Effects of P-glycoprotein on cell volume regulation in mouse
proximal tubule
Yukio
Miyata,
Yasushi
Asano, and
Shigeaki
Muto
Department of Nephrology, Jichi Medical School, Minamikawachi,
Tochigi 329 - 0498, Japan
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ABSTRACT |
The role of P-glycoprotein (P-gp) in cell volume
regulation was examined in isolated nonperfused proximal tubule S2
segments from wild-type (WT) mice and those in which both
mdr1a and mdr1b genes were knocked out (KO). When
the osmolality of the bathing solution was rapidly decreased from 300 to 180 mosmol/kgH2O, the tubules from both the WT and KO
mice exhibited regulatory volume decrease (RVD) by a similar magnitude
after the initial cell swelling. The peritubular addition of two P-pg
inhibitors (verapamil and cyclosporin A) to either group of the tubules
had no effect on RVD. When the tubules from the WT mice were rapidly
exposed to a hyperosmotic solution (500 mosmol/kgH2O)
including 200 mM mannitol, they abruptly shrank to 82.1% of their
control volume but remained in a shrunken state during the experimental
period, indicating a lack of regulatory volume increase (RVI). The
addition of the two P-gp inhibitors, but not the inhibitor of the renal
organic cation transport system (tetraethylammonium), to the tubules
from the WT mice resulted in RVI. Surprisingly, when the tubules from the KO mice were exposed to the hyperosmotic solution, they abruptly shrank to 79.9% of their control volume, and then gradually swelled to
87.7% of their control volume, showing RVI. However, exposure of the
tubules from the KO mice to the hyperosmotic solution in the presence
of the two P-gp inhibitors had no effect on RVI. When the tubules of
the WT mice were exposed to the hyperosmotic solution including either
of the two P-gp inhibitors, in the absence of peritubular
Na+ or in the presence of peritubular
ethylisopropylamiloride (EIPA; the specific inhibitor of
Na+/H+ exchange), they did not exhibit RVI. In
the tubules of the KO mice, both removing peritubular Na+
and adding peritubular EIPA inhibited RVI induced by the hyperosmotic solution. We conclude that 1) in mouse proximal tubule, P-gp
modulates RVI during hyperosmotic stress but not RVD during hyposmotic
stress and 2) basolateral membrane
Na+/H+ exchange partly contributes to the
P-gp-induced modulation of RVI under hyperosmotic stress.
cell volume regulation; nonperfused tubule; mdr1a; mdr1b; basolateral sodium-hydrogen exchange
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INTRODUCTION |
MOST ANIMAL CELLS
REGULATE their volume in response to changes in the osmolality of
the environment (reviewed in Refs. 17 and 20). After cell swelling in
hyposmotic medium, regulatory volume decrease (RVD) is generally
achieved by the loss of ions and other osmolytes and the concomitant
loss of water. Of the various efflux systems that can contribute to
RVD, swelling-activated Cl
channels are present in a
number of cell types (17, 20).
P-glycoprotein (P-gp) was initially identified through its ability to
confer multidrug resistance (MDR) in mammalian tumor cells (reviewed in
Ref. 8). P-gp is a member of the ATP-binding cassette
superfamily of transporters (8) and utilizes ATP to pump
hydrophobic drugs out of the cells, decreasing their intracellular concentrations and hence their toxicity. Humans have one gene encoding
drug-transporting P-gp (MDR1), whereas mice have two genes,
mdr1a (also called mdr3) and mdr1b
(also called mdr1) (8, 12, 24). In the
mouse, the mdr1a gene is predominantly expressed in
intestine, liver, and blood capillaries of brain and testis, whereas
the mdr1b is predominantly expressed in adrenal glands, placenta, and ovarium (8). Similar levels of
mdr1a and mdr1b gene expression are observed in
the kidney (2, 25). In the mouse, mdr1a and
mdr1b together fulfill the same function as MDR1 does in humans.
The expression of P-gp in the kidney was thought to occur exclusively
in the apical membrane of the proximal tubule epithelium, where P-gp
was expected to participate in the excretion of xenobiotics (5,
27). Consistent with a role for P-gp as an excretory transporter, cell lines that express many properties of proximal tubule
cells, including LLC-PK1 (13, 23, 26), opossum
kidney (11), and cultured mouse proximal tubule cells
(4), exhibit net secretion of several MDR substrates,
including verapamil and cyclosporin A. Also, very recently we used
isolated perfused mouse proximal tubules to demonstrate that
P-gp-mediated drug efflux capacity indeed exists in the apical membrane
of proximal tubule S2 segments from wild-type (WT) mice but is lacking
in that of mice in which both mdr1a and mdr1b
genes were disrupted (KO) (28). In addition, after
administration of digoxin, drug accumulation in kidneys of KO mice was
greater than in those of WT mice (24, 28).
