Hyperosmotic urea activates basolateral NHE in proximal
tubule from P-gp null and wild-type mice
Yukio
Miyata,
Yasushi
Asano, and
Shigeaki
Muto
Department of Nephrology, Jichi Medical School, Tochigi 329-0498, Japan
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ABSTRACT |
Using the pH-sensitive fluorescent
dye BCECF, we compared the effects of hyperosmotic urea on basolateral
Na+/H+ exchange (NHE) with those of
hyperosmotic mannitol in isolated nonperfused proximal tubule S2
segments from mice lacking both the mdr1a and
mdr1b genes (KO) and wild-type (WT) mice. All the experiments were performed in
CO2/HCO
-free HEPES solutions. Osmolality
of the peritubular solution was raised from 300 to 500 mosmol/kgH2O by adding mannitol or urea. NHE activity was
assessed by the Na+-dependent acid extrusion rate
(JH) after an acid load with NH4Cl prepulse. In WT mice, hyperosmotic mannitol had no effect on
JH at over the entire range of intracellular pH
(pHi) studied (6.20-6.90), whereas in KO mice it
increased JH at a pHi range of
6.20-6.45. In contrast, in both WT and KO mice, hyperosmotic urea
increased JH at a pHi range of
6.20-6.90. In KO mice, JH in a hyperosmotic urea solution were similar to those in a hyperosmotic mannitol solution
at a pHi range of 6.20-6.40 but were greater than in a
hyperosmotic mannitol solution at a pHi range of
6.45-6.90. In WT mice, hyperosmotic urea caused an increase in
Vmax without changing Km
for peritubular Na+. Staurosporine (the PKC inhibitor)
inhibited hyperosmotic mannitol-induced NHE activation in KO mice,
whereas it had no effect on hyperosmotic urea-induced NHE activation in
WT or KO mice. Genistein (the tyrosine kinase inhibitor) inhibited
hyperosmotic urea-induced NHE activation in WT and KO mice, whereas it
caused no effect on hyperosmotic mannitol-induced NHE activation in KO
mice. We conclude that hyperosmotic urea activates basolateral NHE via
tyrosine kinase in tubules from both WT and KO mice, whereas
hyperosmotic mannitol activates it via PKC only in tubules from KO mice.
mdr1a; mdr1b; isolated nonperfused tubule; intracellular pH measurement; PKC; tyrosine kinase
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INTRODUCTION |
P-GLYCOPROTEIN
(P-gp) IS A member of the
ATP-binding cassette superfamily of transporters and utilizes ATP to
pump hydrophobic drugs out of the cells, decreasing their intracellular
concentrations and hence their toxicity (reviewed in Ref.
15). Humans have one drug-transporting P-gp
(MDR1), whereas mice have two genes encoding
drug-transporting P-gps, mdr1a (also called mdr3)
and mdr1b (also called mdr1) (15, 18,
32). The mdr1a and mdr1b genes in the
mouse together fulfill the same function as MDR1 in humans,
and similar levels of mdr1a and mdr1b expression
have been observed in the kidney (11, 33). In the kidney,
the apical membrane of the proximal tubule epithelium is particularly
rich in P-gp (12, 38), placing this pump in the correct
location to mediate the active excretion of xenobiotics. Consistent
with a role for P-gp as an excretory transporter, our laboratory
(40) previously reported in the isolated perfused mouse
proximal tubule that P-gp-mediated drug efflux capacity indeed exists
in the apical membrane of the proximal tubule S2 segment from wild-type
(WT) mice but is lacking in that of mice in which both mdr1a
and mdr1b genes were disrupted (KO mice).
In a variety of cell types, Na+/H+ exchange
(NHE) is activated by shrinkage of a cell in a hyperosmotic solution,
resulting in Na+ entry into the cell. Osmotic uptake of
water due to this Na+ entry leads to cell swelling, a
process termed regulatory volume increase (RVI) (reviewed in Refs.
19 and 23). In sharp contrast, when rabbit proximal tubule
cells are suddenly exposed to hyperosmotic mannitol, NaCl, or
raffinose, they rapidly shrink but remain reduced in size (13,
22). However, the underlying mechanisms for the lack of RVI have
not fully been understood. Recently, our laboratory (24)
used isolated nonperfused proximal tubule S2 segments from WT and KO
mice to demonstrate that exposure of tubules from WT mice to
hyperosmotic mannitol did not result in RVI, whereas exposure of
tubules from KO mice to hyperosmotic mannitol elicited RVI. We also
observed that in tubules from WT mice, peritubular addition of P-gp
inhibitors (verapamil and cyclosporin A) resulted in RVI, whereas in
tubules from KO mice it had no effect on RVI (24). Therefore, in the mouse proximal tubule, P-gp modulates RVI during exposure to hyperosmotic mannitol. We have also shown that P-gp-induced modulation of RVI occurs via PKC activation (27).
Furthermore, we demonstrated that basolateral NHE partly contributes to
P-gp-induced modulation of RVI (24). Recently, we reported
in the mouse proximal tubule that in the absence of P-gp activity,
hyperosmotic mannitol actually activates basolateral NHE via PKC,
whereas in the presence of P-gp activity it does not (25).
In that report, we observed that in KO mice exposure to hyperosmotic
mannitol significantly increased Na+-dependent acid
extrusion rates (JH) via NHE only at a
pHi range of 6.20-6.45 and shifted
JH vs. pHi by ~0.15 pH units in
the alkaline direction at the low pHi (25).
Here, the question arises of whether the stimulatory effect on NHE is
specific to mannitol, because in a variety of cell types exposed to
hyperosmotic stress, the alkaline shift in pHi sensitivity
of NHE occurs along a wide range of pHi (17, 26, 34,
35).
Unlike the poorly permeating solutes (NaCl, mannitol, and raffinose),
urea is relatively membrane permeant and has traditionally been
considered to play a passive role in renal epithelial cell function
(reviewed in Ref. 14). In fact, we previously observed that in the mouse proximal tubule, the effects of urea on the change in
cell volume differ markedly from those of mannitol; when proximal
tubules of both WT and KO mice were exposed to hyperosmotic urea, the
reduction in cell volume was smaller compared with hyperosmotic mannitol and transient, and cells immediately returned to their control
volume without RVI (24). Our laboratory also reported that
in cultured rat inner medullary collecting duct cells, hyperosmolality induced by NaCl, mannitol, and raffinose stimulates both
Na+-K+-ATPase
1- and
1-subunit mRNA expression and
Na+-K+-ATPase activity, whereas hyperosmolality
induced by urea does not (29). However, the effect of
hyperosmotic urea on NHE has received less attention. In addition, it
is unknown whether the effect is dependent on P-gp. To solve the above
problems, we used isolated nonperfused proximal tubule S2 segments from
KO and WT mice to address the following issues: 1) whether
hyperosmotic urea activates basolateral NHE in proximal tubules of WT
and/or KO mice, and if so 2) how hyperosmotic urea modulates
basolateral NHE. For this purpose, we compared the effects of
hyperosmotic urea on basolateral NHE with those of hyperosmotic mannitol.
