Hyperosmotic mannitol activates basolateral NHE in proximal tubule from P-glycoprotein null mice

Yukio Miyata, Yasushi Asano, and Shigeaki Muto

Department of Nephrology, Jichi Medical School, Tochigi 329-0498, Japan


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Using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we examined the effects of hyperosmotic mannitol on basolateral Na+/H+ exchange (NHE) activity in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type mice (WT). All experiments were performed in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH2O by the addition of mannitol. NHE activity was assessed by Na+-dependent acid extrusion rates (JH) after an acid load with NH4Cl prepulse. Under isosmotic conditions, JH values at a wide intracellular pH (pHi) range of 6.20-6.90 were not different between the two groups. In WT mice, hyperosmotic mannitol had no effect on JH at the wide pHi range. In contrast, in KO mice, hyperosmotic mannitol increased JH at a pHi range of 6.20-6.45 and shifted the JH-pHi relationship by 0.15 pH units in the alkaline direction. In KO mice, hyperosmotic mannitol caused an increase in maximal velocity without changing the Michaelis-Menten constant for peritubular Na+. Exposure of cells from WT mice to the hyperosmotic mannitol solution including the P-gp inhibitor cyclosporin A increased JH (at pHi 6.30) to an extent similar to that in cells from KO mice exposed to hyperosmotic mannitol alone. In KO mice, staurosporine and calphostin C inhibited the hyperosmotic mannitol-induced increase in JH. The stimulatory effect of hyperosmotic mannitol on JH was mimicked by addition to the isosmotic control solution, including phorbol 12-myristate 13-acetate (PMA; the PKC activator). In WT mice, hyperosmotic mannitol with PMA increased JH. We conclude that, in the absence of P-gp activity, hyperosmotic mannitol activates basolateral NHE via protein kinase C, whereas in the presence of P-gp activity, it does not.

isolated nonperfused tubule; regulatory volume increase; intracellular pH measurement; sodium-hydrogen exchange


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P-GLYCOPROTEIN (P-GP) WAS initially identified through its ability to confer multidrug resistance (MDR) in mammalian tumor cells (reviewed in Ref. 11). P-gp is a member of the ATP-binding cassette superfamily of transporters (11) and utilizes ATP to pump hydrophobic drugs out of the cells, decreasing their intracellular concentrations and hence their toxicity. 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) (11, 14, 27). In the mouse, mdr1a and mdr1b together fulfill the same function as MDR1 in humans, and similar levels of mdr1a and mdr1b expression are observed in the kidney (5, 28). In the kidney, the apical membrane of the proximal tubule epithelium is particularly rich in P-gp (7, 30), 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 (31) recently reported that in the isolated perfused mouse proximal tubule, 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 the mdr1a and mdr1b genes were disrupted (KO mice).

In a variety of cell types, Na+/H+ exchange (NHE) is activated by shrinkage of cells 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. 17 and 20). In sharp contrast, when rabbit proximal tubule cells are suddenly exposed to hyperosmotic mannitol, NaCl, or raffinose solutions, they rapidly shrink but remain reduced in size (9, 19). However, the underlying mechanisms responsible for the lack of RVI have not been fully demonstrated. Recently, our laboratory (21) 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 observed that in tubules from WT mice, the peritubular addition of P-gp inhibitors (verapamil and cyclosporin A) resulted in RVI, whereas in the tubules of the KO mice it had no effect on RVI (21). Therefore, in the mouse proximal tubule, P-gp modulates RVI during the exposure to hyperosmotic mannitol. We also observed that the P-gp-induced modulation of RVI was abolished by both removing peritubular Na+ and adding peritubular ethylisopropylamiloride (EIPA; the specific NHE inhibitor) (21). These findings indicate that basolateral NHE contributes to the P-gp-induced modulation of RVI. However, it is not known whether basolateral NHE is actually activated in P-gp-induced cell volume regulation during exposure to hyperosmotic mannitol. NHE in the basolateral membrane of the mouse proximal tubule under isosmotic conditions also has not yet been characterized.

Therefore, we used isolated nonperfused proximal tubule S2 segments from KO and WT mice to examine 1) whether the basolateral membrane of mouse proximal tubule cells possesses an NHE under isosmotic conditions and, if so, 2) whether and how basolateral NHE is modulated by hyperosmotic mannitol.


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Solutions. The compositions of solutions are given in Table 1. All solutions were nominally CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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. A hyperosmotic mannitol solution (500 mosmol/kgH2O) was made by adding mannitol to solutions 1 or 2. The nigericin-calibrating solutions were 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. 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was prepared as a 10 mM stock solution and diluted 1:1,000 to a final concentration of 10 µM. EIPA was prepared as a 100 mM stock solution in methanol and diluted 1:1,000 to a final concentration of 100 µM. Nigericin was prepared as a 10 mM stock solution in ethanol and diluted 1:1,000 into solution 4 (Table 1) to a final concentration of 10 µM. Cyclosporin A was dissolved in ethanol at 0.1% final concentration. Phorbol 12-myristate 13-acetate (PMA), staurosporine, and calphostin C were each dissolved in DMSO at 0.1% final concentrations. Equivalent concentrations of vehicle were added as a control for individual protocols.

                              
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Table 1.   Composition of solutions

Animals. KO mice used in this study were originally described by Schinkel et al. (27). Male KO and FVB (WT) mice, serving as controls (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 standard rodent chow and tap water ad libitum until the experiments began. Ages of KO animals were matched with their WT controls.

In vitro microperfusion. Both groups of mice 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 with 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 tubules were drawn into and crimped by glass micropipettes as described by Dellasega and Grantham (6) and Miyata et al. (21). In this preparation, both ends of the tubules are occluded so that the lumen becomes and remains collapsed. A flow-through bath system was utilized to permit rapid exchange of 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. After the equilibration period, intracellular pH (pHi) was measured as described below.