In addition to drug-transporting activity, P-gp itself has been
reported to have intrinsic channel activity in cultured NIH3T3 fibroblasts transfected with human MDR1 cDNA
(30). Subsequent data have shown that P-gp regulates RVD
through its effects on Cl
channel activation in HTC-R
hepatoma cells overexpressing P-gp activity (21) and in
Chinese hamster ovary cells transfected with the mdr1a gene
(LR73-1a cells) (29). Recently, it has been demonstrated that P-gp has a separate mechanism of drug transport and
Cl
channel function during hyposmotic stress in NIH3T3
fibroblasts transfected with human MDR1 (7) and
in cultured human breast cancer cells transfected with human
MDR1 cDNA (1). However, it is not known whether
P-gp actually regulates RVD in intact cells, including renal tubule
cells. Also, in most studies using cultured cells overexpressing P-gp
activity, cell volume under hyposmotic conditions has not yet been
measured, although swelling-activated Cl
channels have
been well characterized (1, 7, 30, 31).
In most animal cells, after cell shrinkage in hyperosmotic medium,
regulatory volume increase (RVI) is generally achieved by the gain of
ions and other osmolytes (17, 20). In sharp contrast, when
rabbit proximal tubule cells are suddenly exposed to hyperosmotic
mannitol, NaCl, and raffinose solutions, they rapidly shrink but remain
reduced in size (6, 14). However, the mechanisms
responsible for the lack of RVI have not been fully demonstrated. Also,
it is not known whether hyperosmotic stress affects cell volume in
mouse proximal tubule, and if so, whether and how P-gp contributes to
cell volume regulation under hyperosmotic conditions.
Therefore, we used isolated nonperfused proximal tubule S2 segments
from KO and WT mice to examine the role of P-gp in cell volume
regulation during hyposmotic and hyperosmotic stress.
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METHODS |
Animals.
KO mice used in this study were originally described by Schinkel et al.
(24). Male KO and FVB (WT) mice serving as the control (body wt 25-40 g) were purchased from Taconic Engineering
(Germantown, NY). The animals were maintained in a controlled
environment and had free access to a standard rodent chow and tap water
ad libitum until the experiments. Ages of the KO animals were matched
with their WT controls.
In vitro microperfusion.
Mice from both groups were anesthetized with an intraperitoneal
injection of pentobarbital sodium (4 mg/100 g body wt), and both
kidneys were removed. Slices of 1-2 mm were taken from the coronal
section of each kidney and transferred to a dish containing a cold
intracellular fluid-like solution having the following composition (in
mM): 14 KH2PO4, 44 K2HPO4, 15 KCl, 9 NaHCO3, and 160 sucrose. Proximal tubule S2 segments were then dissected by fine
forceps under a stereomicroscope and transferred to a bath chamber
mounted on an inverted microscope (IMT-2, Olympus, Tokyo, Japan).
Both proximal and distal ends of the tubule were drawn into and crimped
by glass micropipettes as originally described by Dellasega and
Grantham (3). In this preparation, both ends of the tubule
are occluded so that the lumen becomes and remains collapsed. A
flow-through bath system was utilized to permit rapid exchange of the
bath fluid. The bathing solution flowed by gravity at 4.5-5.5
ml/min from the reservoirs through a water jacket to stabilize the bath
temperature at 37°C.
Solutions.
The composition of all bathing solutions is given in Table
1. The osmolality of all the solutions
was measured by freezing-point depression before the experiments. All
the solutions were continuously gassed with a 5% CO2-95%
O2 mixture.
After tubules were tightly crimped between the two glass micropipettes,
they were bathed in the isosmotic control solution (solution
1) at 37°C for 10-15 min to allow them to equilibrate. After the equilibration period, the cell volume was measured as described below.
Measurements of tubule cell volume.
Before, during, and after exposure of the tubules to hyposmotic or
hyperosmotic solutions, we took serial photographs, at a magnification
of ×400, that focused on the center of the tubule. The photographs
were magnified, and the outer diameter of the tubule (d) was
estimated from similar photographs of a calibrated micrometer slide as
reference. Assuming the tubule is cylindrical, the apparent cell volume
could then be calculated according to the following equation
where V is the apparent tubule volume, and L is a
constant tubule length. The values obtained are expressed as a
percentage relative to control isosmotic tubule cell volume.