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METHODS |
Solutions.
The composition of the solutions is given in Table
1. All solutions were nominally
CO2/HCO
free. Solutions 1-3
were adjusted to pH 7.4 at 37°C. The osmolality of all solutions
was measured before the experiment and was verified to be within a
range of 300 ± 5 mosmol/kgH2O. Hyperosmotic mannitol or urea solutions (500 mosmol/kgH2O) were made by adding
mannitol or urea to solution 1 or 2,
respectively. The nigericin calibrating solution was titrated to
different pH values at 37°C with either HCl or
N-methyl-D-glucamine (NMDG). For Na+
free solutions (solutions 2-4, Table 1),
Na+ was replaced with NMDG titrated with the appropriate
acid. BCECF-acetoxymethyl ester (AM) was prepared as a 10 mM stock
solution and was diluted 1:1,000 to a final concentration of 10 µM.
Nigericin was prepared as a 10 mM stock solution in ethanol and was
diluted 1:1,000 into solution 4 (Table 1) to a final
concentration of 10 µM. EIPA was prepared as a 100 mM stock solution
in methanol and was diluted 1:1,000 to a final concentration of 100 µM. Staurosporine and genistein were dissolved in DMSO at 0.1% final
concentrations. Equivalent concentrations of vehicle were added as a
control for individual protocols.
Animals.
KO mice used in this study were originally described by Schinkel et al.
(32). Male KO and FVB (WT) mice, serving as a control (body wt 25-40 g), were purchased from Taconic Engineering
(Germantown, NY). The animals were maintained under a controlled
environment and had free access to a standard rodent chow and tap water
ad libitum until the beginning of the experiments. Ages of the KO animals were matched with their WT controls.
In vitro microperfusion.
Both groups of the mice were anesthetized with an injection of
pentobarbital sodium (4 mg/100 g body wt ip), 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 described by Miyata et al. (24, 25,
27). 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. After tubules were tightly
crimped between the two glass micropipettes, they were bathed in the
isosmotic control solution (solution 1, Table 1) at 37°C
for 10-15 min to allow them to equilibrate.
Optical measurement of pHi.
The details of our techniques for measuring pHi in mouse
proximal tubules have been published elsewhere (25).
Briefly, both groups of tubules in the bath were exposed to the
isosmotic control HEPES-buffered solution (solution 1, Table
1) containing BCECF-AM (10 µM). After a 15-min dye-loading period at
37°C, the dye was washed out. pHi was then measured
microfluorometrically by alternately exciting the dye with a 7.5-µm
diameter light at 440 and 490 nm while the emission was monitored at
530 nm (25, 26). The resulting fluorescence-to-excitation
ratios were converted to pHi values as described (25,
26), using the high-K+/nigericin technique
(39). We used the same intracellular dye calibration
coefficients described for the proximal tubule cells from WT and KO
mice (25).
Computation of JH.
In the proximal tubule cells from the WT and KO mice, we computed
JH as the product of the previously measured
intrinsic buffering power (
I) (25), which
varies with pHi, and the rate of Na+-dependent
pHi increase (dpHi/dt). To obtain
the rate of Na+-dependent pHi increase, both
groups of proximal tubule cells were incubated in the
Na+-containing isosmotic solution (solution 1,
Table 1) and then exposed to the Na+-free solution
containing 20 mM NH4Cl for 2 min (solution 3,
Table 1). During the pulse, pHi increased due to the entry
of NH3 into the cell and then tended to decrease toward
baseline due to a slower influx of NH
. In the
continued presence of the Na+-free solution,
NH4Cl was abruptly removed from the peritubular solution,
causing pHi to decrease. Afterward, the readdition of Na+ to the peritubular solution gave rise to a rapid
increase in pHi. The Na+-dependent
pHi increase was divided into two or three sections, each
of which was fitted to a third- or fourth-order polynomial. The
derivatives of these equations (i.e., dpHi/dt
values) were calculated at pHi intervals of 0.05. At each
pHi, data from four or more experiments were averaged to
produce a plot of mean JH vs. pHi
(25, 26, 31).
Drugs and chemicals.
All chemicals were obtained from Wako (Osaka, Japan) unless noted as
follows: HEPES and BCECF-AM were from Dojindo (Kumamoto, Japan); and
EIPA, staurosporine, genistein, and nigericin were from Sigma (St.
Louis, MO).
Data analysis and statistics.
The data are expressed as means ± SE. Comparisons were performed
by Student's t-test or one-way ANOVA in combination with Fisher's protected least significant difference test where
appropriate. P values <0.05 were considered significant.
 |
RESULTS |
Na+-dependent pHi
recovery from an acid load in proximal tubule cells from WT and KO mice
under isosmotic and hyperosmotic conditions.
First, we observed Na+-dependent pHi recovery
from an acid load with NH4Cl in proximal tubule cells from
WT and KO mice. Representative pHi recordings in WT and KO
mice are shown in Figs. 1A and
2A, respectively.
Both groups of cells were first bathed in the
Na+-containing, HEPES-buffered solution (solution
1, Table 1) and then incubated in the Na+-free,
HEPES-buffered solution containing 20 mM NH4Cl
(solution 3, Table 1). At this time, pHi rapidly
increased because of the rapid diffusion of NH3 into the
cells. During the exposure to NH4Cl for 2 min,
pHi tended to decrease toward baseline due to the slow
inward diffusion of NH
. Afterward, removing
NH4Cl from the Na+-free, HEPES-buffered
solution rapidly decreased pHi, because accumulated
internal NH
dissociates into H+ and
NH3 (which exits from the cell). At this time,
pHi values of proximal tubule cells from WT and KO mice
were 6.14 ± 0.04 (n = 10) and 6.15 ± 0.03 (n = 8), respectively, and were not significantly different between the two groups. Subsequent addition of
Na+ to the peritubular solution caused pHi to
rapidly increase to values similar to those for initial steady-state
pHi. In fact, final steady-state pHi values in
cells from WT and KO mice were 6.95 ± 0.03 (n = 10) and 7.01 ± 0.02 (n = 8), respectively, and were not significantly different from those for the initial
steady-state (WT mice: 6.99 ± 0.03, n = 10; KO
mice: 7.00 ± 0.01, n = 8). In both groups of
cells, pretreatment with EIPA (the specific NHE inhibitor; 100 µM)
inhibited the Na+-dependent pHi recovery rate
by ~80%. These findings confirmed our previous observations
(25). To further characterize basolateral NHE in proximal
tubules from WT and KO mice, dose-response experiments were performed
with EIPA. As shown in Fig. 3, inhibition
curves for tubules from both WT and KO mice indicated a single NHE
activity, with IC50 of 11.3 and 10.8 µM, respectively.
