Chloride dependency of renal brush-border membrane phosphate
transport
Norimoto
Yanagawa,
Chi
Pham,
Remi N. J.
Shih,
Stephen
Miao, and
Oak
Don
Jo
Division of Nephrology, Medical and Research Services, Sepulveda
Veterans Affairs Medical Center, University of California at Los
Angeles School of Medicine, Los Angeles, California 91343
 |
ABSTRACT |
In our present
study, we examined the effect of
Cl
on rabbit renal
brush-border membrane (BBM) phosphate
(Pi) uptake. It was found that
the Na+-dependent BBM
32P uptake was significantly
inhibited by Cl
replacement
in the uptake solution with other anions, or by
Cl
transport inhibitors,
including DIDS, SITS, diphenylamine-2-carboxylate (DPC), niflumic acid
(NF), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB).
Intravesicular formate or
Cl
increased BBM
36Cl
uptake but did not affect BBM 32P
uptake. BBM
22Na+
uptake was lowered by Cl
replacement in the uptake solution but not by
Cl
transport inhibitors.
Changes in transmembrane electrical potential altered BBM
36Cl
and 32P uptake in directions
consistent with a net inward movement of negative and positive charges,
respectively. However, the
Cl
-dependent BBM
Pi uptake was not affected by
changes in transmembrane electrical potential. Finally, a similar
Cl
dependency of
Pi uptake was also found with BBM
derived from rat and mouse kidneys. In summary, our study showed that a
component of Na+-dependent
Pi uptake was also
Cl
dependent in rabbit,
rat, and mouse renal BBM. The mechanism underlying this
Cl
dependency remains to be identified.
proximal tubule; chloride channel; chloride channel inhibitor; sodium cotransport; hereditary nephrolithiasis
 |
INTRODUCTION |
THE KIDNEY plays a pivotal role in maintaining body
phosphate (Pi) homeostasis
through regulation of urinary Pi
excretion (2). Pi reabsorption in
the kidney occurs mainly at the proximal tubule, where the uptake of
Pi across the apical brush-border membrane (BBM) represents the rate-limiting step and the main target of
regulation (10). BBM Pi uptake is
an active process that occurs together with sodium
(Na+) along the inwardly
directed Na+ gradient via the
Na-Pi cotransport (23). Recently,
however, several lines of evidence have been reported to suggest that
chloride (Cl
) may in some
way interact with this cotransport process.
First, recent cloning studies have identified two distinctive types of
mammalian renal Na-Pi
cotransporters, i.e., type I Na-Pi
cotransporters in the kidneys of rabbit (NaPi-1) (29), rat (rNaPi-1)
(19), mouse (Npt-1) (8), and human (NPT-1) (7), and type II
Na-Pi cotransporters in the
kidneys of rat (NaPi-2) (21), human (NaPi-3) (21), opossum (NaPi-4)
(26), flounder (NaPi-5) (30), rabbit (NaPi-6) (27), and mouse (NaPi-7) (9, 14). Although the type II
Na-Pi cotransporters are generally considered to be the BBM Na-Pi
cotransporter system responsible for dietary adaptation and hormonal
regulation (18), the functional role of type I
Na-Pi cotransporters remains less
well defined. In addition to its role as a
Na-Pi cotransporter, the rabbit
type I Na-Pi cotransporter,
NaPi-1, was also found to function as an anion channel permeable to
Cl
and other organic anions
(6). It is not known whether this unique function of NaPi-1 as an anion
channel affects BBM Pi transport.
Another line of evidence supporting a potential role of
Cl
in renal
Pi reabsorption derives from
studies on a group of hereditary kidney stone diseases, including
Dent's disease (31), X-linked recessive nephrolithiasis (XRN) (12),
and X-linked recessive hypophosphatemic rickets (XLRH) (3). These
disorders are associated with Fanconi-like proximal tubular dysfunction
such as low-molecular-weight proteinuria, hypercalciuria, and
hyperphosphaturia (24). These disorders have recently been found to be
associated with a common defect in a gene,
CLCN5, which encodes a
Cl
channel, CLC-5,
expressed predominantly in the kidney (20). The function of CLC-5 and
the mechanism whereby CLC-5 defect leads to the characteristic proximal
tubular transport dysfunction remain unknown (15).
Our present study was designed to examine the possible
effect of Cl
on
Pi transport by renal BBM. We
found that up to 40% of the Na+-dependent
Pi uptake by rabbit renal BBM was
Cl
dependent and was
sensitive to inhibition by
Cl
transport inhibitors. A
similar Cl
dependency of
Pi uptake was also found with BBM
prepared from rat and mouse kidneys.
 |
METHODS |
Animals. New Zealand White male
rabbits, weighing 1.5-2.0 kg, were used in these studies. The
animals were maintained on an ad libitum diet of standard chow with
free access to tap water for drinking.
