Effects of P-glycoprotein on cell volume regulation in mouse proximal tubule

Yukio Miyata, Yasushi Asano, and Shigeaki Muto

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The role of P-glycoprotein (P-gp) in cell volume regulation was examined in isolated nonperfused proximal tubule S2 segments from wild-type (WT) mice and those in which both mdr1a and mdr1b genes were knocked out (KO). When the osmolality of the bathing solution was rapidly decreased from 300 to 180 mosmol/kgH2O, the tubules from both the WT and KO mice exhibited regulatory volume decrease (RVD) by a similar magnitude after the initial cell swelling. The peritubular addition of two P-pg inhibitors (verapamil and cyclosporin A) to either group of the tubules had no effect on RVD. When the tubules from the WT mice were rapidly exposed to a hyperosmotic solution (500 mosmol/kgH2O) including 200 mM mannitol, they abruptly shrank to 82.1% of their control volume but remained in a shrunken state during the experimental period, indicating a lack of regulatory volume increase (RVI). The addition of the two P-gp inhibitors, but not the inhibitor of the renal organic cation transport system (tetraethylammonium), to the tubules from the WT mice resulted in RVI. Surprisingly, when the tubules from the KO mice were exposed to the hyperosmotic solution, they abruptly shrank to 79.9% of their control volume, and then gradually swelled to 87.7% of their control volume, showing RVI. However, exposure of the tubules from the KO mice to the hyperosmotic solution in the presence of the two P-gp inhibitors had no effect on RVI. When the tubules of the WT mice were exposed to the hyperosmotic solution including either of the two P-gp inhibitors, in the absence of peritubular Na+ or in the presence of peritubular ethylisopropylamiloride (EIPA; the specific inhibitor of Na+/H+ exchange), they did not exhibit RVI. In the tubules of the KO mice, both removing peritubular Na+ and adding peritubular EIPA inhibited RVI induced by the hyperosmotic solution. We conclude that 1) in mouse proximal tubule, P-gp modulates RVI during hyperosmotic stress but not RVD during hyposmotic stress and 2) basolateral membrane Na+/H+ exchange partly contributes to the P-gp-induced modulation of RVI under hyperosmotic stress.

cell volume regulation; nonperfused tubule; mdr1a; mdr1b; basolateral sodium-hydrogen exchange


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MOST ANIMAL CELLS REGULATE their volume in response to changes in the osmolality of the environment (reviewed in Refs. 17 and 20). After cell swelling in hyposmotic medium, regulatory volume decrease (RVD) is generally achieved by the loss of ions and other osmolytes and the concomitant loss of water. Of the various efflux systems that can contribute to RVD, swelling-activated Cl- channels are present in a number of cell types (17, 20).

P-glycoprotein (P-gp) was initially identified through its ability to confer multidrug resistance (MDR) in mammalian tumor cells (reviewed in Ref. 8). P-gp is a member of the ATP-binding cassette superfamily of transporters (8) and utilizes ATP to pump hydrophobic drugs out of the cells, decreasing their intracellular concentrations and hence their toxicity. Humans have one gene encoding drug-transporting P-gp (MDR1), whereas mice have two genes, mdr1a (also called mdr3) and mdr1b (also called mdr1) (8, 12, 24). In the mouse, the mdr1a gene is predominantly expressed in intestine, liver, and blood capillaries of brain and testis, whereas the mdr1b is predominantly expressed in adrenal glands, placenta, and ovarium (8). Similar levels of mdr1a and mdr1b gene expression are observed in the kidney (2, 25). In the mouse, mdr1a and mdr1b together fulfill the same function as MDR1 does in humans.

The expression of P-gp in the kidney was thought to occur exclusively in the apical membrane of the proximal tubule epithelium, where P-gp was expected to participate in the excretion of xenobiotics (5, 27). Consistent with a role for P-gp as an excretory transporter, cell lines that express many properties of proximal tubule cells, including LLC-PK1 (13, 23, 26), opossum kidney (11), and cultured mouse proximal tubule cells (4), exhibit net secretion of several MDR substrates, including verapamil and cyclosporin A. Also, very recently we used isolated perfused mouse proximal tubules to demonstrate that P-gp-mediated drug efflux capacity indeed exists in the apical membrane of proximal tubule S2 segments from wild-type (WT) mice but is lacking in that of mice in which both mdr1a and mdr1b genes were disrupted (KO) (28). In addition, after administration of digoxin, drug accumulation in kidneys of KO mice was greater than in those of WT mice (24, 28).