These data are consistent with the notion that basolateral NHE in both
groups of proximal tubule cells is highly amiloride resistant (5,
7, 28).

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Fig. 1.
Na+-dependent intracellular pH
(pHi) recovery from an acid load when the proximal tubule
cells from wild-type (WT) mice were exposed to an isosmotic solution
(A), hyperosmotic mannitol solution (B), and
hyperosmotic urea solution (C). The cells were first bathed
in a Na+-containing HEPES-buffered solution (solution
1, Table 1) and then incubated in a Na+-free
HEPES-buffered solution containing 20 mM NH4Cl
(solution 3, Table 1) for 2 min. During the pulse,
pHi increased (due to the entry of NH3 into the
cell) and then tended to decrease toward baseline due to a slower
influx of NH . Thereafter, the abrupt removal of
NH4Cl from the Na+-free solution caused a
substantial and sustained decrease in pHi. Then, readdition
of peritubular Na+ to the cells caused pHi to
increase. When the cells were exposed to the hyperosmotic mannitol
solution (500 mosmol/kgH2O), Na+-dependent
pHi recovery was similar to that when the cells were
exposed to the isosmotic solution. In cells exposed to the isosmotic
solution and the hyperosmotic mannitol solution, final steady-state
pHi values were similar to the initial steady-state
pHi values. However, when cells was exposed to the
hyperosmotic urea solution (500 mosmol/kgH2O),
Na+-dependent pHi recovery was faster than when
they were exposed to the isosmotic solution and the hyperosmotic
mannitol solution. In cells treated with the hyperosmotic urea
solution, the final steady-state pHi values were greater
than the initial steady-state pHi values.
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Fig. 2.
Na+-dependent pHi recovery from
an acid load when proximal tubule cells from KO mice were exposed to an
isosmotic solution (A), hyperosmotic mannitol solution
(B), and hyperosmotic urea solution (C). Cells
were first bathed in the Na+-containing HEPES-buffered
solution (solution 1, Table 1) and then incubated in the
Na+-free HEPES-buffered solution containing 20 mM
NH4Cl (solution 3, Table 1) for 2 min. During
the pulse, pHi increased (due to the entry of
NH3 into the cell) and then tended to decrease toward
baseline due to a slower influx of NH . Thereafter,
the abrupt removal of NH4Cl from the Na+-free
solution caused a substantial and sustained decrease in
pHi. Then, readdition of peritubular Na+ to the
cells caused pHi to increase. When cells were exposed to
the hyperosmotic mannitol or urea solutions (500 mosmol/kgH2O), Na+-dependent pHi
recovery was faster than when they were exposed to the isosmotic
solution. In cells exposed to the hyperosmotic mannitol solution, the
final steady-state pHi values were similar to the initial
steady-state pHi values, whereas in cells exposed to the
hyperosmotic urea solution, the final steady-state pHi
values were greater than the initial steady-state pHi
values.
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Fig. 3.
Dose-response curves for inhibition of basolateral
Na+/H+ exchange (NHE) activity of proximal
tubule cells from WT and KO mice under isosmotic conditions by EIPA.
NHE activity was assessed by Na+-dependent acid extrusion
rates (JH) at a pHi of 6.30 on
readdition of Na+ to the peritubular side after
Na+ removal and an acid load with NH4Cl
prepulse, in the presence of EIPA at various concentrations. Values are
means ± SE of at least 3 tubules from WT and KO mice, expressed
as the percentage of JH compared with those
observed in the absence of EIPA (control).
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Next, we examined whether the hyperosmotic mannitol solution affects
Na+-dependent pHi recovery from an acid load in
proximal tubule cells from WT (Fig. 1B) and KO (Fig.
2B) mice. Both groups of proximal tubule cells were first
incubated in the Na+-containing HEPES-buffered solution
(solution 1, Table 1) and then bathed in the
Na+-free solution containing 20 mM NH4Cl
(solution 3, Table 1) for 2 min. Afterward,
NH4Cl was rapidly removed from the Na+-free
HEPES-buffered solution, leading to a rapid decrease in pHi. Thereafter, both groups of cells were immediately
exposed to the Na+-free HEPES-buffered solution
(solution 2, Table 1) treated with 200 mM mannitol (500 mosmol/kgH2O). At this time, pHi values in both
groups of cells were unchanged. Afterward, when external Na+ was readded in the presence of hyperosmotic mannitol,
pHi recovery in cells from KO mice was substantially faster
than in its absence, although pHi values for the final
steady state (6.99 ± 0.03, n = 13) were not
significantly different from those for the initial steady state
(7.02 ± 0.02, n = 13) (Fig. 2B). At
this time, Na+-dependent pHi recovery rates in
the presence of hyperosmotic mannitol (132.5 ± 10.6 pH/s × 104, n = 13, P < 0.001)
were significantly greater than in its absence (74.6 ± 5.0 pH/s × 104, n = 8). Furthermore,
under hyperosmotic conditions, Na+-dependent
pHi recovery was faster in KO mice than in WT mice (see
Figs. 1B and 2B). At this time,
Na+-dependent pHi recovery rates in KO mice
(132.5 ± 10.6 pH/s × 104, n = 13, P < 0.001) were also significantly greater than
those in WT mice (90.3 ± 7.7 pH/s × 104,
n = 15). In sharp contrast to KO mice, in WT mice
Na+-dependent pHi recovery and
Na+-dependent pHi recovery rates under
hyperosmotic conditions were similar to those under isosmotic
conditions. Therefore, these findings were compatible with our previous
report (25).
Next, we examined whether the hyperosmotic urea solution affects
Na+-dependent pHi recovery from an acid load in
proximal tubule cells from WT (Fig. 1C) and KO (Fig.
