, indicating that, under physiological
conditions, a significant fraction of proximal tubule
HCO
3 absorption is mediated by the
H+-ATPase. This result is also
consistent with previous findings that a similar fraction of
HCO
3 absorption in the proximal tubule of
the rat is mediated by a
Na+-independent and/or
bafilomycin-sensitive mechanism (11, 32).
Fractional inhibition of
JHCO3 by
bafilomycin was far greater (59%) in NHE3 null mice compared with
wild-type controls, indicating that the major fraction of the
JHCO3 that
persists in the absence of NHE3 activity is mediated by the
H+-ATPase. However, the absolute
decrement in
JHCO3 (25 vs. 30 pmol · min1 · mm
1)
was virtually the same in NHE3 knockout and wild-type animals, indicating a lack of significant compensatory upregulation of H+-ATPase in the NHE3 null mice.
Interestingly, although there was a trend for bafilomycin to reduce
Jv, this
inhibition did not reach statistical significance in either wild-type
or NHE3 null mice.
Finally, we studied the effect of the inhibitor Sch-28080 to assess the
role of
H+-K+-ATPase
(14) in mediating proximal tubule HCO3 and
fluid absorption in wild-type and NHE3 null mice. The data in Table 2
and Fig. 1 demonstrate that 10 µM SCH-28080 failed to inhibit
JHCO3 and
Jv in either
wild-type or NHE3 knockout mice. These findings indicate that
H+-K+-ATPase
activity does not contribute significantly to proximal tubule
HCO
3 absorption in wild-type or NHE3 null mice.
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DISCUSSION |
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We have evaluated the relative contributions of apical
H+ extrusion mechanisms to
mediating HCO3 reabsorption in the
proximal tubules of NHE3 null mice. First, we confirmed the previous
findings (27) that a large fraction (50-60%) of HCO
3 and fluid absorption in the
microperfused mouse proximal tubule is dependent on the operation of
the apical NHE isoform NHE3. These findings are also in accord with
free-flow micropuncture data indicating marked impairment of fluid
reabsorption in the proximal tubule of NHE3 null mice (20).
Second, we could detect no EIPA-sensitive component of
JHCO3 or
Jv in the
proximal tubules of NHE3 null mice. Because NHE2 is more sensitive to
amiloride analogs than is NHE3 (12), these findings indicate that NHE2
does not appreciably contribute to HCO3
absorption, although its expression has been detected in the proximal
tubule (39, 40).
Third, we found a significant component of
HCO3 absorption mediated by
bafilomycin-sensitive H+-ATPase in
the proximal tubules of both wild-type and NHE3 null mice, consistent
with previous demonstrations of
Na+-independent and/or
bafilomycin-sensitive acidification in this nephron segment (11, 13,
32, 33). The contribution of apical membrane
H+-ATPase to proximal tubule
acidification is also supported by studies of
H+ transport in isolated
brush-border vesicles (17), the observation of
Na+-independent acid extrusion
from intact cells (19, 34), and the expression of
H+-ATPase on the apical membrane
as detected by immunostaining (8). However, we detected no upregulation
of the bafilomycin-sensitive component of
HCO
3 absorption in the proximal tubule of
the NHE3 null mice.
Fourth, we were unable to detect a component of Sch-28080-sensitive
HCO3 absorption, arguing against a
significant role for
H+-K+-ATPase
in mediating proximal tubule acidification in either wild-type or NHE3
null mice, although Sch-28080-sensitive
K+-ATPase activity has been
detected in this nephron segment (41). It should be noted that the
concentration of Sch-28080 used in our experiments, 10 µM, should
have been sufficient to inhibit at least 80% of the
K+-ATPase activity that had been
identified in the proximal tubule (41).