BBM vesicle preparation and uptake
measurements. Purified BBM vesicles were prepared from
renal cortex by the conventional magnesium-precipitation method.
Purification of BBM preparation, as assessed by the enrichment of BBM
enzyme markers, was monitored routinely as reported previously (22).
Final BBM vesicles were suspended in a medium that comprised (in mM)
300 mannitol, 10 MgSO4, 10 Tris,
and 16 HEPES, pH 7.4. Uptake was measured by a Millipore
rapid-filtration procedure at 24°C and was initiated by mixing 10 µl BBM vesicle suspension with 90 µl of uptake medium that
comprised (in mM) 100 NaCl, 80 mannitol, and 20 HEPES-Tris, pH 7.4. For
Pi or glucose uptake, the uptake
solution also contained 0.1 mM of
32Pi
or [3H]glucose,
respectively. For Na+ or
Cl
uptake, the uptake
solution also contained 100 mM
22Na+
or
36Cl
.
To examine the effect of Cl
replacement, BBM uptake was measured with
Cl
-free uptake solutions
where NaCl was replaced by sodium gluconate. Incubation was terminated
at indicated times by adding 2 ml of ice-cold stop medium that
comprised (in mM) 100 NaCl, 100 mannitol, and 20 K2HPO4,
pH 7.4. The suspension was filtered and washed twice with 2 ml of stop
solution. The filter membrane was then dissolved in 5 ml of
scintillation fluid (UltimaGold, Packard) and counted for radioactivity
in a liquid scintillation counter (model 1600 TR, Packard). All
measurements were carried out in triplicate and expressed as nanomoles
per milligram protein per unit time. The protein concentration was
assayed using Coomassie brilliant blue G250 with bovine serum albumin
as the reference protein (25). For those studies where outward formate
or Cl
gradients were
imposed, BBM vesicles were preequilibrated for 3 h before uptake
measurements with a solution that comprised (in mM) 100 mannitol, 100 K, 100 gluconate, 10 Tris, and 16 HEPES, pH 7.4, as control, or with 40 gluconate replaced by formate or Cl
.
Materials. Radioisotopes including
[32P]phosphoric acid,
22Na,
36Cl, and
[3H]glucose were
purchased from Amersham (Arlington Heights, IL). All other chemicals
were purchased from Sigma (St. Louis, MO).
Data analysis. Measurements made in
triplicate were averaged. These values were pooled, and means ± SE
were determined for the number of experiments indicated. Significance
of difference was determined by two-tailed Student's
t-test for paired and unpaired data as appropriate.
 |
RESULTS |
Effect of
Cl
replacement. To examine the effect of
Cl
replacement, BBM
32P uptake was measured using
Cl
-free uptake solution
where NaCl was replaced by sodium gluconate. As shown in Fig.
1,
Cl
replacement
significantly lowered BBM 32P
uptake. With Na+-free uptake
solution where Na+ was replaced by
tetramethylammonium, the BBM 32P
uptake was significantly lowered, and the replacement of
Cl
with gluconate did not
further lower the 32P uptake. Thus
Cl
replacement affected
only the Na+-dependent BBM
32P uptake. The effect of
Cl
on BBM
32P uptake was concentration
dependent, so that a stepwise decrease in
Cl
concentration from 150 mM caused a gradual decrease in BBM
32P uptake, reaching a maximal
inhibition of up to 40% at 0 mM
Cl
(Fig.
2). The effect of
Cl
replacement on BBM
32P uptake kinetic parameters was
analyzed by varying Pi
concentrations in the uptake solution (0.01-3 mM). These studies
revealed an effect of Cl
replacement to lower the
Vmax values (from
2.18 ± 0.22 to 1.11 ± 0.10 nmol · min
1 · mg
protein
1;
n = 5, P < 0.01) without altering the
apparent Km for
Pi (from 0.24 ± 0.03 to 0.22 ± 0.02 mM; n = 5, P > 0.1). To test whether there is a
reciprocal effect of Pi on
Cl
uptake, we examined the
effect of Pi on BBM
36Cl
uptake. As shown in Fig. 3, varying
Pi concentrations in the uptake
solution from 0 to 10 mM had no significant effect on BBM 36Cl
uptake. Similar to gluconate, replacement of
Cl
in the uptake solution
with other anions, such as NO
3, SO
4, isethionate, or
SCN
, also caused a
significant decrease in BBM 32P
uptake (Table 1). In contrast,
Cl
replacement with these
anions had varying effects on BBM
[3H]glucose uptake. As
shown in Table 1, BBM
[3H]glucose uptake was
significantly lowered with gluconate,
SO
4 or isethionate, but was
significantly increased with
SCN
and not altered with
NO
3.