2C) mice. Both groups of cells were first incubated in the
Na+-containing HEPES-buffered solution (solution
1, Table 1) and then bathed in the Na+-free solution
containing 20 mM NH4Cl (solution 3, Table 1) for 2 min. Afterward, NH4Cl was rapidly removed from the
Na+-free HEPES-buffered solution, leading to a rapid
decrease in pHi. Thereafter, both groups of cells were
immediately exposed to the Na+-free HEPES-buffered solution
(solution 2, Table 1) treated with 200 mM urea (500 mosmol/kgH2O). At this time, pHi values in both groups of cells were unchanged. Afterward, when external
Na+ was readded in the presence of hyperosmotic mannitol,
pHi recovery in cells from WT mice was substantially faster
than in its absence, and pHi values for the final steady
state (7.13 ± 0.04, n = 8, P < 0.005) were also significantly greater than those for the initial
steady state (6.97 ± 0.02, n = 8) (see Fig.
1C). At this time, Na+-dependent pHi
recovery rates in the presence of hyperosmotic urea (125.2 ± 13.9 pH/s × 104, n = 8, P < 0.005) were also significantly greater than in its absence
(81.8 ± 9.8 pH/s × 104, n = 10). Similarly, in cells from KO mice, Na+-dependent
pHi recovery was substantially faster than in its absence, and pHi values for the final steady state (7.04 ± 0.02, P < 0.01, n = 7) were also
significantly greater than those for the initial steady state
(6.95 ± 0.02, n = 7) (see Fig. 2C). At
this time, Na+-dependent pHi recovery rates in
the presence of hyperosmotic urea (107.8 ± 12.2 pH/s × 104, n = 7, P < 0.05) were
also significantly greater than in its absence (74.6 ± 5.0 pH/s × 104, n = 8). Furthermore, for
the hyperosmotic urea solution, Na+-dependent
pHi recovery rates in cells from KO mice (107.8 ± 12.2 pH/s × 104, n = 7) were not
significantly different from those in cells from WT mice (125.2 ± 13.9 pH/s × 104, n = 8).
Next, we examined the effects on Na+-dependent
pHi recovery of adding EIPA to both groups of cells treated
with the hyperosmotic mannitol and urea solutions. Representative
pHi recordings are shown in Fig.
4. When both groups of cells were exposed
to EIPA (100 µM) alone, steady-state pHi values
significantly decreased by ~0.05. In both groups of cells treated
with the hyperosmotic mannitol or urea solutions,
Na+-dependent pHi recovery from an acid load in
the presence of EIPA was substantially slower than in its absence (see
Figs. 1, B and C, and 2, B and
C, and 3). When cells from WT mice were treated with the
hyperosmotic mannitol or urea solutions, Na+-dependent
pHi recovery rates in the presence of EIPA (mannitol: 33.0 ± 5.6 pH/s × 104, n = 6, P < 0.001; urea: 18.5 ± 2.4 pH/s × 104, n = 4, P < 0.001)
were also significantly smaller than in its absence (mannitol:
90.3 ± 7.7 pH/s × 104, n = 15;
urea: 125.2 ± 13.9 pH/s × 104,
n = 8). Similar findings were observed in cells from KO
mice that were exposed to the hyperosmotic mannitol or urea solutions. When cells from KO mice were exposed to the hyperosmotic mannitol or
urea solutions, Na+-dependent pHi recovery
rates in the presence of EIPA (mannitol: 25.2 ± 7.4 pH/s × 104, n = 7, P < 0.001;
urea: 15.9 ± 0.4 pH/s × 104, n = 4, P < 0.001) were also significantly smaller than
in its absence (mannitol: 132.5 ± 10.6 pH/s × 104, n = 13; urea: 107.8 ± 12.1 pH/s × 104, n = 7).

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Fig. 4.
Na+-dependent pHi recovery from
an acid load when proximal tubule cells from WT (A and
C) mice and KO (B and D) mice were
treated with hyperosmotic mannitol or urea in the presence of
peritubular EIPA. It should be noted that in both groups of cells
treated with the hyperosmotic mannitol or urea solutions, EIPA (100 µM) alone decreased pHi, indicating that in the nominal
absence of CO2/HCO , basolateral NHE in
proximal tubule cells from both WT and KO mice must be active in the
normal steady-state pHi to balance a substantial rate of
intracellular acid loading. In both groups of cells exposed to the
hyperosmotic mannitol or urea solutions (500 mosmol/kgH2O),
Na+-dependent pHi recovery in the presence of
EIPA was substantially slower than in its absence.
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From the Na+-dependent pHi recovery rate
(dpHi/dt) and
I, we calculated
the relationship between JH and pHi
in both groups of proximal tubule cells under isosmotic and
hyperosmotic conditions. Results from WT and KO mice are shown in Fig.
5, A and B,
respectively. In both groups of cells under isosmotic and hyperosmotic
conditions, JH decreased as pHi
increased. Under isosmotic conditions, JH values
in cells from KO mice were not significantly different from those from
WT mice at over the entire range of pHi studied (6.20-6.90). In WT mice, JH values in cells
exposed to the hyperosmotic mannitol solution were not significantly
different from those in cells exposed to the isosmotic solution over
the entire range of pHi studied (6.20-6.90) (Fig.
5A). In sharp contrast, in cells from KO mice,
JH values at a pHi range of
6.20-6.45 were significantly (P < 0.05) greater
in those exposed to the hyperosmotic mannitol solution than in those
exposed to the isosmotic solution. Furthermore, hyperosmotic mannitol
shifted JH vs. pHi by ~0.15 pH
units in the alkaline direction at the low-pHi range (Fig.
5B). In cells from KO mice, maximal
JH values in those exposed to the hyperosmotic mannitol solution (747.9 ± 40.9 µM/s at a pHi of
6.20, n = 13, P < 0.05) were also
significantly greater than those in cells exposed to the isosmotic
solution (458.1 ± 34.3 µM/s at pHi of 6.20, n = 8) (Fig. 5B). In sharp contrast to
results for the hyperosmotic mannitol solution, in both WT and KO mice,
JH values over the entire range of
pHi studied (6.20-6.90) were significantly
(P < 0.05) greater in cells exposed to the
hyperosmotic urea solution than in those exposed to the isosmotic
solution (Fig. 5). Furthermore, hyperosmotic urea shifted
JH vs. pHi by ~0.20 pH units in
the alkaline direction at the wide pHi range. In cells from
WT and KO mice exposed to the hyperosmotic urea solution, maximal
JH values at a pHi of 6.20 were
712.6 ± 73.8 (n = 8, P < 0.05)
and 682.7 ± 56.4 µM/s (n = 5, P < 0.05), respectively, both values that were also significantly
greater than those in the cells exposed to the isosmotic solution (WT
mice: 430.6 ± 61.3 µM/s, n = 10; KO mice:
458.1 ± 34.3 µM/s, n = 8). It should be noted
that in KO mice, JH in cells exposed to the
hyperosmotic urea solution were not significantly different from those
in cells exposed to the hyperosmotic mannitol solution at a
pHi range between 6.20 and 6.40 but were significantly
(P < 0.05) greater than with the hyperosmotic mannitol
solution at a pHi range between 6.45 and 6.90. In KO mice,
maximal JH values in cells exposed to the
hyperosmotic urea solution were not significantly different from those
exposed to the hyperosmotic mannitol solution. Therefore, we conclude that in WT mice, exposure to hyperosmotic urea enhanced
JH after an intracellular acid load through
basolateral NHE over the entire wide pHi range examined
(6.20-6.90), whereas exposure to hyperosmotic mannitol did not. In
marked contrast to WT mice, in KO mice treatment with hyperosmotic
mannitol increased JH through basolateral NHE only at the low pHi range (6.20-6.45), but treatment
with hyperosmotic urea resulted in an increased
JH via basolateral NHE over the entire wide
pHi range examined (6.20-6.90).