Chemicals.
Verapamil, cyclosporin A, and ethylisopropylamiloride (EIPA) were
purchased from Sigma (St. Louis, MO). Other high-grade chemicals were
obtained from Wako (Osaka, Japan).
Verapamil was dissolved in dimethylsulfoxide at 0.1% final
concentration. Cyclosporin A was dissolved in ethanol at 0.1% final concentration. Tetraethylammonium (TEA) was dissolved in distilled water at 0.1% final concentration. EIPA was dissolved in methanol at
0.1% final concentration. Equivalent concentrations of vehicle were
added as a control for individual protocols.
Statistics.
The data are expressed as means ± SE. Comparisons were performed
by Student's t-test or one-way analysis of variance in
combination with Fisher's protected least significant differences
test, where appropriate. P values <0.05 were considered significant.
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RESULTS |
Effect of hyposmotic solutions on cell volume.
At first, we examined the effect of hyposmotic solutions on cell volume
in proximal tubules from WT and KO mice. For this purpose, we used the
isosmotic control solution containing 110 mM NaCl (solution
1; 300 mosmol/kgH2O, Table 1), the isosmotic control
solution containing 50 mM NaCl plus 120 mM mannitol (solution 2; 300 mosmol/kgH2O, Table 1), and the hyposmotic
solution (solution 3; 180 mosmol/kgH2O, Table
1), which was made by removal of mannitol from solution 2.
Results are shown in Fig. 1. When both
groups of tubules were initially bathed in solution 1 and
were then perfused with solution 2 for 10 min, there was no
appreciable effect on cell volume (data not shown). However, when the
tubules from the WT mice were initially incubated in solution
2 and were then treated with solution 3 for 10 min,
they abruptly swelled to 133.0 ± 1.8% (n = 6) of
their control volume and then gradually shrank over the next 4-6
min to 115.0 ± 2.2% (n = 6) (Fig.
1A). Similarly, after the osmolality of the bathing solution
was abruptly reduced from 300 to 180 mosmol/kgH2O for 10 min, the tubules from the KO mice rapidly swelled to 129.5 ± 1.4% (n = 6) of their control volume and then
gradually shrank to 111.8 ± 1.1% (n = 6) of
their control volume (Fig. 1B). The changes in cell volume
in the tubules from the KO mice under hyposmotic conditions were
comparable to those from the WT mice. Therefore, both groups of tubules
exhibited RVD during hyposmotic stress.

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Fig. 1.
Effects of hyposmotic solution on cell volume in isolated
nonperfused proximal tubule S2 segments from wild-type (WT;
A) and knockout (KO; B) mice. Both groups of
tubules were initially bathed in solution 2 and were then
treated with solution 3 for 10 min. Values are means ± SE of 6 tubules from WT and KO mice and are expressed as a percentage
relative to the control isosmotic tubule cell volume treated with
solution 2. *P < 0.05 vs. maximum cell
volume increase.
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Next, we examined whether the hyposmolality-induced cell volume changes
in both groups of tubules are affected by two inhibitors of P-gp
(verapamil and cyclosporin A). For this purpose, we monitored cell
volume of both groups of tubules in the absence and presence of bath
verapamil (100 µM) or cyclosporin A (5 µM). Results are shown in
Fig. 2. The addition of verapamil or
cyclosporin A alone had no effect on cell volume in the tubules from
the WT or KO mice (Fig. 2). When the tubules from the WT mice were
treated with solution 3 in the presence of verapamil or
cyclosporin A for 10 min, they swelled to 130.7 ± 3.9 (n = 4) or 130.6 ± 1.1% (n = 7)
of their control volume and then shrank to 114.5 ± 2.0 (n = 4) or 115.9 ± 1.6% (n = 7)
of their control volume, respectively (Fig. 2, A and
B). Similarly, exposure of the tubules from the KO mice to
solution 3 in the presence of verapamil or cyclosporin A for
10 min caused them to swell to 127.6 ± 2.3 (n = 9) or 130.0 ± 1.3% (n = 6) of their control
volume and then to shrink to 112.2 ± 1.3 (n = 9)
or 116.1 ± 2.5% (n = 6) of their control volume, respectively (Fig. 2, C and D). In both groups of
tubules, changes in cell volume under hyposmotic conditions in the
presence of the two inhibitors of P-gp were not significantly different
from those in their absence. Therefore, in both groups of tubules, the
two inhibitors of P-gp caused no effect on RVD during hyposmotic stress.