Measurement of pHi. Both groups of tubules were exposed from the bath to the isosmotic control HEPES-buffered solution (solution 1, Table 1) containing the fluorescent pH-sensitive dye BCECF-AM (10 µM). After a 15-min dye-loading period at 37°C, the dye was washed out.

Single-cell measurements of pHi were performed using a microscopic fluorometer (OSP3, Olympus) as described previously (3, 22, 25). Measurements were made at ×100 magnification, and the diameter of the beam of light focused on the cells was ~7.5 µm. The light source was a 75-W xenon lamp. The fluorescent dye was excited alternatively at 440 and 490 nm by spinning the sector mirror at 300 rpm and measured at a wavelength of 530 nm. Because it takes 10 ms to obtain one fluorescence-excitation ratio (I490/I440) with this apparatus, each cell was exposed to light for 1 s to obtain one mean I490/I440. We used only cells that had at least a 20-fold greater fluorescence intensity than that of the background. To minimize dye bleaching and cell damage, protocols were made as short as possible.

Calibration of pHi. pHi was calculated using the nigericin calibration technique as described previously (3, 22, 25). At the end of each experiment, cells were exposed to a Na+-free solution (solution 4, Table 1) containing 10 µM of the H+/K+ exchanger (nigericin) and 105 mM K+. If internal and external K+ concentration are equal, then nigericin should equalize pHi and extracellular pH. Figure 1A is a plot of I490/I440 vs. time for a single cell exposed to a series of nigericin solutions at different pH values. I490/I440 and pH data from this experiment are summarized in Fig. 1B. The data were normalized to make the ratio at pH 7.0 equal to unity. The ratio data can be described by a pH titration curve of the form
I<SUB>490</SUB>/I<SUB>440</SUB>=a+b[10<SUP>(pH−p<IT>K</IT>)</SUP>]<IT>/</IT>[1<IT>+</IT>10<SUP>pH−p<IT>K</IT></SUP>)] (1)
Inasmuch as the curve was constrained to pass through the point having the coordinates I490/I440 = 1.0 and pH = 7.0, we fitted the data to a variant of the pH titration equation that forces the curve through this standard point
I<SUB>490</SUB>/I<SUB>440</SUB>=1+b<FENCE><FR><NU>10<SUP>(pH−p<IT>K</IT>)</SUP></NU><DE>1<IT>+</IT>10<SUP>(pH−p<IT>K</IT>)</SUP></DE></FR><IT>−</IT><FR><NU>10<SUP>(7−p<IT>K</IT>)</SUP></NU><DE>1<IT>+</IT>10<SUP>(7−p<IT>K</IT>)</SUP></DE></FR></FENCE> (2)
where a and b are the lower asymptote of the curve and the distance between the upper and lower asymptotes of the curve, respectively, and pK is the dissociation constant.


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Fig. 1.   pH calibration of intracellular dye. A: time course of fluorescence-excitation ratio (I490/I440) during exposure to high-extracellular K+ concentration-nigericin solutions at different pH values. At the beginning of recording, proximal tubule cells from wild-type (WT) and mdr1a and mdr1b null (KO) mice were exposed to a pH 7.0 nigericin-containing solution (solution 4, Table 1, with 10 µM nigericin added). At indicated times, bathing solution was switched to nigericin-containing solutions at different pH values. pHo, extracellular pH. B: dependence of normalized fluorescence-excitation ratio (I490/I440) on intracellular pH (pHi). , Data from 5 experiments in WT mice; open circle , data from 7 experiments in KO mice. Curves are results of a nonlinear least-squares fit of data to pH titration curve that forces curve to pass through (I490/I440) = 1.0 at pHi of 7.0.

The curve drawn through the points in Fig. 1B is the result of a nonlinear least-squares fit of the normalized data to Eq. 2. The fitted values in the WT mice were 7.15 ± 0.01 (n = 10) and 1.56 ± 0.01 (n = 10) for pK and b, respectively. The corresponding values in KO mice for pK and b were 7.15 ± 0.04 (n = 10) and 1.55 ± 0.03 (n = 10). The advantage of this normalization procedure is that it allows us to obtain a one-point nigericin calibration for a cell. At the end of each experiment, the cell was exposed to a nigericin solution at pH 7.0, and the I490/I440 data from the entire experiment were divided by the value at pH 7.0. We used Eq. 2 to calculate pHi from these normalized I490/I440 values and the fitted values for pK and b.

Determination of intracellular buffering power. Intrinsic buffering power (beta I) of proximal tubule cells from WT and KO mice was determined by the method described previously (3, 22, 25). As shown in Fig. 2A, acid-loaded cells were exposed to a series of nominally Na+-free solutions (solution 2, Table 1) that contained 20, 10, 5, 2.5, 1.0, 0.5, and 0 mM total ammonium (NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>). Total NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-containing solutions were prepared by adding NH4Cl, with replacement of NMDG in Na+-free solution. With each stepwise decrease in extracellular NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> concentration ([NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>]o), the amount of protons delivered to the cytoplasm (Delta [acid]i) was considered equal to the resultant change in intracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> concentration ([NH<UP><SUB>4</SUB><SUP>+</SUP></UP>]i). If it is assumed that [NH3]i equals [NH3]o, and that the acidic pK governing NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> equilibrium (8.9 at 37°C) is the same in the cytoplasm as in the extracellular fluid, [NH<UP><SUB>4</SUB><SUP>+</SUP></UP>]i can be calculated from the observed pHi. Delta pHi was taken as the change in pHi produced by the stepwise decrease in [NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP>]o. beta I was then calculated as -Delta [acid]i/Delta pHi (3, 22, 25). beta I was assigned to the mean of the two pHi values used for its calculation. beta I vs. pHi data were fitted by a straight line for both groups of cells.