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Fig. 5.
pHi dependence of JH
when proximal tubule cells from WT (A) and KO (B)
mice were exposed to an isosmotic solution, hyperosmotic mannitol
solution, and hyperosmotic urea solution. The plots were computed from
experiments such as those illustrated in Figs. 1 and 2. Values are
means ± SE. Isosmo., isosmolality. *P < 0.05 compared with cells exposed to the isosmotic solution, at comparable
pHi values. P < 0.05 compared with cells
from KO mice exposed to the hyperosmotic mannitol solution, at
comparable pHi values.
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|
Next, we examined whether, in cells from WT mice, changes in
Km for peritubular Na+
concentrations and/or Vmax are involved in
basolateral NHE activation induced by the hyperosmotic urea solution.
For this purpose, the kinetics of basolateral NHE were determined by
measuring JH at a pHi of 6.30 on
readdition of varying concentrations of Na+ (0, 7, 14, 43, 100, and 142 mM) to the peritubular side of the tubules from the WT
mice after peritubular Na+ removal and an acid load with
NH4Cl prepulse in the absence and presence of hyperosmotic
urea. Results are shown in Fig.
6A. As shown in Fig.
6B, a Lineweaver-Burk plot of the data obtained showed that
under isosmotic conditions, apparent Km values
for peritubular Na+ concentrations were 22.9 mM with a
Vmax of 346.8 µM/s. Under hyperosmotic
conditions, the apparent Km for peritubular
Na+ was similar (21.6 mM), but Vmax
increased to 480.8 µM/s.

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Fig. 6.
Kinetics of basolateral NHE when proximal tubule cells from WT mice
were exposed to isosmotic solution and hyperosmotic urea (Urea)
solution. A: after removal of peritubular Na+
and an acid load with NH4Cl prepulse, varying
concentrations of Na+ (0, 7, 14, 43, 100, and 142 mM) were
added to the peritubular side of the tubules from WT mice, and
JH at a pHi of 6.30 were estimated.
Each point represents the mean ± SE of at least 4 determinations.
B: a Lineweaver-Burk analysis of the data indicates that
exposure of cells from WT mice to the hyperosmotic urea solution had no
effect on the apparent Km values for the
peritubular Na+ (from 22.9 to 21.6 mM) but increased
Vmax from 346.8 to 480.8 µM/s.
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Next, we examined whether hyperosmotic urea dose dependently activates
basolateral NHE in cells from WT mice. For this purpose, we added the
Na+-free HEPES-buffered solution (solution 2,
Table 1) containing 0, 100, 200, or 400 mM urea to cells from WT mice
and then observed the Na+-dependent pHi
recovery. JH values at a pHi of 6.30 when cells were treated with different concentrations of urea are
summarized in Fig. 7. Urea at 100 mM (400 mosmol/kgH2O) had no effect on JH.
On the other hand, urea at 200 (500 mosmol/kgH2O) and 400 mM (700 mosmol/kgH2O) significantly increased
JH by a similar magnitude.

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Fig. 7.
JH at a pHi of 6.30 when proximal tubule cells from WT mice were exposed to different
concentrations of urea. Values are means ± SE. The no. of tubules
examined is in parentheses. NS, not significant.
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Next, we examined whether the structural analogs of urea mimic the
stimulatory effect of hyperosmotic urea on basolateral NHE in cells
from WT mice. The urea analogs tested were methylurea, thiourea, and
acetamide. For this purpose, we added the Na+-free
HEPES-buffered solution (solution 2, Table 1) involving 200 mM methylurea, thiourea, and acetamide (500 mosmol/kgH2O) to cells from WT mice and then observed the Na+-dependent
pHi recovery. JH values at a
pHi of 6.30 when cells from WT mice were exposed to
hyperosmotic urea, methylurea, thiourea, or acetamide are summarized in
Fig. 8. In the cells treated with hyperosmotic methylurea, JH values were
563.0 ± 61.5 µM/s (n = 4), a value
significantly greater than those in cells treated with the isosmotic
solution (336.8 ± 30.2 µM/s, n = 8, P < 0.05) but not significantly different from those
in cells treated with hyperosmotic urea (525.2 ± 42.0 µM/s,
n = 7). On the contrary, thiourea and acetamide were
without effect.

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Fig. 8.
JH at a pHi of 6.30 when
proximal tubule cells from WT mice were exposed to an isosmotic
solution (300 mosmol/kgH2O), hyperosmotic urea, methylurea,
thiourea, acetamide, and glycerol (200 mM; 500 mosmol/kgH2O). Values are means ± SE. The no. of
tubules examined is in parentheses.
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|
Next, we examined whether another membrane-permeant solute, glycerol,
mimics the stimulatory effect of hyperosmotic urea on basolateral NHE
in cells from WT mice. For this purpose, we added the
Na+-free HEPES-buffered solution (solution 2,
Table 1), including 200 mM glycerol (500 mosmol/kgH2O), to
cells from WT mice and then observed the Na+-dependent
pHi recovery. JH values at a
pHi of 6.30 when cells from WT mice were exposed to
hyperosmotic glycerol are also summarized in Fig. 8. Hyperosmotic
glycerol had no effect on JH.
Role of PKC in hyperosmotic mannitol- and urea-induced NHE
activation.