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Fig. 2.
Effects of hyposmotic solution on cell volume in isolated
nonperfused proximal tubule S2 segments from WT (A and
B) and KO mice (C and D) in the
presence of verapamil or cyclosporin A, respectively. Values are
means ± SE of 9 or 6 tubules of WT mice in the presence of
verapamil or cyclosporin A, respectively, and of 4 or 7 tubules of KO
mice in the presence of verapamil or cyclosporin A, respectively, and
are expressed as a percentage relative to the control isosmotic tubule
cell volume treated with the solution 2. Both groups of
tubules were initially bathed in solution 2 and were then
perfused with solution 2 containing verapamil (100 µM) or
cyclosporin A (5 µM) for 2 min. Thereafter, the tubules were treated
with solution 3 in the presence of verapamil or cyclosporin
A for 10 min. *P < 0.05 vs. maximum cell volume
increase.
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Effect of hyperosmotic solutions on cell volume.
Next, we examined the effect of hyperosmotic solutions on cell volume
in the proximal tubules from the WT and KO mice. For this purpose, the
tubules were initially bathed in the isosmotic control solution
(solution 1; 300 mosmol/kgH2O, Table 1) and were
then perfused with hyperosmotic solutions (500 mosmol/kgH2O) containing 200 mM mannitol (solution
4; Table 1) or urea (solution 5; Table 1). Results are
shown in Fig. 3. When the tubules from the WT mice were rapidly exposed to the hyperosmotic mannitol solution
for 10 min, they abruptly shrank to 82.1 ± 1.4%
(n = 6) of their control volume but remained in a
shrunken state during the experimental period, indicating a lack of RVI
(Fig. 3A). Surprisingly, when the tubules from the KO mice
were rapidly treated with the hyperosmotic mannitol solution for 10 min, they abruptly shrank to 79.9 ± 1.1% (n = 6)
of their control volume and then gradually swelled to 87.7 ± 1.1% (n = 6) of their control volume, showing a
presence of RVI (Fig. 3C).

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Fig. 3.
Effects of hyperosmotic solution on cell volume in isolated
nonperfused proximal tubule S2 segments from WT and KO mice. Both
groups of tubules were initially bathed in the solution 1 and were then treated with solution 4 or 5 for 10 min. Values are means ± SE of 6 or 8 tubules from the WT mice in
the presence of mannitol (A) or urea (B),
respectively, and 6 or 7 tubules from the KO mice in the presence of
mannitol (C) or urea (D), respectively, and are
expressed as a percentage relative to the control isosmotic tubule cell
volume treated with solution 1. *P < 0.05 vs. maximum cell volume decrease.
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The response of cell volume to the hyperosmotic urea solution was
strikingly different from that to the hyperosmotic mannitol solution
but was similar in both groups of tubules (Fig. 3). In the tubules of
the WT and KO mice, the reduction in cell volume was smaller
[88.5 ± 0.7 (n = 8) and 87.0 ± 1.0%
(n = 7) of their control volume, respectively] and
transient, tubules returning to their control volume within 2.5 min of
the experimental period (Fig. 3, B and D,
respectively). In the recovery period, the initial transient large
swelling of the cells was rapidly dissipated.
Next, we examined whether the hyperosmotic mannitol-induced cell volume
changes in both groups of tubules are influenced by the two inhibitors
of P-gp. For this purpose, both groups of tubules were treated with
solution 4 in the absence and presence of verapamil (100 µM) or cyclosporin A (5 µM). Results are shown in Fig.
4. The peritubular addition of verapamil
or cyclosporin A alone caused no effect on cell volume in either group
of tubules (Fig. 4). On the other hand, when the tubules from the WT
mice were rapidly treated with the hyperosmotic mannitol solution in
the presence of verapamil or cyclosporin A, they immediately shrank to
81.7 ± 1.9 (n = 7) or 80.2 ± 2.2%
(n = 4) of their control volume and then gradually
swelled to 89.7 ± 1.5 (n = 7) or 89.2 ± 1.6% (n = 4) of their control volume, respectively
(Fig. 4, A and B). In sharp contrast to treatment
with the inhibitors of P-gp, the peritubular addition of TEA (1 mM; an
inhibitor of the renal organic cation transport system) to the tubules
from the WT mice had no effect at all on RVI (Fig.