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Fig. 1.
Effect of Cl replacement on
brush-border membrane (BBM) 32P
uptake. BBM 32P uptake measured at
indicated time periods was significantly lower with
Cl -free uptake solution
where NaCl was replaced with sodium gluconate ( ), compared with the
corresponding control uptake ( ). BBM
32P uptake was significantly
lowered when Na+ in uptake
solution was replaced by tetramethylammonium (+) and was not further
lowered when Cl was also
replaced with gluconate ( ). Results are means ± SE
(n = 5).
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Fig. 2.
Inhibition of BBM 32P uptake by
lowering Cl concentrations.
BBM 32P uptake at 1 min was
measured with uptake solutions where a stepwise decrease in
Cl concentrations was
induced by replacing NaCl with increasing concentrations of sodium
gluconate. BBM 32P uptake was
expressed as the percentage of uptake at 150 mM
Cl . Lowering
Cl concentration below 150 mM caused a gradual decrease in BBM
32P uptake, reaching a maximal
inhibition up to 40% at 0 mM
Cl . Results are means ± SE (n = 6).
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Fig. 3.
Effect of Pi on BBM
36Cl uptake. BBM
36Cl uptake at 1 min was measured
in uptake solutions containing varying concentrations of
Pi. Presence of
Pi at concentrations up to 10 mM
had no significant effect on BBM
36Cl
uptake. Results are means ± SE (n = 4).
|
|
Effect of
Cl
transport
inhibitors. To examine whether the
Cl
dependency of BBM
32P uptake involves
Cl
uptake, we tested the
effect of Cl
transport
inhibitors. As shown in Table 2, BBM
36Cl
uptake was significantly lowered by anion transport inhibitors including DIDS (1 mM), SITS (1 mM), and by
Cl
-channel
inhibitors including diphenylamine-2-carboxylate (DPC, 1 mM), niflumic acid (NF, 1 mM), and
5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 0.1 mM). As shown in
Fig. 4, these
Cl
transport inhibitors
also significantly lowered BBM 32P
uptake, which occurred in a
Cl
-dependent manner so that
no further inhibition on BBM 32P
uptake occurred when Cl
was
replaced with gluconate in the uptake solution. These results thus
indicate that the Cl
dependency of BBM 32P uptake
involves not only the presence, but also the uptake of Cl
.

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Fig. 4.
Effect of Cl transport
inhibitors on BBM 32P uptake. BBM
32P uptake at 1 min was measured
in control uptake solution (open bars) or in
Cl -free uptake solution
where NaCl was replaced with sodium gluconate (hatched bars). Addition
of Cl transport inhibitors,
including DIDS (1 mM), SITS (1 mM), diphenylamine-2-carboxylate (DPC, 1 mM), niflumic acid (NF, 1 mM), and
5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 0.1 mM), significantly
lowered BBM 32P uptake in control,
but not in Cl -free uptake
solution. Results are means ± SE
(n = 5).
* P < 0.05 vs. control.
|
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Effects of intravesicular formate and
Cl
. Since BBM
Cl
uptake has been shown to
involve anion-exchange mechanism, such as Cl
/formate exchange (16),
we studied the effect of intravesicular formate on BBM
Pi uptake. In addition, to test
whether the Cl
dependency
of BBM 32P uptake involves a
Pi/Cl
exchange transport process that may occur subsequent to
Cl
uptake, we studied the
effect of intravesicular
Cl
. As shown in Fig.
5, preloading BBM vesicles with 40 mM of
either formate or Cl
increased BBM
36Cl
uptake but did not affect BBM 32P
uptake. These results therefore provide no evidence for the involvement
of anion-exchange mechanisms in the effect of
Cl
on BBM
Pi uptake.

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Fig. 5.
Effects of intravesicular formate and
Cl . BBM vesicles were
preequilibrated for 3 h before uptake measurements with a solution that
comprised (in mM) 100 mannitol, 100 K, 100 gluconate, 10 Tris, and 16 HEPES, pH 7.4, as control, or with 40 gluconate replaced by formate or
Cl . Outward formate and
Cl gradients increased BBM
uptake of
36Cl
(top) but not
32P
(bottom). Results are means ± SE
(n = 4).