In addition to drug-transporting activity, P-gp itself has been reported to have intrinsic channel activity in cultured NIH3T3 fibroblasts transfected with human MDR1 cDNA (30). Subsequent data have shown that P-gp regulates RVD through its effects on Cl- channel activation in HTC-R hepatoma cells overexpressing P-gp activity (21) and in Chinese hamster ovary cells transfected with the mdr1a gene (LR73-1a cells) (29). Recently, it has been demonstrated that P-gp has a separate mechanism of drug transport and Cl- channel function during hyposmotic stress in NIH3T3 fibroblasts transfected with human MDR1 (7) and in cultured human breast cancer cells transfected with human MDR1 cDNA (1). However, it is not known whether P-gp actually regulates RVD in intact cells, including renal tubule cells. Also, in most studies using cultured cells overexpressing P-gp activity, cell volume under hyposmotic conditions has not yet been measured, although swelling-activated Cl- channels have been well characterized (1, 7, 30, 31).

In most animal cells, after cell shrinkage in hyperosmotic medium, regulatory volume increase (RVI) is generally achieved by the gain of ions and other osmolytes (17, 20). In sharp contrast, when rabbit proximal tubule cells are suddenly exposed to hyperosmotic mannitol, NaCl, and raffinose solutions, they rapidly shrink but remain reduced in size (6, 14). However, the mechanisms responsible for the lack of RVI have not been fully demonstrated. Also, it is not known whether hyperosmotic stress affects cell volume in mouse proximal tubule, and if so, whether and how P-gp contributes to cell volume regulation under hyperosmotic conditions.

Therefore, we used isolated nonperfused proximal tubule S2 segments from KO and WT mice to examine the role of P-gp in cell volume regulation during hyposmotic and hyperosmotic stress.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. KO mice used in this study were originally described by Schinkel et al. (24). Male KO and FVB (WT) mice serving as the control (body wt 25-40 g) were purchased from Taconic Engineering (Germantown, NY). The animals were maintained in a controlled environment and had free access to a standard rodent chow and tap water ad libitum until the experiments. Ages of the KO animals were matched with their WT controls.

In vitro microperfusion. Mice from both groups were anesthetized with an intraperitoneal injection of pentobarbital sodium (4 mg/100 g body wt), and both kidneys were removed. Slices of 1-2 mm were taken from the coronal section of each kidney and transferred to a dish containing a cold intracellular fluid-like solution having the following composition (in mM): 14 KH2PO4, 44 K2HPO4, 15 KCl, 9 NaHCO3, and 160 sucrose. Proximal tubule S2 segments were then dissected by fine forceps under a stereomicroscope and transferred to a bath chamber mounted on an inverted microscope (IMT-2, Olympus, Tokyo, Japan).

Both proximal and distal ends of the tubule were drawn into and crimped by glass micropipettes as originally described by Dellasega and Grantham (3). In this preparation, both ends of the tubule are occluded so that the lumen becomes and remains collapsed. A flow-through bath system was utilized to permit rapid exchange of the bath fluid. The bathing solution flowed by gravity at 4.5-5.5 ml/min from the reservoirs through a water jacket to stabilize the bath temperature at 37°C.

Solutions. The composition of all bathing solutions is given in Table 1. The osmolality of all the solutions was measured by freezing-point depression before the experiments. All the solutions were continuously gassed with a 5% CO2-95% O2 mixture.

                              
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Table 1.   Composition of solutions

After tubules were tightly crimped between the two glass micropipettes, they were bathed in the isosmotic control solution (solution 1) at 37°C for 10-15 min to allow them to equilibrate. After the equilibration period, the cell volume was measured as described below.

Measurements of tubule cell volume. Before, during, and after exposure of the tubules to hyposmotic or hyperosmotic solutions, we took serial photographs, at a magnification of ×400, that focused on the center of the tubule. The photographs were magnified, and the outer diameter of the tubule (d) was estimated from similar photographs of a calibrated micrometer slide as reference. Assuming the tubule is cylindrical, the apparent cell volume could then be calculated according to the following equation
V<IT>=&pgr;</IT>(<IT>d/2</IT>)<SUP><IT>2</IT></SUP><IT>L</IT>
where V is the apparent tubule volume, and L is a constant tubule length. The values obtained are expressed as a percentage relative to control isosmotic tubule cell volume.