5). Accordingly, in the WT mice, addition of the two inhibitors of P-gp, but not the inhibitor of the renal organic cation transport system, resulted in RVI during hyperosmotic stress. In sharp contrast to WT mice, when the tubules from the KO mice
were rapidly exposed to the hyperosmotic mannitol solution in the
presence of verapamil or cyclosporin A, they abruptly shrank to
79.2 ± 1.9 (n = 6) or 81.4 ± 1.3%
(n = 6) of their control volume and then gradually
swelled to 89.7 ± 1.8 (n = 6) or 88.2 ± 1.2% (n = 6) of their control volume, respectively
(Fig. 4, C and D). In the KO mice, changes in
cell volume in the presence of the two inhibitors of P-gp were not
significantly different from those in their absence (see Figs.
3C and 4, C and D). Therefore, exposure of the tubules from the KO mice to hyperosmotic mannitol in
the presence of two inhibitors of P-gp had no effect on RVI during
hyperosmotic stress.

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Fig. 4.
Effects of hyperosmotic solution with mannitol on cell
volume in isolated nonperfused proximal tubule S2 segments from the WT
and KO mice in the presence of verapamil or cyclosporin A. Values are
means ± SE of 7 or 4 tubules of WT mice in the presence of
verapamil (A) or cyclosporin A (B), respectively,
and of 6 tubules of KO mice in the presence of verapamil (C)
or cyclosporin A (D) and are expressed as a percentage
relative to the control isosmotic tubule cell volume treated with the
solution 1. Both groups of tubules were initially bathed in
solution 1 and were then perfused with solution 1 containing verapamil (100 µM) or cyclosporin A (5 µM) for 2 min.
Thereafter, the tubules were treated with solution 4 in the
presence of verapamil or cyclosporin A for 10 min. *P < 0.05 vs. maximum cell volume decrease.
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Fig. 5.
Effects of hyperosmotic solution with mannitol on cell
volume in isolated nonperfused proximal tubule S2 segments from WT mice
in the presence of tetraethylammonium (TEA). Values are means ± SE of 5 tubules and are expressed as a percentage relative to the
control isosmotic tubule cell volume treated with solution
1. The tubules were initially bathed in solution 1 and
were then perfused with solution 1 containing TEA (1 mM) for
2 min. Thereafter, the tubules were treated with solution 4 in the presence of TEA for 10 min.
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Transport mechanisms responsible for hyperosmotic cell volume
regulation.
When most animal cells are subjected to hyperosmotic stress, activation
of ion transport systems, followed by a later accumulation of organic
osmolytes, is known to occur (reviewed in Refs. 17 and
20). The activation of ion transport systems includes influx of NaCl,
which is mediated by parallel Na+/H+ and
Cl
/HCO
antiports or
Na+-K+-2Cl
cotransport (reviewed
in Refs. 17 and 20). Accordingly, we examined whether, in
the tubules of the WT mice, RVI induced by the hyperosmotic mannitol
solution containing the two P-gp inhibitors occurs through
Na+ entry into the cell. For this purpose, the tubules of
the WT mice were initially incubated in the control isosmotic solution (solution 1; Table 1) and were then bathed in the
Na+-free isosmotic solution (solution 6; Table
1). Thereafter, they were treated with solution 6 containing
verapamil (100 µM) or cyclosporin A (5 µM), and were finally
exposed to the Na+-free hyperosmotic mannitol solution
(solution 7; 500 mosmol/kgH2O, Table 1) in the
presence of verapamil or cyclosporin A. Results are shown in Fig.
6. The exposure of the tubules to either
the isosmotic Na+-free solution alone or the isosmotic
Na+-free solution containing verapamil or cyclosporin A had
no effect on cell volume (Fig. 6). On the other hand, when the tubules
of the WT mice were treated with the Na+-free hyperosmotic
mannitol solution including verapamil, they initially shrank to
81.0 ± 1.2% (n = 5) of their control volume but
remained reduced in size (Fig. 6A). Similarly, when the
tubules of the WT mice were exposed to the Na+-free
hyperosmotic mannitol solution containing cyclosporin A, they initially
shrank to 80.0 ± 1.6% (n = 5) of their control volume but remained in a shrunken state during the experimental period
(Fig. 6B). Next, we examined whether, in proximal tubules from the WT mice, basolateral membrane Na+/H+
exchange contributes to the RVI response induced by the hyperosmotic mannitol solution containing the two P-gp inhibitors. For this purpose,
the tubules of the WT mice were initially incubated in solution
1 and were then bathed in solution 1 containing EIPA (the specific inhibitor of Na+/H+ exchange; 100 µM). Thereafter, they were treated with solution 1 containing verapamil (100 µM) or cyclosporin A (5 µM) in the continued presence of EIPA and were finally exposed to the hyperosmotic mannitol solution (solution 4; 500 mosmol/kgH2O, Table 1) containing EIPA and verapamil
or cyclosporin A. Results are shown in Fig. 7. The peritubular addition of either
EIPA alone or EIPA including verapamil or cyclosporin A to the tubules
of the WT mice had no effect on cell volume (Fig. 7). On the other
hand, when the tubules of the WT mice were exposed to the hyperosmotic
mannitol solution containing verapamil and EIPA, they initially shrank
to 81.2 ± 0.9% (n = 4) of their control volume
but remained in a shrunken state during the experimental period (Fig.