Previously, we have demonstrated that exposure to the hyperosmotic
mannitol solution actually activated PKC in proximal tubules from KO
mice but not in those from WT mice, whereas exposure of proximal
tubules from WT mice to the hyperosmotic mannitol solution containing
PMA (the PKC activator) activated PKC (27). Recently, we
observed that in the cells from KO mice, PKC inhibitors (staurosporine and calphostin C) inhibited hyperosmotic mannitol-induced NHE activation, whereas PMA under isosmotic conditions mimicked the stimulatory effect of hyperosmotic mannitol on basolateral NHE (25). On the basis of this observation, we concluded that
a hyperosmotic mannitol solution activates basolateral NHE via PKC in
tubules from KO mice but not in those from WT mice (25). Therefore, we examined whether PKC is also involved in hyperosmotic urea-induced NHE activation. For this purpose, we added the
hyperosmotic mannitol or urea solutions to both groups of the cells in
the presence of staurosporine (100 or 500 nM) and then observed the Na+-dependent pHi recovery.
JH values at a pHi of 6.30 when
cells from WT and KO mice were exposed to the hyperosmotic mannitol or
urea solutions (500 mosmol/kgH2O) in the absence and
presence of staurosporine are summarized in Fig.
9. When cells from KO mice were treated
with the hyperosmotic mannitol solution plus staurosporine (100 nM),
the JH were 376.6 ± 44.1 µM/s
(n = 10), which were significantly smaller than those
in cells treated with the hyperosmotic mannitol solution alone
(490.4 ± 25.1 µM/s, n = 12) but were not
significantly different from those in cells treated with the isosmotic
solution (336.8 ± 30.2 µM/s, n = 8). These
findings were in good accord with our previous report
(25). When the cells of the WT mice were exposed to the
hyperosmotic urea solution plus staurosporine (100 or 500 nM),
JH values at a pHi of 6.30 were not
significantly changed. When the cells of the KO mice were exposed to
the hyperosmotic urea solution plus staurosporine (500 nM),
JH values at a pHi of 6.30 were not
significantly influenced, either.

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Fig. 9.
JH at a pHi of 6.30 when proximal tubule cells from WT and KO mice were exposed to an
isosmotic solution, hyperosmotic mannitol solution, hyperosmotic
mannitol solution plus staurosporine, hyperosmotic urea solution, and
hyperosmotic urea solution plus staurosporine. Values are means ± SE. Stauro, staurosporine. The no. of tubules examined is in
parentheses.
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|
Role of tyrosine kinase in hyperosmotic mannitol- and urea-induced
NHE activation.
Cohen et al. (10) reported that in a murine inner
medullary collecting duct cell line, hyperosmotic urea induced
transcription of the zinc finger-containing transcription factor
Egr-1 and urea-induced Egr-1 transcription was
sensitive to genistein (the tyrosine kinase inhibitor). Their findings
suggest the possibility that tyrosine kinase may be involved in
hyperosmotic urea-induced NHE activation. To demonstrate this
possibility, we added our hyperosmotic mannitol or urea solutions in
the presence of genistein to both groups of cells and then observed the
Na+-dependent pHi recovery.
JH values at a pHi of 6.30 when
cells from WT and KO mice were exposed to the hyperosmotic mannitol or
urea solutions (500 mosmol/kgH2O) in the absence and
presence of genistein (10 µM) are summarized in Fig.
10. When cells from WT mice were
treated with the hyperosmotic urea solution plus genistein,
JH values were 411.4 ± 10.7 µM/s
(n = 9). The values were significantly smaller than
those in cells treated with the hyperosmotic urea solution alone
(527.2 ± 39.9 µM/s, n = 13) but were not
significantly different from those in the cells treated with the
isosmotic solution (357.5 ± 41.3 µM/s, n = 10).
Similarly, when cells from KO mice were treated with the hyperosmotic
urea solution plus genistein, JH values were
354.4 ± 28.5 µM/s (n = 7), values that were
significantly smaller than those in cells treated with the hyperosmotic
urea solution alone (511.4 ± 38.9 µM/s, n = 8)
but were not significantly different from those in cells treated with
the isosmotic solution (336.8 ± 30.2 µM/s, n = 8). In sharp contrast, when cells from KO mice were exposed to the
hyperosmotic mannitol solution plus genistein,
JH values at a pHi of 6.30 were not
significantly changed at all.

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Fig. 10.
JH at a pHi of 6.30 when proximal tubule cells fromWT and KO mice were exposed to an
isosmotic solution, hyperosmotic mannitol solution, hyperosmotic
mannitol solution plus genistein, hyperosmotic urea solution, and
hyperosmotic urea solution plus genistein. Values are means ± SE.
The no. of tubules examined is in parentheses.
|
|
 |
DISCUSSION |
Our laboratory has previously reported that exposure of isolated
nonperfused proximal tubules from WT mice to hyperosmotic mannitol did
not elicit RVI after initial cell shrinkage (24). On the
other hand, RVI was observed with hyperosmotic mannitol, when P-gp
activity is acutely suppressed by the P-gp inhibitors (verapamil and
cyclosporin A) or when both mdr1a and mdr1b genes are genetically knocked out (24). The P-gp-induced
modulation of RVI during exposure to hyperosmotic mannitol occurs via
PKC activation (27). Basolateral NHE partly contributes to
the P-gp-induced modulation of RVI (24). Furthermore, we
demonstrated that in the absence of P-gp activity, hyperosmotic
mannitol actually activates basolateral NHE via PKC, but in the
presence of P-gp activity it does not (25). In the present
study, we extend our previous study to compare the effects of
hyperosmotic urea on basolateral NHE with those of hyperosmotic
mannitol in mouse proximal tubule cells.
Basolateral NHE activity in both groups of cells treated with
isosmotic, hyperosmotic mannitol, and hyperosmotic urea solutions.
As shown in Fig. 5, in cells from WT and KO mice under both isosmotic
and hyperosmotic conditions, JH decreased
through a NHE process as pHi increased from the acidic to
the normal range. Under isosmotic conditions, JH
values in cells from KO mice were not significantly different from
those in cells from WT mice over the entire range of pHi
studied (6.20-6.90), consistent with the notion that under these
conditions, basolateral membranes of proximal tubule cells from KO mice
have a NHE activity similar to that in cells from WT mice. However, in
KO mice, both JH values at a pHi
range of 6.20-6.45 and maximal JH values
were significantly greater in cells exposed to the hyperosmotic
mannitol solution than in those exposed to the isosmotic solution. In
KO mice, hyperosmotic mannitol shifted JH vs.
pHi by ~0.15 pH units in the alkaline direction at the
low pHi values. In sharp contrast to KO mice, when cells
from WT mice were treated with the hyperosmotic mannitol solution and
the isosmotic solution, JH values over the
entire range of pHi examined (6.20-6.90) were not
significantly different. In marked contrast to cells exposed to
hyperosmotic mannitol, in WT and KO mice, both
JH values over the entire range of
pHi examined and maximal JH values
were significantly greater in cells exposed to the hyperosmotic urea
solution than in those exposed to the isosmotic solution. For the
hyperosmotic urea solution, maximal JH values
were not significantly different between the two groups. In both groups
of cells, hyperosmotic urea shifted JH vs.
pHi by 0.15-0.20 pH units in the alkaline direction
for the wide range of pHi . In both groups of cells treated
with the hyperosmotic mannitol or urea solutions,
Na+-dependent pHi recovery was blocked by
pretreatment with EIPA (see Fig. 4). Taken together, we conclude that
in both groups of cells, hyperosmotic urea activates basolateral NHE by
a similar magnitude, whereas only in cells from KO mice does
hyperosmotic mannitol activate basolateral NHE. In other words,
hyperosmotic urea-induced NHE activation is independent of P-gp,
whereas hyperosmotic mannitol-induced NHE activation is dependent on
P-gp. In contrast to the stimulatory effect of urea on NHE in the mouse
proximal tubule, Leviel et al. (21) reported that in rat
medullary thick ascending limb, hyperosmotic urea inhibits NHE activity.