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Fig. 2.   pHi dependence of intrinsic intracellular buffering power (beta I). A: a typical experiment in which we determined the buffering power of proximal tubule cells from WT mice in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-free, HEPES-buffered solution. Na+ was removed, and NMDGCl was then replaced with 20 mM NH4Cl. NH4Cl concentration was decreased step by step (horizontal bar). Restoration of extracellular Na+ concentration to 142 mM caused pHi to increase, indicating Na+/H+ exchange (NHE) activity. From these data, beta I was calculated as described in METHODS. B: pHi dependence of beta I for proximal tubule cells from WT () and KO (open circle ) mice. Straight lines fit data from 8 proximal tubule cells from 3 WT mice and 6 proximal tubule cells from 3 KO mice. The equations of the best fit line in the proximal tubule cells from WT and KO mice were beta I = 441.0 - 61.5 × pHi (r = 0.985) and beta I = 429.6 - 60.0 × pHi (r = 0.992) at a range of physiological pHi, respectively.

Computation of Na+-dependent acid extrusion rates. Na+-dependent acid extrusion rates (JH) in proximal tubule cells from WT and KO mice were calculated from rates of pHi increase (dpHi/dt) and beta I in the following experiment: proximal tubule cells from WT and KO mice 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 slower influx of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. In the continued presence of the Na+-free solution, NH4Cl was abruptly removed from the peritubular solution, causing pHi to decrease. After that, readdition of Na+ to the peritubular solution gave rise to a rapid increase in pHi. For the experiment, the 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-pHi data (3, 22, 25). The H+ flux due to NHE (i.e., JH) was then determined from the equation JH = dpHi/dt × beta I.

Drugs and chemicals. All chemicals were obtained from Wako (Osaka, Japan) except HEPES and BCECF-AM, which were from Dojindo (Kumamoto, Japan), and EIPA, NMDG, PMA, staurosporine, nigericin, and cyclosporin A, which 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.


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Steady-state pHi in proximal tubule cells from WT and KO mice. The study of pHi was carried out in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free HEPES-buffered solutions to minimize the contribution of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent transport mechanisms. When collapsed proximal tubule cells from WT and KO mice were peritubularly perfused with the Na+-containing HEPES-buffered solution (solution 1, Table 1) at a rate of 4.5-5.5 ml/min at 37°C, the steady-state pHi values were 7.00 ± 0.01 (n = 52) and 6.98 ± 0.01 (n = 54), respectively, and were not statistically significant between the two groups.

beta I in proximal tubule cells from WT and KO mice. The pHi dependence of beta I was determined from experiments such as those illustrated in Fig. 2A. The pHi dependence of such beta I data is summarized in Fig. 2B. The equations of the best fit line of data for proximal tubule cells from WT and KO mice were beta I = 441.0 - 61.5 × pHi (r = 0.985) and beta I = 429.6 - 60.0 × pHi (r = 0.992) at a range of physiological pHi, respectively. These data indicate that in both groups of proximal tubule cells, beta I decreases with increasing pHi. Furthermore, beta I at comparable pHi values was not significantly different between the two groups of proximal tubule cells.

Na+-dependent pHi recovery from an acid load in proximal tubule cells from WT and KO mice under isosmotic conditions. Next, we observed Na+-dependent pHi recovery from an acid load with NH4Cl in proximal tubule cells from WT (Fig. 3A) and KO (Fig. 3B) mice. Proximal tubule cells from WT and KO mice were first bathed in the solution 1 (Table 1) and were then incubated in the Na+-free HEPES-buffered solution containing 20 mM NH4Cl (solution 3, Table 1). At this time, the pHi increased rapidly due to the rapid diffusion of NH3 into the cell. During the exposure to NH4Cl for 2 min, pHi tended to decrease toward baseline due to slow inward diffusion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. After that, removing NH4Cl from the Na+-free HEPES-buffered solution rapidly decreased the pHi, because accumulated internal NH<UP><SUB>4</SUB><SUP>+</SUP></UP> 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 6.16 ± 0.05 (n = 8) and 6.15 ± 0.03 (n = 10), 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 of the initial steady-state pHi. In fact, the pHi values of the final steady state in cells from WT and KO mice were 6.95 ± 0.03 (n = 8) and 6.97 ± 0.03 (n = 10), respectively, and were not significantly different from those of the initial steady-state (WT mice: 7.03 ± 0.02, n = 8; KO mice: 6.97 ± 0.02, n = 10). Figure 4, A and B, shows the effects of peritubular addition of EIPA (100 µM) to proximal tubule cells from WT and KO mice on Na+-dependent pHi recovery from an acid load, respectively. When proximal tubule cells from WT and KO mice were exposed to EIPA, steady-state pHi values significantly decreased from 6.97 ± 0.04 (n = 4) and 7.02 ± 0.05 (n = 6) to 6.95 ± 0.04 (n = 4, P < 0.05) and 6.99 ± 0.05 (n = 6, P < 0.05), respectively. In the continued presence of EIPA, readdition of Na+ produced a much smaller pHi increase (WT mice: 0.27 ± 0.02, n = 4, P < 0.001; KO mice: 0.31 ± 0.07, n = 6, P < 0.001) than in its absence (WT mice: 0.79 ± 0.03, n = 8; KO mice: 0.82 ± 0.04, n = 10). These findings indicate that basolateral NHE indeed exists in proximal tubule cells from both WT and KO mice and contributes to Na+-dependent cell alkalinization from an acid load.