* P < 0.05 vs. control.
|
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Effect of transmembrane electrical
potential. The effect of transmembrane electrical
potential on BBM Cl
and
Pi uptake was examined by imposing
a negative or positive intravesicular potential with the use of
nigericin (10 µg/mg protein) and an outwardly or inwardly directed
K+ gradient, respectively
(intracellular and extracellular
K+ concentrations are indicated by
[K+]in
and
[K+]out,
respectively). The uptake of
36Cl
at voltage-clamp control condition
([K+]in/[K+]out,
50/50 mM: 79.6 ± 5.8 nmol · min
1 · mg
protein
1,
n = 5) was decreased with a negative
intravesicular potential ([K+]in/[K+]out,
50/0 mM: 63.4 ± 9.4 nmol · min
1 · mg
protein
1;
n = 5, P < 0.02) and increased with a
positive intravesicular potential
([K+]in/[K+]out,
0/50 mM: 113.5 ± 11.9 nmol · min
1 · mg
protein
1;
n = 5, P < 0.005), consistent with a net
inward movement of negative charges with
Cl
uptake. In contrast, as
shown in Fig. 6, BBM
32P uptake was higher with a
negative intravesicular potential
([K+]in/[K+]out,
50/0 mM) and lower with a positive intravesicular potential ([K+]in/[K+]out,
0/50 mM), consistent with a net inward movement of positive charges
with Pi uptake. Nevertheless,
Cl
replacement continued to
lower BBM 32P uptake irrespective
of changes in transmembrane electrical potential (Fig. 6). The net
inward movement of negative charges induced by
Cl
uptake therefore cannot
be accountable for the Cl
dependency of BBM Pi uptake. Also
evident from Fig. 6, the
Cl
-dependent component of
BBM Pi uptake, i.e., the
difference between BBM 32P uptake
in the presence and absence of
Cl
, remained constant at
different transmembrane electrical potentials (1.32 ± 0.29 nmol · min
1 · mg
protein
1 at
[K+]in/[K+]out
of 50/0 mM; 1.44 ± 0.32 nmol · min
1 · mg
protein
1 at
[K+]in/[K+]out
of 50/50 mM; and 1.40 ± 0.32 nmol · min
1 · mg
protein
1 at
[K+]in/[K+]out
of 0/50 mM; n = 5, P > 0.6). This observation suggests
that the Cl
-dependent BBM
Pi uptake is an electroneutral
transport process. In contrast,
Cl
-independent component of
BBM Pi uptake, i.e., BBM
32P uptake in the absence of
Cl
, is electrogenic with a
net inward movement of positive charges (Fig. 6).

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Fig. 6.
Effect of transmembrane electrical potential. BBM vesicles were
preequilibrated for 3 h before uptake measurements with a solution that
comprised (in mM) 200 mannitol, 50 K+, 50 gluconate, 20 HEPES, pH 7.4 (for intracellular K+
concentration,
[K+]in,
of 50 mM); or 300 mannitol, 20 HEPES, pH 7.4 (for
[K+]in = 0). Nigericin (10 µg/mg protein) was added during preincubation.
BBM 32P uptake at 1 min was
measured in control uptake solution (open bars) that comprised (in mM)
50 Na+, 50 K+, 100 Cl , 100 mannitol, 20 HEPES,
pH 7.4 (for extracellular K+
concentration,
[K+]out,
of 50 mM); or 50 Na+, 50 Cl , 200 mannitol, 20 HEPES,
pH 7.4 (for
[K+]out = 0); or in Cl -free uptake
solutions with similar compositions except for the replacement of
Cl with gluconate (hatched
bars). Compared with voltage-clamp control condition
([K+]in/[K+]out,
50/50 mM), BBM 32P uptake was
higher with a negative intravesicular potential
([K+]in/[K+]out,
50/0 mM) and lower with a positive intravesicular potential
([K+]in/[K+]out,
0/50 mM). Cl replacement
significantly lowered BBM 32P
uptake (P < 0.05) by similar extent
regardless of changes in transmembrane electrical potential. Results
are means ± SE (n = 6).
* P < 0.05 vs. respective
group at
[K+]in/[K+]out,
50/50 mM.
|
|
Effect of
Cl
replacement on BBM
22Na+
uptake.