In both groups of cells treated with hyperosmotic urea, final
steady-state pHi values after Na+-dependent
pHi recovery were significantly greater than initial steady-state pHi values before the NH4Cl pulse.
The exposure of both groups of cells to hyperosmotic urea caused an
alkaline shift in pHi sensitivity of NHE at a wide
pHi range. Similar to the effect of hyperosmotic urea,
Miyata et al. (26) showed in cultured rat mesangial cells
that hyperosmotic mannitol shifted JH vs. pHi by 0.15-0.3 pH units in the alkaline direction at
a wide pHi range of 6.40-6.95. Similarly, Seo et al.
(34) reported in perfused rat mandibular salivary gland
that hyperosmotic sucrose shifted H+ flux via the NHE vs.
pHi relationship in the alkaline direction at a
pHi range of 7.05-7.45. Grinstein et al.
(17) also studied the Na+-dependent component
of pHi recovery from an acid load in thymic lymphocytes,
finding that shrinkage shifts the flux vs. pHi relationship by 0.2-0.3 pH units in the alkaline direction over the entire range of pHi between ~6.2 and ~7.2. In addition, Shrode
et al. (35) reported in C6 glioma cells
treated with hyperosmotic mannitol that shrinkage caused an alkaline
shift in pHi sensitivity of NHE at a wide range of
pHi. Therefore, the stimulatory effect of hyperosmotic urea
on NHE is common to many cell types. Because the pHi
sensitivity of the exchange system appears to be largely determined by
an allosteric modifier site located on the cytoplasmic surface of the
membrane (2, 16), one of the mechanisms responsible for
hyperosmotic urea-induced NHE activation in both groups of cells is a
shift in pHi dependence of the antiport. In marked contrast
to cells exposed to hyperosmotic urea, in cells from KO mice that were
exposed to hyperosmotic mannitol, final steady-state pHi
values after Na+-dependent pHi recovery were
similar to initial steady-state pHi values before the
NH4Cl pulse, and the alkaline shift in pHi
sensitivity of NHE occurred only at the low pHi range.
Therefore, this phenomenon is specific to mannitol only in proximal
tubule cells from KO mice, although the underlying mechanisms remain unknown.
In many cell types, NHE, in addition to its important role in
pHi homeostasis, contributes to RVI after a hyperosmotic
stress such as mannitol (17, 19, 23, 26, 33, 35). In fact, we previously observed that only in proximal tubules from KO mice did
hyperosmotic mannitol elicit RVI, whereas hyperosmotic mannitol-induced RVI was abolished by pretreatment with EIPA (24). On the
other hand, we previously reported that in both groups of tubules
exposed to hyperosmotic urea, the reduction in cell volume was smaller compared with hyperosmotic mannitol and transient, and washout of urea
caused a large transient overshoot. This indicates that the volume
change during the presence of urea is the result of simple diffusion
but not RVI. Therefore, the effect of urea on basolateral NHE is not
associated with RVI. We further examined whether another
membrane-permeant solute, glycerol, activates basolateral NHE like
urea, but we failed to demonstrate it. Of the structural analogs of
urea, thiourea or acetamide had no effect on NHE, although methylurea
activates NHE by a similar magnitude to urea. Accordingly, the
stimulatory effect on basolateral NHE is relatively specific to urea.
Similarly, Cohen and Gullans (9) observed that in
Madin-Darby canine kidney (MDCK) cells, hyperosmotic urea increased
[3H]thymidine incorporation, but hyperosmotic glycerol,
acetamide, thiourea, or methylurea did not, and concluded that
hyperosmotic urea selectively induced DNA synthesis in the cells.
Under isosmotic conditions, the apparent Km
(22.9 mM) for peritubular Na+ of NHE and
Vmax (346.8 µM/s) in basolateral membranes of
proximal tubule cells from WT mice were similar to those reported in
basolateral membranes of proximal tubule cells from KO mice (21.1 mM
and 370.6 µM/s, respectively) (25). In the present
study, we found that the exposure of cells from WT mice to hyperosmotic
urea increased Vmax from 346.8 to 480.8 µM/s
without changing the Km for peritubular Na+, indicating that in cells exposed to hyperosmotic urea,
the affinity for peritubular Na+ remained the same, but the
number of NHEs increased. This is also one of the mechanisms for
hyperosmotic urea-induced NHE activation. Similar results were observed
in cells from KO mice that were exposed to the hyperosmotic mannitol
(25).
Functional and pharmacological properties of basolateral NHE.
Amiloride and its analogs, including EIPA, have been widely used for
pharmacological characterization of distinct NHE isoforms (7,
28). In the present experiments, IC50 values of
basolateral NHE in proximal tubules from WT and KO mice suggested that
basolateral NHE in both groups of proximal tubules is a highly
amiloride-resistant type (NHE3 or NHE4) (7, 28).
Furthermore, it should be noted that dose-response curves with EIPA are
consistent with the presence of only one form of NHE. Although other
NHE isoforms might be present at the basolateral membrane, the present
results are compatible with the inhibition of a single population of
NHE. These findings are unique because most epithelial cells, including
proximal tubule cells, express NHE1 at the basolateral membrane
(3, 28); however, our results clearly do not support the
existence of a highly-sensitive NHE isoform at the basolateral membrane
of both groups of proximal tubules.
Further studies concerning the effects of hyperosmolality on NHE
activity were performed in an effort to discriminate between the
drug-resistant isoforms, NHE3 and NHE4. Hyperosmolality-induced cell shrinkage has been shown to stimulate NHE4 activity
(4), whereas it inhibits NHE3 activity (35).