A component of proximal tubule HCO3
reabsorption (21 pmol · min
1 · mm
1)
persisted in the NHE3 null mice in the presence of bafilomycin (see
Table 2 and Fig. 1), corresponding to 19% of the control rate of
HCO
3 absorption in wild-type mice. There are several possible explanations for this small remaining component of
inhibitor-insensitive HCO
3 absorption in the NHE3 null mice. First, a passive driving force for
JHCO3 was present
in these experiments because plasma
HCO
3 concentration was reduced to
~20 mM in NHE3 null mice, whereas the concentration of
HCO
3 in the tubule microperfusion solution was 25 mM. Assuming a mean transtubular
HCO
3 gradient of 3 mM (Table 2), a
negligible transtubular potential difference (15), and a
HCO
3 permeability of 1.6 nl · mm
1 · min
1
as found in the rat proximal tubule (2), we calculate that only a minor
fraction (5 pmol · min
1 · mm
1)
of the remaining
JHCO3 can be
attributed to passive transport.
A second possible explanation for the remaining component of
inhibitor-insensitive HCO3 absorption in
NHE3 null mice is the use of insufficient inhibitor concentrations to
abolish H+-ATPase,
H+-K+-ATPase,
or non-NHE3-mediated
Na+/H+
exchange. In the case of bafilomycin, a concentration of 15 nM is
sufficient to abolish ATP-stimulated
H+ transport in rat renal
brush-border vesicles (17), suggesting that the concentration of 1 µM
used in the present study should have been adequate to block
H+-ATPase activity completely.
However, it is possible that the inhibitor is absorbed in the perfused
segment so that its concentration decreases, thereby resulting in
incomplete inhibition of H+-ATPase
activity. As mentioned above, 10 µM Sch-28080 should have been
sufficient to reduce
H+-K+-ATPase
>80% in the proximal tubule (41), yet no inhibition by this agent
was observed. NHE2, the only apical NHE isoform other than NHE3 so far
identified, should have been significantly inhibited by 100 µM EIPA
(12), but no EIPA inhibition of
JHCO3 was
observed in NHE3 null mice. Taken together, these considerations indicate that
H+-K+-ATPase
and non-NHE3-mediated
Na+/H+
exchange are not likely to have contributed substantially to the
inhibitor-insensitive
JHCO3 in NHE3
null mice.
Third, it should be noted that acetate was present in the
microperfusion solution used in these studies. Absorption of acetate via Na+-acetate cotransport in
parallel with recycling of acetate back into the lumen by nonionic
diffusion has been shown to effect net acid extrusion across the apical
membrane of proximal tubule cells (22). However, acetate was found to
inhibit rather than stimulate transtubular
HCO3 absorption (16), so that this
mechanism is unlikely to account for significant HCO
3 absorption in NHE3 null mice.
Alternatively, it is possible that secretion from blood to lumen of
some other organic anion that crosses the apical membrane by nonionic
diffusion or anion/OH
exchange may contribute to proximal acidification in these experiments (4).
Finally, despite a profound impairment of proximal tubule
HCO3 absorption capacity in NHE3 null
mice, we confirmed previous findings (27) that only a mild metabolic acidosis is present in these animals. Although, as described above, we
detected no upregulation of acidification mechanisms in the proximal
tubule itself, at least two other compensatory responses to limit
acidosis have so far been identified. First, there is a marked
reduction in glomerular filtration rate in NHE3 null mice due to
tubuloglomerular feedback (20). This in turn would be expected to
reduce the filtered load of HCO
3 and
thereby limit the delivery of HCO
3 out
of the proximal tubule even in the presence of a reduced capacity for
HCO
3 absorption. Second,
HCO
3 absorption capacity is increased
in cortical and outer medullary collecting ducts of NHE3 null mice due
to upregulation of
H+-K+-ATPase
isoforms (21).
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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-33793, DK-17433, and DK-50594.
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FOOTNOTES |
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Portions of the study were previously published in abstract form (37).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. S. Aronson, Dept. of Medicine/Nephrology, Yale School of Medicine, 333 Cedar St., PO Box 208029, New Haven, CT 06520-8029 (E-mail: peter.aronson{at}yale.edu).
Received 19 January 1999; accepted in final form 12 May 1999.
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