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Fig. 3.   Na+-dependent pHi recovery from an acid load under is- and hyperosmotic conditions in proximal tubule cells from WT (A and C) and KO (B and D) mice. Both groups of cells were first bathed in Na+-containing HEPES-buffered solution (solution 1, Table 1) and were then incubated in Na+-free HEPES-buffered solution containing 20 mM NH4Cl (solution 3, Table 1) for 2 min. During the pulse, pHi increased (due to entry of NH3 into the cell) and then tended to decrease toward baseline due to slower influx of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Thereafter, the abrupt removal of NH4Cl from the Na+-free solution caused a substantial and sustained decrease in pHi. In the absence and presence of the hyperosmotic mannitol solution, readdition of peritubular Na+ to both groups of cells caused pHi to rapidly increase to values similar to initial steady-state pHi values. In both groups of cells in the absence of peritubular Na+, hyperosmotic mannitol had no effect on pHi. However, when cells from KO mice were exposed to the hyperosmotic mannitol solution, the Na+-dependent pHi recovery was faster than when cells from WT mice were exposed to hyperosmotic mannitol or when cells from KO mice were exposed to the isosmotic solution.



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Fig. 4.   Na+-dependent pHi recovery from an acid load in the presence of peritubular ethylisopropylamiloride (EIPA) under is- and hyperosmotic conditions in proximal tubule cells from WT (A and C) and KO (B and D) mice. It should be noted that in both groups of cells under isosmotic conditions, EIPA (100 µM) alone decreased pHi. In both groups of cells under is- and hyperosmotic conditions, Na+-dependent pHi recovery in the presence of EIPA was substantially slower, and final steady-state pHi values after the addition of EIPA were much smaller than those of the initial steady state.

Na+-dependent pHi recovery from an acid load in proximal tubule cells from WT and KO mice under hyperosmotic conditions. Next, we examined whether the hyperosmotic mannitol solution affected Na+-dependent pHi recovery from an acid load in the proximal tubule cells from WT (Fig. 3C) and KO (Fig. 3D) mice. Both groups of proximal tubule cells were first incubated in solution 1 and were then bathed in solution 3 for 2 min. After that, NH4Cl was rapidly removed from solution 3, leading to a rapid decrease in pHi. Thereafter, both groups of cells were exposed to the Na+-free HEPES-buffered solution (solution 2, Table 1) treated with 200 mM mannitol (500 mosmol/kgH2O). At this time, the pHi values in either group of cells were unchanged (WT mice: from 6.11 ± 0.03 to 6.13 ± 0.03, n = 18; KO mice: from 6.13 ± 0.04 to 6.16 ± 0.03, n = 15) (Fig. 3, C and D). After that, when external Na+ was readded in the presence of hyperosmotic mannitol, pHi recovery in proximal tubule cells from KO mice was substantially faster than in its absence, although final steady-state pHi values (7.01 ± 0.03, n = 15) were not significantly different from those in the initial steady state (7.01 ± 0.02, n = 15) (see Fig. 3, B and D). At this time, the Na+-dependent pHi recovery rate in the presence of hyperosmotic mannitol (124.4 ± 10.7 pH/s × 104, P < 0.001, n = 15) was significantly greater than that in its absence (76.8 ± 4.6 pH/s × 104, n = 10). Furthermore, under hyperosmotic conditions, the Na+-dependent pHi recovery in proximal tubule cells from KO mice was faster than that in cells from WT mice (see Fig. 3, C and D). At this time, the Na+-dependent pHi recovery rate in KO mice (124.4 ± 10.7 pH/s × 104, P < 0.001, n = 15) was also significantly greater than that in cells from WT mice (88.8 ± 7.2 pH/s × 104, n = 18). In sharp contrast to proximal tubule cells of KO mice, in proximal tubule cells from WT mice, Na+-dependent pHi recovery under hyperosmotic conditions was similar to that under isosmotic conditions, and final steady-state pHi values (6.94 ± 0.04, n = 18) were not significantly different from those of the initial steady state (6.99 ± 0.03, n = 18) (see Fig. 3, A and C).

Effects of peritubular addition of EIPA to proximal tubule cells of WT and KO mice on Na+-dependent pHi recovery under hyperosmotic conditions are shown in Fig. 4, C and D, respectively. In both groups of proximal tubule cells treated with hyperosmotic mannitol, Na+-dependent pHi recovery from an acid load in the presence of EIPA (100 µM) was substantially slower than in its absence (see Figs. 3, C and D, and 4, C and D). In cells from WT and KO mice under hyperosmotic conditions, final steady-state pHi values after readdition of Na+ in the presence of EIPA were 6.51 ± 0.03 (n = 6) and 6.45 ± 0.06 (n = 7), respectively, and were not significantly different between the two groups. Also, in both groups of cells, the Na+-dependent pHi increase in the presence of EIPA (WT mice: 0.31 ± 0.04, n = 6, P < 0.001; KO mice: 0.25 ± 0.05, n = 7, P < 0.001) was much smaller than in its absence (WT mice: 0.81 ± 0.05, n = 18; KO mice: 0.84 ± 0.03, n = 15). At this time, in both groups of cells, the Na+-dependent pHi recovery rate in the presence of EIPA (WT mice: 29.1 ± 2.9 pH/s × 104, n = 6, P < 0.001; KO mice: 28.8 ± 6.2 pH/s × 104, n = 7, P < 0.001) was also significantly smaller than in its absence (WT mice: 88.8 ± 7.2 pH/s × 104, n = 18; KO mice: 124.4 ± 10.7 pH/s × 104, n = 15).