To examine whether the effect of
Cl
on BBM
Pi uptake was mediated through its
effect on BBM Na+ uptake, we
studied the effects of Cl
replacement and Cl
transport inhibitors on BBM
22Na+
uptake. We found that replacement of
Cl
in the uptake solution
with gluconate lowered BBM
22Na+
uptake from 208 ± 4.5 to 154 ± 7.5 nmol · min
1 · mg
protein
1
(n = 5, P < 0.005). However, as shown in
Table 2,
Cl
transport inhibitors,
including DIDS, SITS, DPC, NF, and NPPB, did not affect BBM
22Na+
uptake. Thus the Cl
dependency of BBM Na+ uptake may
involve the presence of Cl
and/or the uptake of Cl
through mechanisms not inhibited by these
Cl
transport inhibitors.
Combined with the fact that these
Cl
transport inhibitors
significantly lowered BBM 32P
uptake, it seems unlikely that
Cl
interacts with BBM
Pi uptake through its effect on
Na+ uptake.
Effect of
Cl
replacement on rat and mouse
BBM Pi uptake.
The effect of Cl
replacement on
32Pi
uptake was also examined with BBM vesicles isolated from rat and mouse
kidneys. In these studies, kidneys from at least two rats
(Sprague-Dawley, 250-300 g) or four mice (C57BL/6J, 25-30 g)
were pooled, and BBM vesicles were prepared from kidney cortex with the
enrichment of BBM marker enzymes monitored in a fashion similar to that
with rabbit kidneys. As shown in Fig. 7,
replacement of Cl
in the
uptake solution by gluconate inhibited
32Pi
uptake to a similar extent in both rat and mouse renal BBM compared with rabbit renal BBM.

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Fig. 7.
Effect of Cl replacement on
rat and mouse BBM Pi uptake.
32Pi
uptake at 1 min was measured with BBM vesicles isolated from rat and
mouse kidneys, either in control uptake solution or in
Cl -free uptake solution
where NaCl was replaced with sodium gluconate. Results are means ± SE (n = 3) and are expressed as the
percentage inhibition by Cl
replacement.
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|
 |
DISCUSSION |
In our present study, we found that
Cl
replacement in the
uptake solution with other anions, including gluconate,
NO
3, SO
4, isethionate, and
SCN
, lowered BBM
Pi uptake up to 40% (Table 1;
Figs. 1 and 2). Cl
replacement affected only the
Na+-dependent BBM
Pi uptake, so that a component of
BBM Na+-dependent
Pi uptake appears to be also
Cl
dependent. The
Cl
dependency of BBM
Pi uptake involves the uptake of
Cl
, because inhibition of
BBM Cl
uptake by
Cl
transport inhibitors
similarly lowered BBM Pi uptake in
a Cl
-dependent manner (Fig.
4). In contrast, as shown in Fig. 3,
Pi did not affect BBM
36Cl
uptake. The lack of effect of Pi
on Cl
uptake suggests that
the interaction between BBM Pi and
Cl
uptake consists mainly
of a Cl
dependency of BBM
Pi uptake. However, we cannot
completely exclude the possibility that a component of
Pi-dependent BBM
Cl
uptake may exist that is
sufficiently small compared with the total
Cl
uptake to remain
undetected by our uptake measurements.
It has been well demonstrated that the anion-exchange mechanism such as
Cl
/formate exchange
constitutes an important transport process responsible for
Cl
uptake by renal BBM (1,
16). Our current study supports this notion, as BBM
Cl
uptake was stimulated
when an outwardly directed formate gradient was imposed (Fig. 5).
However, we found that this maneuver did not affect BBM
Pi uptake (Fig. 5), suggesting
that the interaction between BBM
Cl
uptake and
Pi uptake does not involve the
Cl
/formate exchange. We
also found that an outwardly directed
Cl
gradient did not affect
BBM Pi uptake (Fig. 5), suggesting
that the Cl
dependency of
BBM Pi uptake is not mediated by a
transport process such as
Cl
/Pi
exchange following an initial uptake of
Cl
into the intravesicular
space. Our data therefore do not provide evidence for the involvement
of anion exchangers in the interaction between BBM
Cl
uptake and
Pi uptake. Since anion
conductances have been shown to be present on the apical membrane of
the proximal tubule (17, 28), our data may be more in favor of the
involvement of Cl
channels.
Our current study shows that changes in transmembrane electrical
potential affected both BBM
Cl
and
Pi uptake. BBM
Cl
uptake was enhanced by a
positive, and suppressed by a negative, intravesicular potential,
consistent with a net inward movement of negative charges with BBM
Cl
uptake. In contrast, BBM
Pi uptake was enhanced by a
negative, and suppressed by a positive, intravesicular potential,
consistent with a net inward movement of positive charges with BBM
Pi uptake (Fig. 6). These results
raised the possibility that the effect of
Cl
on BBM
Pi uptake may be due to the inward
movement of negative charges with
Cl
uptake. However, this
possibility seems unlikely given the continuing lowering effect of
Cl
replacement on BBM
Pi uptake at varying
intravesicular potentials (Fig. 6).