Chemicals. Verapamil, cyclosporin A, and ethylisopropylamiloride (EIPA) were purchased from Sigma (St. Louis, MO). Other high-grade chemicals were obtained from Wako (Osaka, Japan).

Verapamil was dissolved in dimethylsulfoxide at 0.1% final concentration. Cyclosporin A was dissolved in ethanol at 0.1% final concentration. Tetraethylammonium (TEA) was dissolved in distilled water at 0.1% final concentration. EIPA was dissolved in methanol at 0.1% final concentration. Equivalent concentrations of vehicle were added as a control for individual protocols.

Statistics. The data are expressed as means ± SE. Comparisons were performed by Student's t-test or one-way analysis of variance in combination with Fisher's protected least significant differences test, where appropriate. P values <0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of hyposmotic solutions on cell volume. At first, we examined the effect of hyposmotic solutions on cell volume in proximal tubules from WT and KO mice. For this purpose, we used the isosmotic control solution containing 110 mM NaCl (solution 1; 300 mosmol/kgH2O, Table 1), the isosmotic control solution containing 50 mM NaCl plus 120 mM mannitol (solution 2; 300 mosmol/kgH2O, Table 1), and the hyposmotic solution (solution 3; 180 mosmol/kgH2O, Table 1), which was made by removal of mannitol from solution 2. Results are shown in Fig. 1. When both groups of tubules were initially bathed in solution 1 and were then perfused with solution 2 for 10 min, there was no appreciable effect on cell volume (data not shown). However, when the tubules from the WT mice were initially incubated in solution 2 and were then treated with solution 3 for 10 min, they abruptly swelled to 133.0 ± 1.8% (n = 6) of their control volume and then gradually shrank over the next 4-6 min to 115.0 ± 2.2% (n = 6) (Fig. 1A). Similarly, after the osmolality of the bathing solution was abruptly reduced from 300 to 180 mosmol/kgH2O for 10 min, the tubules from the KO mice rapidly swelled to 129.5 ± 1.4% (n = 6) of their control volume and then gradually shrank to 111.8 ± 1.1% (n = 6) of their control volume (Fig. 1B). The changes in cell volume in the tubules from the KO mice under hyposmotic conditions were comparable to those from the WT mice. Therefore, both groups of tubules exhibited RVD during hyposmotic stress.


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Fig. 1.   Effects of hyposmotic solution on cell volume in isolated nonperfused proximal tubule S2 segments from wild-type (WT; A) and knockout (KO; B) mice. Both groups of tubules were initially bathed in solution 2 and were then treated with solution 3 for 10 min. Values are means ± SE of 6 tubules from WT and KO mice and are expressed as a percentage relative to the control isosmotic tubule cell volume treated with solution 2. *P < 0.05 vs. maximum cell volume increase.

Next, we examined whether the hyposmolality-induced cell volume changes in both groups of tubules are affected by two inhibitors of P-gp (verapamil and cyclosporin A). For this purpose, we monitored cell volume of both groups of tubules in the absence and presence of bath verapamil (100 µM) or cyclosporin A (5 µM). Results are shown in Fig. 2. The addition of verapamil or cyclosporin A alone had no effect on cell volume in the tubules from the WT or KO mice (Fig. 2). When the tubules from the WT mice were treated with solution 3 in the presence of verapamil or cyclosporin A for 10 min, they swelled to 130.7 ± 3.9 (n = 4) or 130.6 ± 1.1% (n = 7) of their control volume and then shrank to 114.5 ± 2.0 (n = 4) or 115.9 ± 1.6% (n = 7) of their control volume, respectively (Fig. 2, A and B). Similarly, exposure of the tubules from the KO mice to solution 3 in the presence of verapamil or cyclosporin A for 10 min caused them to swell to 127.6 ± 2.3 (n = 9) or 130.0 ± 1.3% (n = 6) of their control volume and then to shrink to 112.2 ± 1.3 (n = 9) or 116.1 ± 2.5% (n = 6) of their control volume, respectively (Fig. 2, C and D). In both groups of tubules, changes in cell volume under hyposmotic conditions in the presence of the two inhibitors of P-gp were not significantly different from those in their absence. Therefore, in both groups of tubules, the two inhibitors of P-gp caused no effect on RVD during hyposmotic stress.