From dpHi/dt and beta I, we calculated the relationship between JH and pHi under is- and hyperosmotic conditions in both groups of proximal tubule cells, as shown in Fig. 5. In both groups of cells under is- and hyperosmotic conditions, JH decreased as pHi increased. In cells from WT mice, JH values under hyperosmotic conditions were not significantly different from those under isosmotic conditions over the entire range of pHi studied (6.20-6.90). In sharp contrast, in cells from KO mice, JH values under hyperosmotic conditions were significantly (P < 0.05) greater than those under isosmotic conditions at a pHi range of 6.20-6.45. In cells from KO mice, maximal JH values under hyperosmotic conditions were also significantly greater than under isosmotic conditions. Furthermore, under hyperosmotic conditions, cells from KO mice had significantly (P < 0.05) greater JH values than those of cells from WT mice at pHi values of 6.20-6.45, although, under isosmotic conditions, JH values in cells from KO mice were not significantly different from those in cells of WT mice over the entire range of pHi studied (6.20-6.90). Therefore, we conclude that in proximal tubule cells from KO mice, the hyperosmotic mannitol solution enhanced JH after an intracellular acid load through basolateral NHE at low pHi (6.20-6.45), whereas in proximal tubule cells from WT mice, it had no effect on basolateral NHE activity over the entire wide pHi range examined (6.20-6.90). Under isosmotic conditions, the Na+-dependent acid extrusion rates through basolateral NHE were not different between the two groups of cells.


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Fig. 5.   pHi dependence of the Na+-dependent acid extrusion rate (JH) under is- and hyperosmotic conditions in proximal tubule cells from WT and KO mice. Plots were computed from experiments such as those illustrated in Fig. 3. Values are means ± SE; n = no. of tubules examined. Isosm., isosmolality; Hyperosm., hyperosmolality. *P < 0.05 compared with cells from KO mice that were exposed to the isosmotic control solution, at comparable pHi values. dagger P < 0.05 compared with cells from WT mice that were exposed to the hyperosmotic mannitol solution, at comparable pHi values.

Next, we examined whether the changes in Michaelis-Menten constant (Km) values for peritubular Na+ concentrations and/or maximal velocity (Vmax) are involved in basolateral NHE activation induced by hyperosmotic mannitol. For this purpose, the kinetics of basolateral NHE were determined by measuring JH at pHi of 6.30 on readdition of varying concentrations of Na+ (0, 7, 14, 43, 100, and 142 mM) to the peritubular side of tubules from KO mice after peritubular Na+ removal and an acid load with NH4Cl prepulse under is- and hyperosmotic conditions. Results are shown in Fig. 6A. As shown in Fig. 6B, a Lineweaver-Burk plot of the data obtained showed that, under isosmotic conditions, the apparent Km for peritubular Na+ concentrations was 21.1 mM with a Vmax of 370.6 µM/s. Under hyperosmotic conditions, the apparent Km for peritubular Na+ was similar (22.7 mM), but Vmax increased to 528.4 µM/s.


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Fig. 6.   Kinetics of basolateral NHE under is- and hyperosmotic conditions. A: after the removal of peritubular Na+ and an acid load with NH4Cl prepulse, varying concentrations of Na+ ([Na+]; 0, 7, 14, 43, 100, and 142 mM) were added to the peritubular side of tubules from KO mice, and JH at pHi of 6.30 was estimated. Each point represents the mean ± SE of at least 4 determinations. B: Lineweaver-Burk analysis of data indicates that exposure of cells from KO mice to the hyperosmotic mannitol solution had no effect on the apparent Michaelis-Menten constant values for peritubular Na+ (from 21.1 to 22.7 mM), but increased maximal velocity from 370.6 to 528.4 µM/s.

Na+-dependent pHi recovery from an acid load in proximal tubule cells from WT and KO mice under is- and hyperosmotic conditions in the presence of cyclosporin A. Next, we examined the effects of cyclosporin A (a P-gp inhibitor) on Na+-dependent pHi recovery from an acid load in proximal tubule cells from WT and KO mice under is- and hyperosmotic conditions. Representative pHi recordings are shown in Fig. 7. When cyclosporin A (5 µM) alone, in the presence of Na+, was added to cells from WT or KO mice, pHi values were not influenced at all (WT mice: from 6.96 ± 0.05 to 6.96 ± 0.05, n = 7; KO mice: 7.00 ± 0.05 to 6.98 ± 0.04, n = 6) (Fig. 7, A and B). When cyclosporin A (5 µM) plus hyperosmotic mannitol, in the absence of Na+, were added to cells from WT or KO mice, pHi values were also not affected at all (WT mice: from 6.17 ± 0.04 to 6.20 ± 0.05, n = 11; KO mice: from 6.17 ± 0.04 to 6.17 ± 0.04, n = 8; see Fig. 7, C and D). When external Na+ was readded to cells from WT mice in the continued presence of hyperosmotic mannitol and cyclosporin A, pHi recovery from an acid load was substantially faster than that in the continued presence of cyclosporin A alone, although final steady-state pHi values (7.00 ± 0.04, n = 11) were not significantly different from those in the initial steady state (6.96 ± 0.03, n = 11) (see Fig. 7, A and C). In cells from WT mice, Na+-dependent pHi recovery from an acid load in the continued presence of hyperosmotic mannitol and cyclosporin A was also faster than that in the continued presence of hyperosmotic mannitol alone (see Figs. 3C and 7C). When Na+ was readded to tubules from KO mice in the continued presence of hyperosmotic mannitol and cyclosporin A, pHi recovery from an acid load was also faster than that in the continued presence of cyclosporin A alone, although final steady-state pHi values (7.04 ± 0.04, n = 8) were not significantly different from those of the initial steady state (7.05 ± 0.03, n = 8) (Fig. 7, B and D). In the presence of cyclosporin A under isosmotic conditions, in cells from KO mice, Na+-dependent pHi recovery from an acid load was similar to that in the cells of the WT mice (see Fig. 7, A and B). From dpHi/dt and beta I, we computed JH at pHi of 6.30 and compared them in the absence and presence and cyclosporin A under is- and hyperosmotic conditions in cells from WT and KO mice, as shown in Fig. 8. In the absence of cyclosporin A, in the cells from WT mice, JH values at pHi of 6.30 under hyperosmotic conditions (379.1 ± 32.8 µM/s, n = 17) were not significantly different from those under isosmotic conditions (378.0 ± 57.0 µM/s, n = 7), whereas in cells from KO mice, JH values at pHi of 6.30 under hyperosmotic conditions (565.0 ± 59.5 µM/s, P < 0.05, n = 12) were significantly greater than those under isosmotic conditions (360.0 ± 28.6 µM/s, n = 10). In the presence of cyclosporin A, in cells from WT mice, hyperosmotic mannitol significantly (P < 0.05) increased JH (at pHi of 6.30) to 579.8 ± 45.6 µM/s (n = 5), values that were not different from those in KO mice in the presence of hyperosmotic mannitol alone. Furthermore, in cells from WT mice under hyperosmotic conditions, JH values at pHi of 6.30 in the presence of cyclosporin A were significantly greater than those in its absence, although in cells from WT mice under isosmotic conditions, JH values at pHi of 6.30 in the presence of cyclosporin A (398.3 ± 68.6 µM/s, n = 6) were not different from those in its absence. In cells from KO mice in the presence of cyclosporin A, hyperosmotic mannitol significantly increased JH (at pHi of 6.30) to 570.2 ± 15.9 µM/s (n = 5, P < 0.05), values that were not different from those in KO mice in the presence of hyperosmotic mannitol alone. In cells from KO mice under isosmotic conditions, JH values at pHi of 6.30 in the presence of cyclosporin A were not different from those in its absence.