In our experiments, hyperosmotic mannitol activated basolateral NHE
only in proximal tubules from KO mice, whereas hyperosmotic urea
activated basolateral NHE in both groups of tubules. Previously, we
reported that hyperosmotic mannitol and urea elicited cell shrinkage,
although the response of cell volume to hyperosmotic urea was markedly
different from that to hyperosmotic mannitol (24).
One might argue that in hyperosmotic medium, ubiquitously expressed
NHE1 may become activated and contribute to basolateral NHE activity,
because this isoform has also been shown to be activated by
hyperosmolality (4, 35). However, under hyperosmotic
conditions, addition of EIPA at a concentration that should completely
inhibit NHE1 activity, resulted in a very slight inhibition of
basolateral NHE activity. Considering the very low IC50
value (0.02 µM) (28) of NHE1 for EIPA, we conclude that
NHE1 does not contribute to basolateral NHE activity under either
isosmotic or hyperosmotic conditions. Also, if we compare EIPA data
under hyperosmotic conditions to those in Fig. 3, the dose-response
profile for inhibition of basolateral NHE activity by EIPA is exactly
the same under isosmotic and hyperosmotic conditions. These functional
properties strongly suggest that drug-resistant NHE4 is the isoform
responsible for basolateral NHE activity in both groups of proximal
tubules. This is also supported by the report of Chambrey et al.
(6), in which the basolateral membrane of the rat proximal
tubule was labeled with anti-NHE4 antibody.
Role of PKC and tyrosine kinase in hyperosmotic mannitol- and
urea-induced NHE activation.
In MDCK cells, hyperosmolality induced by NaCl or raffinose has been
shown to enhance inositol 1,4,5-triphosphate levels and thereby
activate PMA-sensitive PKC (37). In Ehrlich mouse ascites tumor cells, PKC is involved in activation of the
Na+/K+/2Cl
cotransporter induced
by hyperosmolality (20). Treatment of NIH/3T3 cells with
hyperosmotic NaCl solution has also been reported to trigger
phospholipase C activation, then induce an increase in diacylglycerol
levels, and as a consequence, PKC activation (41).
Previously, we reported that in KO mice, the effects of hyperosmotic
mannitol on basolateral NHE (25) and RVI (27) indeed occur via PKC activation. In the present study, we confirmed that in KO mice, staurosporine (the PKC inhibitor) at 100 nM eliminated the hyperosmotic mannitol-induced NHE activation (see Fig. 9). Cohen et
al. (8) reported that hyperosmotic urea upregulated expression at the mRNA level of two growth-associated immediate-early genes, Egr-1 and c-fos, in a renal epithelial
cell-specific fashion. They also demonstrated that PKC is implicated in
urea-induced Egr-1 transcription, because both the PKC
inhibitors (staurosporine and calphostin C) and downregulation of PKC
with chronic exposure to O-tetradecanoylphorbol 13-acetate
inhibited the ability of urea to activate transcription of Egr-1
(10). Therefore, we expected that staurosporine might
block hyperosmotic urea-induced NHE activation. However, staurosporine
at 100 and 500 nM failed to do so in both WT and KO mice. Taken
together with our previous and present findings, hyperosmotic
mannitol-induced NHE activation is PKC dependent only in KO mice,
whereas hyperosmotic urea-induced NHE activation is PKC independent in
both WT and KO mice.
Finally, we examined the intracellular signaling mechanisms responsible
for hyperosmotic urea-induced NHE activation. The result of the present
study showed that tyrosine kinase is indeed involved in hyperosmotic
urea-induced NHE activation in both groups of proximal tubule cells.
Evidence supporting the conclusions of this study was obtained
primarily from experiments examining the effects of the tyrosine kinase
inhibitor genistein. Several findings support the view that genistein
influenced basolateral NHE activity via its action on tyrosine kinase
activity. At the concentration studied (10 µM), genistein is a
selective tyrosine kinase inhibitor, with no significant activity
against a variety of other protein kinases or phosphatases
(1). The apparent specificity of this agent was supported
in the present study by the observation that genistein, at the same
concentration that completely abolished hyperosmotic urea-induced NHE
activation in both groups of proximal tubule cells, had no effect on
hyperosmotic mannitol-induced NHE activation in proximal tubule cells
from KO mice. Also, these findings were not the results of a toxic or
nonspecific metabolic effect on proximal tubule cells. Taken together,
these results support the notion that genistein prevents hyperosmotic
urea-induced NHE activation via its targeted action to inhibit tyrosine
kinase activity.
Although our results suggest an important role for tyrosine
phosphorylation in hyperosmotic urea-induced NHE activation, they do
not establish the nature of the link between tyrosine kinase and
hyperosmotic urea. An increase in tyrosine phosphorylation in response
to hyperosmotic urea could result from stimulation of tyrosine kinase
activity, inhibition of protein tyrosine phosphatase activity, or both.
Pewitt et al. (30) have reported that exposure of duck red
blood cells to hyperosmotic conditions stimulates phosphorylation of
the bumetanide-sensitive
Na+-K+-2Cl
cotransporter itself
or a regulatory protein by activation of both cAMP-dependent and
-independent kinases, thereby activating the cotransporter. Bianchini
et al. (3) have shown in lymphocytes that okadaic acid
(the phosphatase inhibitor) induces activation and phosphorylation of
NHE protein. Future studies will be required to clarify whether
hyperosmotic urea actually stimulates tyrosine kinase activity and how
the tyrosine kinase induced by hyperosmotic urea increases basolateral
NHE activity.
As shown in Figs. 9 and 10, in proximal tubule cells from KO mice,
staurosporin suppressed hyperosmotic mannitol-induced NHE activation,
whereas genistein did not. In marked contrast, in both groups of
proximal tubule cells, genistein inhibited hyperosmotic urea-induced
NHE activation, whereas staurosporin did not. Therefore, a
genistein-sensitive tyrosine kinase does not appear to be involved in
hypersomotic mannitol-induced NHE activation. On the basis of the use
of pharmacological agents, we conclude that hyperosmotic mannitol-induced NHE activation occurs via a PKC-dependent pathway that
does not involve tyrosine kinase, whereas hyperosmotic urea-induced NHE
activation occurs via a tyrosine kinase-dependent pathway that
functions independently of PKC. Therefore, hyperosmotic mannitol and
urea differentially activate basolateral NHE in the mouse proximal tubule.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a grant from the Japanese Kidney
Foundation (Jinkenkyukai), a grant from the Salt Science Foundation,
and Grants-in-Aid for Scientific Research from the Ministry of
Education, Science, and Culture (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.
10.1152/ajprenal.00025.2002
Received 18 January 2002; accepted in final form 17 May 2002.
 |
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