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Fig. 2.   Effects of hyposmotic solution on cell volume in isolated nonperfused proximal tubule S2 segments from WT (A and B) and KO mice (C and D) in the presence of verapamil or cyclosporin A, respectively. Values are means ± SE of 9 or 6 tubules of WT mice in the presence of verapamil or cyclosporin A, respectively, and of 4 or 7 tubules of KO mice in the presence of verapamil or cyclosporin A, respectively, and are expressed as a percentage relative to the control isosmotic tubule cell volume treated with the solution 2. Both groups of tubules were initially bathed in solution 2 and were then perfused with solution 2 containing verapamil (100 µM) or cyclosporin A (5 µM) for 2 min. Thereafter, the tubules were treated with solution 3 in the presence of verapamil or cyclosporin A for 10 min. *P < 0.05 vs. maximum cell volume increase.

Effect of hyperosmotic solutions on cell volume. Next, we examined the effect of hyperosmotic solutions on cell volume in the proximal tubules from the WT and KO mice. For this purpose, the tubules were initially bathed in the isosmotic control solution (solution 1; 300 mosmol/kgH2O, Table 1) and were then perfused with hyperosmotic solutions (500 mosmol/kgH2O) containing 200 mM mannitol (solution 4; Table 1) or urea (solution 5; Table 1). Results are shown in Fig. 3. When the tubules from the WT mice were rapidly exposed to the hyperosmotic mannitol solution for 10 min, they abruptly shrank to 82.1 ± 1.4% (n = 6) of their control volume but remained in a shrunken state during the experimental period, indicating a lack of RVI (Fig. 3A). Surprisingly, when the tubules from the KO mice were rapidly treated with the hyperosmotic mannitol solution for 10 min, they abruptly shrank to 79.9 ± 1.1% (n = 6) of their control volume and then gradually swelled to 87.7 ± 1.1% (n = 6) of their control volume, showing a presence of RVI (Fig. 3C).


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Fig. 3.   Effects of hyperosmotic solution on cell volume in isolated nonperfused proximal tubule S2 segments from WT and KO mice. Both groups of tubules were initially bathed in the solution 1 and were then treated with solution 4 or 5 for 10 min. Values are means ± SE of 6 or 8 tubules from the WT mice in the presence of mannitol (A) or urea (B), respectively, and 6 or 7 tubules from the KO mice in the presence of mannitol (C) or urea (D), respectively, and are expressed as a percentage relative to the control isosmotic tubule cell volume treated with solution 1. *P < 0.05 vs. maximum cell volume decrease.

The response of cell volume to the hyperosmotic urea solution was strikingly different from that to the hyperosmotic mannitol solution but was similar in both groups of tubules (Fig. 3). In the tubules of the WT and KO mice, the reduction in cell volume was smaller [88.5 ± 0.7 (n = 8) and 87.0 ± 1.0% (n = 7) of their control volume, respectively] and transient, tubules returning to their control volume within 2.5 min of the experimental period (Fig. 3, B and D, respectively). In the recovery period, the initial transient large swelling of the cells was rapidly dissipated.

Next, we examined whether the hyperosmotic mannitol-induced cell volume changes in both groups of tubules are influenced by the two inhibitors of P-gp. For this purpose, both groups of tubules were treated with solution 4 in the absence and presence of verapamil (100 µM) or cyclosporin A (5 µM). Results are shown in Fig. 4. The peritubular addition of verapamil or cyclosporin A alone caused no effect on cell volume in either group of tubules (Fig. 4). On the other hand, when the tubules from the WT mice were rapidly treated with the hyperosmotic mannitol solution in the presence of verapamil or cyclosporin A, they immediately shrank to 81.7 ± 1.9 (n = 7) or 80.2 ± 2.2% (n = 4) of their control volume and then gradually swelled to 89.7 ± 1.5 (n = 7) or 89.2 ± 1.6% (n = 4) of their control volume, respectively (Fig. 4, A and B). In sharp contrast to treatment with the inhibitors of P-gp, the peritubular addition of TEA (1 mM; an inhibitor of the renal organic cation transport system) to the tubules from the WT mice had no effect at all on RVI (Fig. 5). Accordingly, in the WT mice, addition of the two inhibitors of P-gp, but not the inhibitor of the renal organic cation transport system, resulted in RVI during hyperosmotic stress. In sharp contrast to WT mice, when the tubules from the KO mice were rapidly exposed to the hyperosmotic mannitol solution in the presence of verapamil or cyclosporin A, they abruptly shrank to 79.2 ± 1.9 (n = 6) or 81.4 ± 1.3% (n = 6) of their control volume and then gradually swelled to 89.7 ± 1.8 (n = 6) or 88.2 ± 1.2% (n = 6) of their control volume, respectively (Fig. 4, C and D). In the KO mice, changes in cell volume in the presence of the two inhibitors of P-gp were not significantly different from those in their absence (see Figs. 3C and 4, C and D). Therefore, exposure of the tubules from the KO mice to hyperosmotic mannitol in the presence of two inhibitors of P-gp had no effect on RVI during hyperosmotic stress.