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Fig. 7.   Na+-dependent pHi recovery from an acid load in the presence of peritubular cyclosporin A under is- and hyperosmotic conditions in proximal tubule cells from WT (A and C) and KO (B and D) mice. In the presence of peritubular cyclosporin A (5 µM) under is- and hyperosmotic conditions, readdition of peritubular Na+ to both groups of cells caused pHi to rapidly increase to pHi values similar to those of the initial steady state. In both groups of cells in the absence of peritubular Na+, the hyperosmotic mannitol solution involving cyclosporin A had no effect on pHi. It should be noted that when cells from WT mice were exposed to the hyperosmotic mannitol solution including cyclosporin A, Na+-dependent pHi recovery was faster than when cells from WT mice were exposed to the isosmotic solution including cyclosporin A. Also, when cells from KO mice were exposed to the hyperosmotic mannitol solution including cyclosporin A, Na+-dependent pHi recovery was faster than when cells from KO mice were exposed to the isosmotic solution including cyclosporin A.



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Fig. 8.   JH at pHi of 6.30 under is- and hyperosmotic conditions in the absence and presence of cyclosporin A in proximal tubule cells from WT and KO mice. Values are means ± SE taken from experiments such as those illustrated in Figs. 3 and 7; no. of tubules examined are in parentheses. Isosmotic, isosmolality; Hyperosmotic, hyperosmolality; CyA, cyclosporin A; NS, not significant.

Role of protein kinase C in hyperosmotic mannitol-induced NHE activation. Very recently, Miyata et al. (23) have reported that exposure of tubules from WT mice to a hyperosmotic mannitol solution including PMA elicited RVI, whereas exposure to the hyperosmotic mannitol solution alone did not. Also, we found that the addition of tubules from KO mice to the hyperosmotic mannitol solution including either of two protein kinse C (PKC) inhibitors (staurosporine and calphostin C) abolished RVI (23). Furthermore, we have demonstrated that exposure to the hyperosmotic mannitol solution activated PKC in tubules from KO mice but not in those from WT mice, whereas exposure of tubules from WT mice to the hyperosmotic mannitol solution containing PMA activated PKC (23). These findings suggest the possibility that PKC may be involved in hyperosmotic mannitol-induced NHE activation. To explore this possibility, we added the hyperosmotic mannitol solution in the presence of either staurosporine (100 nM) or calphostin C (500 nM) to cells from KO mice and then observed the Na+-dependent pHi recovery. Representative pHi recordings are shown in Fig. 9, A (for the staurosporine-treated cell) and B (for the calphostin C-treated cell). When staurosporine or calphostin C alone in the presence of Na+ was added to cells from KO mice, pHi values were not influenced at all (staurosporine: from 6.92 ± 0.02 to 6.94 ± 0.02, n = 9; calphostin C: 6.95 ± 0.06 to 6.94 ± 0.05, n = 5). When external Na+ was readded to cells from KO mice in the presence of the hyperosmotic mannitol solution plus either staurosporine or calphostin C, pHi recovery from an acid load was substantially slower than that in the presence of the hyperosmotic mannitol solution alone (see Figs. 3D and 9, A and B). From dpHi/dt and beta I, we computed JH values at pHi of 6.30 and compared them in the absence and presence of the two PKC inhibitors under hyperosmotic conditions in cells from KO mice, as shown in Fig. 10. When cells from KO mice were treated with the hyperosmotic mannitol solution plus either staurosporine or calphostin C, JH values at pHi of 6.30 were 355.1 ± 34.6 (n = 9) or 310.3 ± 29.3 (n = 5) µM/s, respectively. These values were significantly smaller than those in cells treated with the hyperosmotic mannitol solution alone (536.7 ± 58.6 µM/s, n = 13) but were not significantly different from those in cells treated with the isosmotic control solution (332.7 ± 27.9 µM/s, n = 8). Next, we examined whether the exposure of cells from KO mice to PMA (the specific activator of PKC) under isosmotic conditions increases basolateral NHE activity. Representative pHi recordings are shown in Fig. 9C. When PMA (100 nM) in the absence of Na+ was added to cells from KO mice, pHi values were not influenced at all (from 6.19 ± 0.03 to 6.15 ± 0.04, n = 4). When external Na+ was readded to cells from KO mice in the continued presence of PMA, pHi recovery from an acid load was substantially faster than in its absence (see Fig. 3B), although final steady-state pHi values (6.98 ± 0.02, n = 4) were not significantly different from those in the initial steady state (6.96 ± 0.05, n = 4). At this time, JH at pHi of 6.30 was 516.9 ± 29.6 µM/s (n = 4). These values were significantly greater than those in cells treated with the isosmotic control solution alone but were not significantly different from those in cells treated with the hyperosmotic mannitol solution alone (Fig. 10). Finally, we examined whether exposure of cells from WT mice to the hyperosmotic mannitol solution plus PMA increases basolateral NHE activity. Representative pHi recordings are shown in Fig. 9D. When external Na+ was readded to cells from WT mice in the presence of the hyperosmotic mannitol solution plus PMA, pHi recovery from an acid load was substantially faster than that in the presence of the hyperosmotic mannitol solution alone. At this time, JH values at pHi of 6.30 were 524.1 ± 51.2 µM/s (n = 5), values that were significantly greater than those in cells exposed to the hyperosmotic mannitol solution alone (365.9 ± 32.0 µM/s, n = 16) and were significantly greater than those in cells exposed to the isosmotic control solution (330.9 ± 38.0 µM/s, n = 6) ( Fig. 10). These values were not significantly different from those in cells from KO mice in the presence of the hyperosmotic mannitol alone.