7A). Similarly, when the tubules of the WT mice were treated
with the hyperosmotic mannitol solution including cyclosporin A and
EIPA, they initially shrank to 82.2 ± 1.3% (n = 5) of their control volume but remained reduced in size (Fig.
7B).

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Fig. 6.
Effects of hyperosmotic mannitol solution containing
verapamil (A) or cyclosporin A (B) in the absence
of peritubular Na+ on cell volume in isolated nonperfused
proximal tubule S2 segments from WT mice. Values are means ± SE
of 5 tubules from WT mice in the presence of verapamil or cyclosporin A
and are expressed as a percentage relative to the control, isosmotic
tubule cell volume treated with the solution 1. The tubules
were initially bathed in solution 1 and were then perfused
with solution 6 for 2 min. Thereafter, the tubules were
treated with solution 6 containing verapamil (100 µM) or
cyclosporin A (5 µM) for 2 min and were finally exposed to
solution 7 including verapamil or cyclosporin A for 10 min.
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Fig. 7.
Effects of hyperosmotic mannitol solution containing
verapamil or cyclosporin A in the presence of peritubular
ethylisopropylamiloride (EIPA) on cell volume in isolated nonperfused
proximal tubule S2 segments from WT mice. Values are means ± SE
of 4 or 5 tubules from WT mice in the presence of verapamil
(A) or cyclosporin A (B), respectively, and are
expressed as a percentage relative to the control isosmotic tubule cell
volume treated with solution 1. The tubules were initially
bathed in solution 1 and were then perfused with
solution 1 containing EIPA (100 µM) for 2 min. Thereafter,
the tubules were treated with solution 1 containing EIPA and
verapamil (100 µM) or cyclosporin A (5 µM) for 2 min, and were
finally exposed to solution 4 including EIPA and verapamil
(100 µM) or cyclosporin A for 10 min.
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Next, we examined whether, in proximal tubules from the KO mice, the
RVI response induced by the hyperosmotic mannitol solution is mediated
through Na+ entry into the cell. For this purpose, the
tubules of the KO mice were initially incubated in the control
isosmotic solution (solution 1; Table 1) and were then
bathed in the Na+-free isosmotic solution (solution
6; Table 1). Thereafter, they were treated with the
Na+-free hyperosmotic mannitol solution (solution
7; 500 mosmol/kgH2O, Table 1). Results are shown in
Fig. 8. Removing peritubular
Na+ alone had no effect on cell volume (Fig. 8). On the
other hand, when the tubules of the KO mice were exposed to the
Na+-free hyperosmotic mannitol solution, they initially
shrank to 80.7 ± 0.8% (n = 6) of their control
volume but remained in a shrunken state during the experimental period
(Fig. 8). Next, we examined whether, in the proximal tubules from the
KO mice, basolateral membrane Na+/H+ exchange
is involved in the RVI response induced by the hyperosmotic mannitol
solution. For this purpose, the tubules of the KO mice were initially
incubated in solution 1 and were then bathed in solution 1 containing EIPA (100 µM). Thereafter, they were
treated with the hyperosmotic mannitol solution (solution 4;
500 mosmol/kgH2O, Table 1) in the continued presence of
peritubular EIPA. EIPA alone caused no effect on cell volume (Fig.
9). In sharp contrast, when the tubules
of the KO mice were exposed to the hyperosmotic mannitol solution in
the presence of EIPA, they initially shrank to 82.0 ± 0.9%
(n = 6) of their control volume but remained reduced in
size (Fig. 9).

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Fig. 8.
Effects of hyperosmotic solution with mannitol in the
absence of peritubular Na+ on cell volume in isolated
nonperfused proximal tubule S2 segments from KO mice. The tubules were
initially bathed in solution 1 and were then perfused with
solution 6 for 2 min. Thereafter, the tubules were treated
with solution 7 for 10 min. Values are means ± SE of 6 tubules from KO mice and are expressed as a percentage relative to the
control isosmotic tubule cell volume treated with solution
1.