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Fig. 4.   Effects of hyperosmotic solution with mannitol on cell volume in isolated nonperfused proximal tubule S2 segments from the WT and KO mice in the presence of verapamil or cyclosporin A. Values are means ± SE of 7 or 4 tubules of WT mice in the presence of verapamil (A) or cyclosporin A (B), respectively, and of 6 tubules of KO mice in the presence of verapamil (C) or cyclosporin A (D) and are expressed as a percentage relative to the control isosmotic tubule cell volume treated with the solution 1. Both groups of tubules were initially bathed in solution 1 and were then perfused with solution 1 containing verapamil (100 µM) or cyclosporin A (5 µM) for 2 min. Thereafter, the tubules were treated with solution 4 in the presence of verapamil or cyclosporin A for 10 min. *P < 0.05 vs. maximum cell volume decrease.



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Fig. 5.   Effects of hyperosmotic solution with mannitol on cell volume in isolated nonperfused proximal tubule S2 segments from WT mice in the presence of tetraethylammonium (TEA). Values are means ± SE of 5 tubules and are expressed as a percentage relative to the control isosmotic tubule cell volume treated with solution 1. The tubules were initially bathed in solution 1 and were then perfused with solution 1 containing TEA (1 mM) for 2 min. Thereafter, the tubules were treated with solution 4 in the presence of TEA for 10 min.

Transport mechanisms responsible for hyperosmotic cell volume regulation. When most animal cells are subjected to hyperosmotic stress, activation of ion transport systems, followed by a later accumulation of organic osmolytes, is known to occur (reviewed in Refs. 17 and 20). The activation of ion transport systems includes influx of NaCl, which is mediated by parallel Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> antiports or Na+-K+-2Cl- cotransport (reviewed in Refs. 17 and 20). Accordingly, we examined whether, in the tubules of the WT mice, RVI induced by the hyperosmotic mannitol solution containing the two P-gp inhibitors occurs through Na+ entry into the cell. For this purpose, the tubules of the WT mice were initially incubated in the control isosmotic solution (solution 1; Table 1) and were then bathed in the Na+-free isosmotic solution (solution 6; Table 1). Thereafter, they were treated with solution 6 containing verapamil (100 µM) or cyclosporin A (5 µM), and were finally exposed to the Na+-free hyperosmotic mannitol solution (solution 7; 500 mosmol/kgH2O, Table 1) in the presence of verapamil or cyclosporin A. Results are shown in Fig. 6. The exposure of the tubules to either the isosmotic Na+-free solution alone or the isosmotic Na+-free solution containing verapamil or cyclosporin A had no effect on cell volume (Fig. 6). On the other hand, when the tubules of the WT mice were treated with the Na+-free hyperosmotic mannitol solution including verapamil, they initially shrank to 81.0 ± 1.2% (n = 5) of their control volume but remained reduced in size (Fig. 6A). Similarly, when the tubules of the WT mice were exposed to the Na+-free hyperosmotic mannitol solution containing cyclosporin A, they initially shrank to 80.0 ± 1.6% (n = 5) of their control volume but remained in a shrunken state during the experimental period (Fig. 6B). Next, we examined whether, in proximal tubules from the WT mice, basolateral membrane Na+/H+ exchange contributes to the RVI response induced by the hyperosmotic mannitol solution containing the two P-gp inhibitors. For this purpose, the tubules of the WT mice were initially incubated in solution 1 and were then bathed in solution 1 containing EIPA (the specific inhibitor of Na+/H+ exchange; 100 µM). Thereafter, they were treated with solution 1 containing verapamil (100 µM) or cyclosporin A (5 µM) in the continued presence of EIPA and were finally exposed to the hyperosmotic mannitol solution (solution 4; 500 mosmol/kgH2O, Table 1) containing EIPA and verapamil or cyclosporin A. Results are shown in Fig. 7. The peritubular addition of either EIPA alone or EIPA including verapamil or cyclosporin A to the tubules of the WT mice had no effect on cell volume (Fig. 7). On the other hand, when the tubules of the WT mice were exposed to the hyperosmotic mannitol solution containing verapamil and EIPA, they initially shrank to 81.2 ± 0.9% (n = 4) of their control volume but remained in a shrunken state during the experimental period (Fig. 7A). Similarly, when the tubules of the WT mice were treated with the hyperosmotic mannitol solution including cyclosporin A and EIPA, they initially shrank to 82.2 ± 1.3% (n = 5) of their control volume but remained reduced in size (Fig. 7B).