Although Cl
replacement
lowered BBM
22Na+
uptake up to 25%, Cl
transport inhibitors, including DIDS, SITS, DPC, NF, and NPPB, did not
affect BBM
22Na+
uptake (Table 2). Since these
Cl
transport inhibitors
were effective in lowering BBM Pi
uptake, the Cl
dependency
of BBM Pi uptake thus seems
unlikely to be mediated through changes in
Na+ uptake. The diverse effects of
different anions on BBM glucose uptake may also lend support to this
notion. However, it is possible that the portion of BBM
Na+ uptake coupled to
Pi uptake may constitute only a
small fraction of the total Na+
uptake and cannot be easily detected with our uptake measurements using
Cl
transport inhibitors. We
therefore cannot completely exclude the possibility that
Cl
affects BBM
Pi uptake through its effect on
BBM Na+ uptake that is coupled to
Pi uptake.
It is not clear from our current study whether the
Cl
dependency of BBM
Pi uptake is related to the anion
channel function of the rabbit renal type I
Na-Pi cotransporter, NaPi-1. As is evident from Fig. 6, although the
Cl
-independent component of
BBM Pi uptake varied with changes
in intravesicular potential, the
Cl
-dependent component of
BBM Pi uptake remained constant
with varying intravesicular potentials. It thus appears that, although
Cl
-independent BBM
Pi uptake is electrogenic and
carries a net inward movement of positive charges,
Cl
-dependent BBM
Pi uptake is electroneutral. These
results may be relevant to the recently described electrophysiological
characteristics of Na-Pi
cotransporters expressed in Xenopus
oocytes (5, 6). These studies showed that type II
Na-Pi cotransporters exhibited an
electrogenic transport with inward movement of positive charges (5),
whereas type I Na-Pi
cotransporter, NaPi-1, exhibited electrogenic transport only at high
extracellular Pi concentrations (3 mM) (6). In our current studies with rabbit renal BBM at low
Pi concentration (0.1 mM), it is
possible that the type I Na-Pi
cotransporter, NaPi-1, mediated the
Cl
-dependent and
electroneutral component of Pi
uptake while the type II Na-Pi
cotransporter, NaPi-6, mediated the
Cl
-independent and
electrogenic component of Pi
uptake. However, it is also possible that a component of BBM
Pi uptake mediated by type II
Na-Pi cotransporter may be
Cl
dependent, which occurs
in an electroneutral fashion when
Cl
is cotransported through
Cl
channels either related
or unrelated to type I Na-Pi
cotransporter and nullifies the positive charges. Indeed, it has been
reported recently that both Pi
uptake and Cl
conductance
induced by NaPi-1 in Xenopus oocytes
appeared to be separate functions, and the NaPi-1-induced
Pi uptake was not affected by
Cl
channel inhibitor, NPPB
(4). Furthermore, preliminary observations with
Xenopus oocytes expressing type II
Na-Pi cotransporters showed a
decrease in Pi-induced inward
current under voltage-clamp condition when extracellular
Cl
was replaced with
gluconate (I. Forster, personal communication). Resolution of these
issues clearly requires further studies. In separate studies, we also
found that BBM derived from rat and mouse kidneys exhibited a similar
component of Pi uptake which is
Cl
dependent (Fig. 7). Thus
the Cl
dependency is a
feature of BBM Pi uptake not only
limited to rabbit kidneys.
Relevant to our current finding of a
Cl
-dependent component of
BBM Pi uptake is the recent report
that a group of hereditary kidney stone diseases associated with
proximal tubular dysfunctions, including decreased
Pi reabsorption (24), shares a
common defect in the renal
Cl
channel, CLC-5 (20).
CLC-5 belongs to a growing family of
Cl
channels without
similarity in molecular structure with NaPi-1 (11). The function of
CLC-5 and the mechanism whereby its defect leads to proximal tubular
dysfunction are currently not known. However, CLC-5 has recently been
found to be expressed in the proximal tubular cells, where it is
localized closely with H+-ATPase
in a region below BBM densely packed with endocytic vesicles (13).