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Fig. 9.   Role of protein kinase C (PKC) in the Na+-dependent pHi recovery from an acid load under is- and hyperosmotic conditions in proximal tubule cells from KO (A, B, and C) and WT (D) mice. When cells from KO mice were exposed to the hyperosmotic mannitol in the presence of either of 2 PKC inhibitors, staurosporine (A) or calphostin C (B), the Na+-dependent pHi recovery was slower than in its absence (see Fig. 3D). C: when cells from KO mice were exposed to the isosmotic control solution in the presence of phorbol 12-myristate 13-acetate (PMA), Na+-dependent pHi recovery was faster than in its absence (see Fig. 3B) D: when cells from WT mice were exposed to the hyperosmotic mannitol solution in the presence of PMA, the Na+-dependent pHi recovery was faster than in its absence (see Fig. 3C).



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Fig. 10.   JH at pHi of 6.30 under is- and hyperosmotic conditions in the absence and presence of PMA in proximal tubule cells from WT and KO mice. Values are means ± SE taken from experiments such as those illustrated in Figs. 3 and 9; no. of tubules examined are in parentheses. Stauro, staurosporine; Cal, calphostin C.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our laboratory has recently reported that the exposure of isolated nonperfused proximal tubules from WT mice to a hyperosmotic mannitol solution did not elicit RVI after the initial cell shrinkage (21). On the other hand, RVI was observed in tubules in the hyperosmotic mannitol solution when P-gp activity was acutely suppressed by P-gp inhibitors (verapamil and cyclosporin A) or when both the mdr1a and mdr1b genes were genetically knocked out (21). We also reported that when tubules from WT mice were exposed to the hyperosmotic mannitol solution including either of the two P-gp inhibitors, in the absence of peritubular Na+ or in the presence of peritubular EIPA, they did not exhibit RVI (21). Furthermore, in proximal tubules from KO mice, both removing peritubular Na+ and adding peritubular EIPA inhibited RVI induced by the hyperosmotic mannitol solution. These findings indicate that basolateral NHE partly contributes to the P-gp-induced modulation of RVI under hyperosmotic stress. In the present study, we extend our previous study to determine whether and how basolateral NHE is activated in the P-gp-induced modulation of RVI.

Evidence for basolateral NHE in proximal tubule cells from WT and KO mice. In contrast to apical NHE, which has been widely documented in the proximal tubule, basolateral NHE is more controversial. Studies in basolateral membrane vesicles prepared from the renal cortex of rabbits (15) and rats (26) have provided no evidence for basolateral NHE. On the other hand, in intact proximal tubules, previous evidence for basolateral NHE has been described in the salamander (2), rabbit proximal tubule S3 segment (16), and rabbit juxtamedullary S1 and S2 segments (10). However, it has not yet been demonstrated whether the basolateral membrane of the mouse proximal tubule possesses NHE. In the present study, we observed that in proximal tubule cells from both WT and KO mice, readdition of Na+ to the bath after an acid load caused a rapid pHi increase that was significantly inhibited by pretreatment with EIPA, as shown in Figs. 3, A and B, and 4, A and B. Therefore, in proximal tubule cells from both WT and KO mice, basolateral Na+-dependent pHi recovery is mediated by NHE.

When proximal tubule cells from both WT and KO mice were treated with EIPA, steady-state pHi values significantly decreased, as shown in Fig. 4, A and B. These findings indicate that in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 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.