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Fig. 6.   Effects of hyperosmotic mannitol solution containing verapamil (A) or cyclosporin A (B) in the absence of peritubular Na+ on cell volume in isolated nonperfused proximal tubule S2 segments from WT mice. Values are means ± SE of 5 tubules from WT mice in the presence of verapamil or cyclosporin A and are expressed as a percentage relative to the control, isosmotic tubule cell volume treated with the solution 1. The tubules were initially bathed in solution 1 and were then perfused with solution 6 for 2 min. Thereafter, the tubules were treated with solution 6 containing verapamil (100 µM) or cyclosporin A (5 µM) for 2 min and were finally exposed to solution 7 including verapamil or cyclosporin A for 10 min.



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Fig. 7.   Effects of hyperosmotic mannitol solution containing verapamil or cyclosporin A in the presence of peritubular ethylisopropylamiloride (EIPA) on cell volume in isolated nonperfused proximal tubule S2 segments from WT mice. Values are means ± SE of 4 or 5 tubules from WT mice in the presence of verapamil (A) or cyclosporin A (B), respectively, and are expressed as a percentage relative to the control isosmotic tubule cell volume treated with solution 1. The tubules were initially bathed in solution 1 and were then perfused with solution 1 containing EIPA (100 µM) for 2 min. Thereafter, the tubules were treated with solution 1 containing EIPA and verapamil (100 µM) or cyclosporin A (5 µM) for 2 min, and were finally exposed to solution 4 including EIPA and verapamil (100 µM) or cyclosporin A for 10 min.

Next, we examined whether, in proximal tubules from the KO mice, the RVI response induced by the hyperosmotic mannitol solution is mediated through Na+ entry into the cell. For this purpose, the tubules of the KO mice were initially incubated in the control isosmotic solution (solution 1; Table 1) and were then bathed in the Na+-free isosmotic solution (solution 6; Table 1). Thereafter, they were treated with the Na+-free hyperosmotic mannitol solution (solution 7; 500 mosmol/kgH2O, Table 1). Results are shown in Fig. 8. Removing peritubular Na+ alone had no effect on cell volume (Fig. 8). On the other hand, when the tubules of the KO mice were exposed to the Na+-free hyperosmotic mannitol solution, they initially shrank to 80.7 ± 0.8% (n = 6) of their control volume but remained in a shrunken state during the experimental period (Fig. 8). Next, we examined whether, in the proximal tubules from the KO mice, basolateral membrane Na+/H+ exchange is involved in the RVI response induced by the hyperosmotic mannitol solution. For this purpose, the tubules of the KO mice were initially incubated in solution 1 and were then bathed in solution 1 containing EIPA (100 µM). Thereafter, they were treated with the hyperosmotic mannitol solution (solution 4; 500 mosmol/kgH2O, Table 1) in the continued presence of peritubular EIPA. EIPA alone caused no effect on cell volume (Fig. 9). In sharp contrast, when the tubules of the KO mice were exposed to the hyperosmotic mannitol solution in the presence of EIPA, they initially shrank to 82.0 ± 0.9% (n = 6) of their control volume but remained reduced in size (Fig. 9).