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Fig. 9.
Effects of hyperosmotic solution with mannitol in the
presence of peritubular EIPA on cell volume in isolated nonperfused
proximal tubule S2 segments from KO mice. The tubules were initially
bathed in solution 1 and were then perfused with
solution 1 containing EIPA (100 µM) for 2 min. Thereafter,
the tubules were treated with solution 4 including EIPA for
10 min. Values are means ± SE of 6 tubules from KO mice and are
expressed as a percentage relative to the control isosmotic tubule cell
volume treated with solution 1.
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DISCUSSION |
The goal of the present study was to examine the role of P-gp in
isolated nonperfused proximal tubules exposed to anisosmotic media. We
demonstrated that exposure of the tubules from both WT and KO mice to a
hyposmotic solution elicited RVD with a similar magnitude. RVD during
hyposmotic stress was unchanged by the two inhibitors of P-gp
(verapamil and cyclosporin A). In sharp contrast to the hyposmotic
stress, exposure of the tubules from the WT mice to the hyperosmotic
mannitol solution did not produce RVI, whereas exposure of the tubules
from the KO mice to the hyperosmotic mannitol solution elicited RVI. In
the WT mice, the two inhibitors of P-gp, but not the inhibitor of the
renal organic cation transport system (TEA), produced RVI under
hyperosmotic conditions, whereas in KO mice the inhibitors of P-gp did
not affect RVI during hyperosmotic stress at all. This is the first
study demonstrating that, in mouse proximal tubule cells, P-gp
modulates RVI during hyperosmotic stress, but not RVD during hyposmotic stress.
Role of P-gp in cell volume regulation under hyposmotic conditions.
In the present study, we report that when isolated nonperfused proximal
tubules of WT mice were rapidly exposed to hyposmotic medium, cell
swelling was followed by RVD. Similar findings have been reported in
isolated nonperfused rabbit proximal convoluted (6, 16)
and straight (3, 14, 33) tubules and in isolated perfused
mouse proximal straight tubules (32).
Valverde et al. (29) have shown that exposure of Chinese
hamster ovary cells transfected with mdr1a (LR73-1a
cells) to hyposmotic medium elicited RVD after the initial cell
swelling, whereas exposure of the parental cells (LR73 cells) without
expression of P-gp to the hyposmotic medium did not. Roman et al.
(21) also reported that when HTC-R hepatoma cells
expressing P-gp were exposed to hyposmotic solution, RVD was seen after
the initial cell swelling. RVD was then abolished by the inhibitor of
P-gp (verapamil). From these reports, we expected that in the tubules
of the KO mice, RVD in hyposmotic medium would not occur, whereas in
the tubules of the WT mice, it would be suppressed by the addition of
the inhibitors of P-gp (verapamil and cyclosporin A). However,
unexpectedly, in the tubules of the KO mice, RVD under hyposmotic
conditions was indeed observed to a similar extent to that in the WT
mice. Also, in both groups of tubules, RVD was not affected at all by the addition of inhibitors of P-gp. Furthermore, drug-transporting P-gp
activity exists in proximal tubules from WT mice and is suppressed by
verapamil, whereas it is absent in proximal tubules of KO mice (28). Also, in isolated perfused tubules of KO mice, the
magnitude of RVD found after cell swelling was of a magnitude similar
to that in isolated nonperfused tubules of KO mice (data not shown). From our present and previous findings, we conclude that in intact proximal tubule cells, P-gp is not involved in RVD in hyposmotic medium. Therefore, the role of P-gp in RVD is strikingly different from
that in cultured cells and intact cells, although it remains unclear
why in mouse proximal tubule cells, P-gp does not contribute to RVD
during hyposmotic stress.
Role of P-gp in cell volume regulation under hyperosmotic
conditions.