Basolateral NHE activity under is- and hyperosmotic conditions. As shown in Fig. 5, in cells from WT and KO mice under both is- and hyperosmotic conditions, JH, through an NHE process, decreased 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 from WT mice over the entire range of pHi studied (6.20-6.90), which is consistent with the notion that under these conditions, NHE activity in the basolateral membrane of proximal tubule cells from KO mice is similar to that in cells from WT mice. However, in cells from KO mice, JH values under hyperosmotic conditions were significantly greater than those under isosmotic conditions at a pHi range of 6.20-6.45, although in cells from WT mice, JH values under hyperosmotic conditions were not significantly different from those under isosmotic conditions over the entire range of pHi examined (6.20-6.90). In sharp contrast, under hyperosmotic conditions, JH values in cells from KO mice were significantly greater than those in cells from WT mice at pHi values of 6.20-6.45. In cells from KO mice, maximal JH values under hyperosmotic conditions were also significantly greater than under isosmotic conditions. Furthermore, in proximal tubule cells from KO mice, the hyperosmotic mannitol solution shifted the JH-pHi relationship by ~0.15 pH units in the alkaline direction at low pHi values. Therefore, in proximal tubule cells from KO mice, hyperosmolality greatly enhances the pHi sensitivity of the NHE at low pHi, with an increase in maximal JH. Similarly, Parker (24) has demonstrated that NHE in dog erythrocytes is activated by low pHi, although they did not demonstrate that shrinkage-induced activation of NHE is mediated via a shift in pHi sensitivity. In contrast, Miyata et al. (22) reported that in cultured mesangial cells from rat kidneys, a hyperosmotic mannitol solution shifted the JH-pHi relationship by 0.15-0.3 pH units in the alkaline direction at a wide pHi range of 6.40-6.95, with increasing maximal JH. Similarly, Grinstein et al. (13) studied the Na+-dependent component of pHi recovery from an acid load in thymic lymphocytes, finding that shrinkage shifts the JH-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 the present study, we observed that the addition of the hyperosmotic mannitol solution including cyclosporin A (the P-gp inhibitor) to cells from WT mice activated basolateral NHE at magnitude similar to that for the addition of hyperosmotic mannitol alone to cells from KO mice. Cyclosporin A alone had no effect on NHE activity in cells from WT mice exposed to the isosmotic solution or in the cells from KO mice exposed to the hyperosmotic mannitol solution. Taken together, when P-gp activity is acutely inhibited by the P-gp inhibitor (cyclosporin A) or when both the mdr1a and mdr1b genes are genetically disrupted, hyperosmotic mannitol activates basolateral NHE in mouse proximal tubule cells and consequently elicits RVI. However, the mechanisms responsible for the activation of basolateral NHE under the above conditions are presently unclear. Because the pHi sensitivity of the exchange system appears to be largely determined by an allosteric modifier site (1, 12) located on the cytoplasmic surface of the membrane, one of the mechanisms for shrinkage-induced NHE activation in proximal tubule cells from KO mice is a shift in pHi dependence of the antiport.

In Madin-Darby canine kidney cells, hyperosmolality induced by NaCl or raffinose has been shown to enhance inositol 1,4,5-triphosphate levels and thereby activate PMA-sensitive PKC (29). In Ehrlich mouse ascites tumor cells, PKC is involved in activation of the Na+-K+-2Cl- cotransporter induced by hyperosmolality (18). Treatment of NIH/3T3 cells with hyperosmotic NaCl solution has also been reported to trigger phospholipase C activation and then induce an increase in diacylglycerol levels, and as a consequence, PKC activation (32). Very recently, Miyata et al. (23) demonstrated that exposure of proximal tubules from KO mice to staurosporine and calphostin C abolished RVI. We also demonstrated that exposure of proximal tubules from KO mice to a hyperosmotic mannitol solution activated PKC (23). The present studies have shown that exposure of proximal tubule cells from KO mice to staurosporine and calphostin C inhibited basolateral NHE activation induced by the hyperosmotic mannitol solution (see Figs. 9, A and B, and 10). We have also shown that the addition of PMA to cells from KO mice under isosmotic conditions mimicked the stimulatory effects of the hyperosmotic mannitol solution on basolateral NHE activity (see Figs. 9C and 10). In sharp contrast to that in cells from KO mice, exposure of cells from WT mice to hyperosmotic mannitol did not induce PKC activation (23), had no stimulatory effect on NHE activity (see Fig. 5), and did not result in RVI (23). On the other hand, exposure of cells from WT mice to the hyperosmotic mannitol solution including PMA activated PKC (23), increased basolateral NHE activity (see Figs. 9D and 10), and elicited RVI (23). Taking these observations together, we conclude that the effects of a hyperosmotic mannitol solution on basolateral NHE activity and RVI indeed occur via PKC.

The apparent Km (21.1 mM) for peritubular Na+ of NHE in the basolateral membrane of proximal tubule cells from KO mice under isosmotic conditions was lower than that found for the basolateral membrane of the rabbit proximal tubule S3 segment (16) and rat thymic lymphocytes (12) but was similar to that reported for the basolateral membrane of the rabbit cortical collecting duct (4) and human endothelial cells (8). In the present study, we found that exposure of cells from KO mice to the hyperosmotic mannitol solution increased Vmax from 370.6 to 528.4 µM/s without changing the Km for peritubular Na+, indicating that under hyperosmotic conditions, the affinity for peritubular Na+ remained the same, but the number of the NHEs increased. These findings also contribute to the hyperosmotic mannitol-induced increase in basolateral NHE activity. Very recently, we reported that the addition of the microtubule disruptor (colchicine) and the microfilament disruptor (cytochalasin B) to tubules from WT mice inhibited the PMA-induced RVI after the initial cell shrinkage (23). We have also shown that the addition of colchicine and cytochalsin B to tubules from KO mice abolished RVI (23). From our previous and present findings, we propose that under hyperosmotic conditions, PKC and/or NHE itself may be shuttled to the basolateral membrane via colchicine- and cytochalasin B-sensitive processes to activate NHE. Also, it is possible that PKC activation may promote the exocytic insertion into the basolateral membrane of NHE previously stored in the cytoplasm. These possibilities will have to await further investigation.

From the above data, we conclude that under isosmotic conditions, the basolateral membrane of proximal tubule cells from both WT and KO mice possesses an NHE. We also clearly demonstrated that in the absence of P-gp activity, hyperosmotic mannitol activates basolateral NHE via PKC, whereas in the presence of P-gp activity, it does not. Therefore, basolateral NHE indeed contributes to the P-gp-induced modulation of RVI after initial cell shrinkage.


    ACKNOWLEDGEMENTS

This work was supported in part by a grant from the Japanese Kidney Foundation (Jinkenkyukai), by 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.00183.2001

Received 21 March 2001; accepted in final form 1 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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