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Fig. 8.   Effects of hyperosmotic solution with mannitol in the absence of peritubular Na+ on cell volume in isolated nonperfused proximal tubule S2 segments from KO mice. The tubules were initially bathed in solution 1 and were then perfused with solution 6 for 2 min. Thereafter, the tubules were treated with solution 7 for 10 min. Values are means ± SE of 6 tubules from KO mice and are expressed as a percentage relative to the control isosmotic tubule cell volume treated with solution 1.



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Fig. 9.   Effects of hyperosmotic solution with mannitol in the presence of peritubular EIPA on cell volume in isolated nonperfused proximal tubule S2 segments from KO mice. The tubules were initially bathed in solution 1 and were then perfused with solution 1 containing EIPA (100 µM) for 2 min. Thereafter, the tubules were treated with solution 4 including EIPA for 10 min. Values are means ± SE of 6 tubules from KO mice and are expressed as a percentage relative to the control isosmotic tubule cell volume treated with solution 1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of the present study was to examine the role of P-gp in isolated nonperfused proximal tubules exposed to anisosmotic media. We demonstrated that exposure of the tubules from both WT and KO mice to a hyposmotic solution elicited RVD with a similar magnitude. RVD during hyposmotic stress was unchanged by the two inhibitors of P-gp (verapamil and cyclosporin A). In sharp contrast to the hyposmotic stress, exposure of the tubules from the WT mice to the hyperosmotic mannitol solution did not produce RVI, whereas exposure of the tubules from the KO mice to the hyperosmotic mannitol solution elicited RVI. In the WT mice, the two inhibitors of P-gp, but not the inhibitor of the renal organic cation transport system (TEA), produced RVI under hyperosmotic conditions, whereas in KO mice the inhibitors of P-gp did not affect RVI during hyperosmotic stress at all. This is the first study demonstrating that, in mouse proximal tubule cells, P-gp modulates RVI during hyperosmotic stress, but not RVD during hyposmotic stress.

Role of P-gp in cell volume regulation under hyposmotic conditions. In the present study, we report that when isolated nonperfused proximal tubules of WT mice were rapidly exposed to hyposmotic medium, cell swelling was followed by RVD. Similar findings have been reported in isolated nonperfused rabbit proximal convoluted (6, 16) and straight (3, 14, 33) tubules and in isolated perfused mouse proximal straight tubules (32).

Valverde et al. (29) have shown that exposure of Chinese hamster ovary cells transfected with mdr1a (LR73-1a cells) to hyposmotic medium elicited RVD after the initial cell swelling, whereas exposure of the parental cells (LR73 cells) without expression of P-gp to the hyposmotic medium did not. Roman et al. (21) also reported that when HTC-R hepatoma cells expressing P-gp were exposed to hyposmotic solution, RVD was seen after the initial cell swelling. RVD was then abolished by the inhibitor of P-gp (verapamil). From these reports, we expected that in the tubules of the KO mice, RVD in hyposmotic medium would not occur, whereas in the tubules of the WT mice, it would be suppressed by the addition of the inhibitors of P-gp (verapamil and cyclosporin A). However, unexpectedly, in the tubules of the KO mice, RVD under hyposmotic conditions was indeed observed to a similar extent to that in the WT mice. Also, in both groups of tubules, RVD was not affected at all by the addition of inhibitors of P-gp. Furthermore, drug-transporting P-gp activity exists in proximal tubules from WT mice and is suppressed by verapamil, whereas it is absent in proximal tubules of KO mice (28). Also, in isolated perfused tubules of KO mice, the magnitude of RVD found after cell swelling was of a magnitude similar to that in isolated nonperfused tubules of KO mice (data not shown). From our present and previous findings, we conclude that in intact proximal tubule cells, P-gp is not involved in RVD in hyposmotic medium. Therefore, the role of P-gp in RVD is strikingly different from that in cultured cells and intact cells, although it remains unclear why in mouse proximal tubule cells, P-gp does not contribute to RVD during hyposmotic stress.