In the present study, exposure of isolated nonperfused proximal tubules
from WT mice to a hyperosmotic mannitol solution did not elicit RVI
after the initial cell shrinkage. Similar findings have been reported
in nonperfused rabbit proximal convoluted (6) and straight
(14) tubules rapidly treated with hyperosmotic NaCl,
raffinose, or mannitol solutions. In our study, in the tubules from the
WT mice, the two inhibitors of P-gp (verapamil and cyclosporin A)
produced RVI during exposure to the hyperosmotic mannitol solution, whereas the inhibitor of the renal organic cation transport system (TEA) did not. Most exciting, exposure of tubules from KO mice to the
hyperosmotic mannitol solution elicited RVI, and RVI during hyperosmotic stress was not influenced at all by the two inhibitors of
P-gp. Under isosmotic conditions, cell volume in either group of
tubules was unaltered by the two inhibitors of P-gp. Therefore, when
P-gp activity is acutely suppressed by inhibitors of P-gp or when both
mdr1a and mdr1b are genetically disrupted, RVI in hyperosmotic medium occurs. These findings are compatible with the
notion that in mouse proximal tubule, P-gp maintains cell volume at a
constant level under hyperosmotic conditions but not isosmotic conditions.
Studies, including the present work, on cell volume regulation in
mammalian kidneys have used nonperfused tubule segments studied in
vitro (3, 6, 14-16, 19, 22, 33). In this nonpolar
preparation, cell volume regulation primarily reflects the steady-state
transport of solutes and water only across the basolateral membranes
(9, 14, 15, 33). Therefore, we propose that in mouse
proximal tubule, P-gp could keep cell volume at a constant level during
hyperosmotic stress via the basolateral transporters but not via the
apical transporters. To clarify which basolateral transporters are
involved in P-gp-induced cell volume regulation, the effects of
removing peritubular Na+ and adding peritubular EIPA (the
specific inhibitor of Na+/H+ exchange) on cell
volume were examined. In the proximal tubules of the WT mice, both
removal of peritubular Na+ and addition of peritubular EIPA
inhibited RVI induced by the hyperosmotic mannitol solution containing
the two P-gp inhibitors (Figs. 6 and 7). In the tubules of the KO mice,
both removing peritubular Na+ and adding peritubular EIPA
eliminated RVI induced by the hyperosmotic mannitol solution (Figs. 8
and 9). These findings indicate that basolateral
Na+/H+ exchange partly contributes to
P-gp-induced modulation of RVI after initial cell shrinkage.
In a variety of cell types (8, 18), protein kinase C has
been reported to be involved in P-gp-mediated drug efflux. In BALB/c-3T3 cells transfected with human MDR1, it has been
demonstrated that activation of protein kinase C reduced the rate of
increase in swelling-activated Cl
currents, whereas
activation of protein kinase A reduced steady-state swelling-activated
Cl
currents (31). In rabbit proximal tubule,
cell volume regulation under hyperosmotic conditions is regulated by
the availability of metabolizable fatty acids in the medium. Acetate,
butyrate, and valerate have been reported to enhance the ability of
proximal tubule cells to maintain a constant cell volume under mildly
hyperosmotic conditions (15, 22). Grantham et al.
(15, 22) suggested that nonionic acids entering the cell
may support Na+/H+ exchange by supplying
intracellular protons. However, it is not known at present whether
these kinases and/or the availability of metabolizable fatty acids
contributes to P-gp-induced cell volume regulation in mouse proximal
tubule cells. Further studies will be required to clarify the
intracellular signaling mechanisms responsible for cell volume
regulation induced by P-gp under hyperosmotic conditions. Also, whether
basolateral Na+/H+ exchange in the mouse
proximal tubule is actually activated in P-gp-induced cell volume
regulation under hyperosmotic stress will have to await further investigation.
In contrast to the effect of mannitol (an impermeant solute), the
effect of urea (a more permeant solute) on cell volume change was
strikingly different (see Fig. 3). In both groups of tubules, hyperosmotic urea caused only a transient decrease in cell volume. Urea
entry across the basolateral membrane quickly restored cell volume. The
large overshoot observed during washout of urea is consistent with this
urea entry. Similar observations have been made in rabbit proximal
convoluted tubule (6) and cortical collecting duct
(19). Volume changes during the presence of urea were the
result of simple diffusion and not volume regulation.
In conclusion, we clearly demonstrated that, in mouse proximal tubule
S2 segments, P-gp modulates RVI during hyperosmotic stress but not RVD
during hyposmotic stress. We also found that basolateral membrane
Na+/H+ exchange partly contributes to
P-gp-induced modulation of RVI under hyperosmotic stress.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a grant from the Japanese Kidney
Foundation (Jinkenkyukai), the Salt Science Foundation, and
Grants-in-Aid for Scientific Research from the Ministry of Education,
Science, Culture, and Sports, Japan.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: S. Muto,
Dept. of Nephrology, Jichi Medical School, Minamikawachi, Tochigi
329-0498, Japan (E-mail: smuto{at}jichi.ac.jp).
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 9 June 2000; accepted in final form 4 January 2001.
 |
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