Although it is not clear whether CLC-5 is directly involved in BBM
Cl
transport, it seems
likely that CLC-5 may serve as the
Cl
channel of proximal
tubular endosomes. Defect in CLC-5 can thus affect BBM transport
indirectly through the disrupted endocytotic process. Although these
possibilities still need to be examined, it does appear that
Cl
is emerging as an
important anion capable of regulating BBM
Pi transport, either directly or indirectly.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant RO1-DK-47203 and by funds from the
Department of Veterans Affairs.
 |
FOOTNOTES |
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: N. Yanagawa,
Nephrology Division (111R), Sepulveda Veterans Affairs Medical Center,
16111 Plummer St., Sepulveda, CA 91343 (E-mail:
nori{at}ucla.edu).
Received 13 November 1998; accepted in final form 25 May 1999.
 |
REFERENCES |
1.
Aronson, P. S.
Role of ion exchangers in mediating NaCl transport in the proximal tubule.
Kidney Int.
49:
1665-1670,
1996[Medline].
2.
Berndt, T. J.,
and
F. G. Knox.
Renal regulation of phosphate excretion.
In: The Kidney: Physiology and Pathophysiology, edited by D. W. Seldin,
and G. Giebisch. New York: Raven, 1992, p. 2511-2532.
3.
Bolino, A.,
M. Devoto,
G. Enia,
C. Zoccali,
J. Weisenbach,
and
G. Romeo.
Genetic mapping in the Xp11.2 region of a new form of X-linked hypophosphatemic rickets.
Eur. J. Hum. Genet.
1:
269-279,
1993[Medline].
4.
Broer, S.,
A. Schuster,
C. A. Wanger,
A. Broer,
I. Forster,
J. Biber,
H. Murer,
A. Werner,
F. Lang,
and
A. E. Busch.
Chloride conductance and Pi transport are separate functions induced by the expression of NaPi-1 in Xenopus Oocytes.
J. Membr. Biol.
164:
71-77,
1998[Medline].
5.
Busch, A. E.,
S. Waldegger,
T. Herzer,
J. Biber,
D. Markovich,
G. Hayes,
H. Murer,
and
F. Lang.
Electrophysiological analysis of Na+/Pi cotransport mediated by a transporter cloned from rat kidney and expressed in Xenopus oocytes.
Proc. Natl. Acad. Sci. USA
91:
8205-8208,
1994[Abstract].
6.
Busch, A. E.,
A. Schuster,
S. Waldegger,
C. A. Wagner,
G. Zempel,
S. Broer,
J. Biber,
H. Murer,
and
F. Lang.
Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions.
Proc. Natl. Acad. Sci. USA
93:
5347-5351,
1996[Abstract/Free Full Text].
7.
Chong, S. S.,
K. Kritsjanson,
H. Y. Zoghbin,
and
M. R. Hughes.
Molecular cloning of the cDNA encoding a human renal sodium phosphate transport protein and its assignment to chromosome 6p21.3-p23.
Genomics
18:
335-339,
1993.
8.
Chong, S. S.,
C. A. Kozak,
L. Liu,
K. Kritsjanson,
S. T. Dunn,
J. E. Bourdeau,
and
M. R. Hughes.
Cloning, genetic mapping, and expression analysis of a mouse renal sodium-dependent phosphate cotransport.
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F1038-F1045,
1995[Abstract/Free Full Text].
9.
Collins, J. F.,
and
F. K. Ghishan.
Molecular cloning, functional expression, tissue distribution and in situ hybridization of the renal sodium phosphate (Na/Pi) transporter in the control and hypophosphatemic mouse.
FASEB J.
8:
862-868,
1994[Abstract/Free Full Text].
10.
Dousa, T. P.,
and
S. A. Kempson.
Regulation of renal brush border membrane transport of phosphate.
Miner. Electrolyte Metab.
7:
113-121,
1982[Medline].
11.
Fisher, S. E.,
I. Black,
S. E. Lloyd,
S. H. S. Pearce,
R. V. Thakker,
and
I. W. Craig.
Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis).
Genomics
29:
598-606,
1995[Medline].
12.
Frymoyere, P. A.,
S. J. Scheinman,
P. B. Dunham,
D. B. Jones,
P. Hueber,
and
E. T. Schroeder.
X-linked recessive nephrolithiasis with renal failure.
N. Engl. J. Med.
325:
681-686,
1991[Abstract].
13.
Gunther, W.,
A. Luchow,
F. Cluzeaud,
A. Vandewalle,
and
T. J. Jentsch.
CLC-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytotically active kidney cells.
Proc. Natl. Acad. Sci. USA
95:
8075-8080,
1998[Abstract/Free Full Text].
14.
Hartmann, C.,
C. A. Wagner,
A. E. Busch,
D. Markovich,
J. Biber,
F. Lang,
and
H. Murer.
Transport characteristics of a murine renal Na/Pi cotransporter.