Role of P-gp in cell volume regulation under hyperosmotic conditions. In the present study, exposure of isolated nonperfused proximal tubules from WT mice to a hyperosmotic mannitol solution did not elicit RVI after the initial cell shrinkage. Similar findings have been reported in nonperfused rabbit proximal convoluted (6) and straight (14) tubules rapidly treated with hyperosmotic NaCl, raffinose, or mannitol solutions. In our study, in the tubules from the WT mice, the two inhibitors of P-gp (verapamil and cyclosporin A) produced RVI during exposure to the hyperosmotic mannitol solution, whereas the inhibitor of the renal organic cation transport system (TEA) did not. Most exciting, exposure of tubules from KO mice to the hyperosmotic mannitol solution elicited RVI, and RVI during hyperosmotic stress was not influenced at all by the two inhibitors of P-gp. Under isosmotic conditions, cell volume in either group of tubules was unaltered by the two inhibitors of P-gp. Therefore, when P-gp activity is acutely suppressed by inhibitors of P-gp or when both mdr1a and mdr1b are genetically disrupted, RVI in hyperosmotic medium occurs. These findings are compatible with the notion that in mouse proximal tubule, P-gp maintains cell volume at a constant level under hyperosmotic conditions but not isosmotic conditions.

Studies, including the present work, on cell volume regulation in mammalian kidneys have used nonperfused tubule segments studied in vitro (3, 6, 14-16, 19, 22, 33). In this nonpolar preparation, cell volume regulation primarily reflects the steady-state transport of solutes and water only across the basolateral membranes (9, 14, 15, 33). Therefore, we propose that in mouse proximal tubule, P-gp could keep cell volume at a constant level during hyperosmotic stress via the basolateral transporters but not via the apical transporters. To clarify which basolateral transporters are involved in P-gp-induced cell volume regulation, the effects of removing peritubular Na+ and adding peritubular EIPA (the specific inhibitor of Na+/H+ exchange) on cell volume were examined. In the proximal tubules of the WT mice, both removal of peritubular Na+ and addition of peritubular EIPA inhibited RVI induced by the hyperosmotic mannitol solution containing the two P-gp inhibitors (Figs. 6 and 7). In the tubules of the KO mice, both removing peritubular Na+ and adding peritubular EIPA eliminated RVI induced by the hyperosmotic mannitol solution (Figs. 8 and 9). These findings indicate that basolateral Na+/H+ exchange partly contributes to P-gp-induced modulation of RVI after initial cell shrinkage.

In a variety of cell types (8, 18), protein kinase C has been reported to be involved in P-gp-mediated drug efflux. In BALB/c-3T3 cells transfected with human MDR1, it has been demonstrated that activation of protein kinase C reduced the rate of increase in swelling-activated Cl- currents, whereas activation of protein kinase A reduced steady-state swelling-activated Cl- currents (31). In rabbit proximal tubule, cell volume regulation under hyperosmotic conditions is regulated by the availability of metabolizable fatty acids in the medium. Acetate, butyrate, and valerate have been reported to enhance the ability of proximal tubule cells to maintain a constant cell volume under mildly hyperosmotic conditions (15, 22). Grantham et al. (15, 22) suggested that nonionic acids entering the cell may support Na+/H+ exchange by supplying intracellular protons. However, it is not known at present whether these kinases and/or the availability of metabolizable fatty acids contributes to P-gp-induced cell volume regulation in mouse proximal tubule cells. Further studies will be required to clarify the intracellular signaling mechanisms responsible for cell volume regulation induced by P-gp under hyperosmotic conditions. Also, whether basolateral Na+/H+ exchange in the mouse proximal tubule is actually activated in P-gp-induced cell volume regulation under hyperosmotic stress will have to await further investigation.

In contrast to the effect of mannitol (an impermeant solute), the effect of urea (a more permeant solute) on cell volume change was strikingly different (see Fig. 3). In both groups of tubules, hyperosmotic urea caused only a transient decrease in cell volume. Urea entry across the basolateral membrane quickly restored cell volume. The large overshoot observed during washout of urea is consistent with this urea entry. Similar observations have been made in rabbit proximal convoluted tubule (6) and cortical collecting duct (19). Volume changes during the presence of urea were the result of simple diffusion and not volume regulation.

In conclusion, we clearly demonstrated that, in mouse proximal tubule S2 segments, P-gp modulates RVI during hyperosmotic stress but not RVD during hyposmotic stress. We also found that basolateral membrane Na+/H+ exchange partly contributes to P-gp-induced modulation of RVI under hyperosmotic stress.


    ACKNOWLEDGEMENTS

This work was supported in part by a grant from the Japanese Kidney Foundation (Jinkenkyukai), the Salt Science Foundation, and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports, Japan.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Muto, Dept. of Nephrology, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan (E-mail: smuto{at}jichi.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9 June 2000; accepted in final form 4 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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