Pflügers Arch.
430:
830-836,
1995[Medline].
15.
Jentsch, T. J.,
and
W. Gunther.
Chloride channels: an emerging molecular picture.
Bioessays
19:
117-126,
1997[Medline].
16.
Karniski, L. P.,
and
P. S. Aronson.
Chloride-formate exchange with formic acid recycling: a mechanism of active Cl transport across epithelial membrane.
Proc. Natl. Acad. Sci. USA
82:
6362-6265,
1985[Abstract].
17.
Krick, W.,
A. Dolle,
Y. Hagos,
and
G. Burckhardt.
Characterization of the chloride conductance in porcine renal brush-border membrane vesicles.
Pflügers Arch.
435:
415-421,
1998[Medline].
18.
Levi, M.,
M. Lotscher,
V. Sorribas,
M. Custer,
M. Arar,
B. Kaissling,
H. Murer,
and
J. Biber.
Cellular mechanisms of acute and chronic adaptation of rat renal Pi transporter to alterations in dietary Pi.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F900-F908,
1994[Abstract/Free Full Text].
19.
Li, H.,
and
Z. Xie.
Molecular cloning of two rat Na+/Pi cotransporters: evidence for differential tissue expression of transcripts.
Cell Mol. Biol. Res.
41:
451-460,
1995[Medline].
20.
Lloyd, S. E.,
S. H. S. Pearce,
S. E. Fisher,
K. Steinmeyer,
B. Schwappach,
S. J. Scheinman,
B. Harding,
A. Bolino,
M. Devoto,
and
P. Goodyer.
A common molecular basis for three inherited kidney stone diseases.
Nature
379:
445-449,
1996[Medline].
21.
Magagnin, S.,
A. Werner,
D. Markovich,
V. Sorribas,
J. Biber,
and
H. Murer.
Expression cloning of human and rat renal cortex Na/Pi cotransport.
Proc. Natl. Acad. Sci. USA
90:
5979-5983,
1993[Abstract].
22.
Morduchowicz, G. A.,
and
N. Yanagawa.
Ca2+ dependency of Na+ transport by rabbit renal brush border membrane.
J. Membr. Biol.
109:
105-112,
1989[Medline].
23.
Murer, H.,
and
J. Biber.
Molecular mechanisms of renal apical Na/phosphate cotransport.
Annu. Rev. Physiol.
58:
607-618,
1996[Medline].
24.
Scheinman, S. J.
X-linked hypercalciuric nephrolithiasis: Clinical syndromes and chloride channel mutations.
Kidney Int.
53:
3-17,
1998[Medline].
25.
Sedmak, J. J.,
and
S. E. Grossberg.
A rapid, sensitive and versatile assay for protein using Coomassie Brilliant blue G250.
Anal. Biochem.
79:
544-552,
1977[Medline].
26.
Sorribas, V.,
D. Markovich,
G. Hayes,
G. Stange,
J. Forgo,
J. Biber,
and
H. Murer.
Cloning of a Na/Pi cotransporter from opossum kidney cells.
J. Biol. Chem.
269:
6615-6621,
1994[Abstract/Free Full Text].
27.
Verri, T.,
D. Markovich,
C. Perego,
F. Norbis,
G. Stange,
V. Sorribas,
J. Biber,
and
H. Murer.
Cloning and regulation of a rabbit Na-Pi cotransporter.
Am. J. Physiol.
269 (Renal Physiol. 38):
F626-F633,
1995.
28.
Warnock, D. G.,
and
V. J. Yee.
Chloride uptake by brush border membrane vesicles isolated from rabbit renal cortex.
J. Clin. Invest.
67:
103-115,
1981[Medline].
29.
Werner, A.,
M. L. Moore,
N. Mantei,
J. Biber,
G. Semenza,
and
H. Murer.
Cloning and expression of cDNA for a Na/Pi-cotransport system of kidney cortex.
Proc. Natl. Acad. Sci. USA
88:
9608-9612,
1991[Abstract/Free Full Text].
30.
Werner, A.,
H. Murer,
and
R. Kinne.
Cloning and expression of a renal Na-Pi cotransport system from flounder.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F311-F317,
1994[Abstract/Free Full Text].
31.
Wrong, O. M.,
A. G. W. Norden,
and
T. G. Feest.
Dent's disease; a familial proximal renal tubular syndrome with low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, metabolic bone disease, progressive renal failure and a marked male predominance.
QJM
87:
473-493,
1994[Abstract].
Am J Physiol Renal Physiol 277(4):F506-F512
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