P-gp-induced modulation of regulatory volume increase occurs via PKC in mouse proximal tubule

Yukio Miyata1, Koji Okada2, Shun Ishibashi2, Yasushi Asano1, and Shigeaki Muto1

Departments of 1 Nephrology and 2 Endocrinology and Metabolism, Jichi Medical School, Tochigi, 329-0498 Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The present study examined the role of protein kinase C (PKC) in the P-glycoprotein (P-gp)-induced modulation of regulatory volume increase (RVI) in the isolated nonperfused proximal tubule S2 segments from mice lacking both mdr1a and mdr1b genes (KO) and wild-type (WT) mice. The hyperosmotic solution (500 mosmol/kgH2O) involving 200 mM mannitol activated PKC and elicited RVI in the tubules from KO mice but not from WT mice. The addition of the hyperosmotic solution including the PKC activator phorbol 12-myristate 13-acetate (PMA) to the tubules of the WT mice activated PKC and elicited RVI. The hyperosmotic solution in the presence of the P-gp inhibitors (verapamil or cyclosporin A) elicited RVI in the tubules from the WT mice but not from the KO mice. The PMA- and the P-gp inhibitors-induced RVI was abolished by cotreatment with the PKC inhibitors (staurosporine or calphostin C). In the tubules of the KO mice, the PKC inhibitors abolished RVI, but PMA did not. In the tubules of the WT mice, the microtubule disruptor (colchicine), the microfilament disruptor (cytochalasin B), the phosphatidylinositol 3-kinase (PI 3-kinase) blocker (wortmannin), but not another PI 3-kinase blocker (LY-294002), inhibited the PMA-induced RVI. In the tubules of the KO mice, colchicine, cytochalsin B, and wortmannin abolished RVI, but LY-294002 did not. We conclude that 1) in the mouse proximal tubule, P-gp-induced modulation of RVI occurs via PKC; and 2) the microtubule, microfilament, and wortmannin-sensitive, LY-294002-insensitive PI 3-kinase contribute to the PKC-induced RVI.

mdr1a gene; mdr1b gene; protein kinase C; microtubule; microfilament; phosphatidylinositol 3-kinase; P-glycoprotein


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

P-GLYCOPROTEIN (P-GP) WAS initially identified through its ability to confer multidrug resistance (MDR) in mammalian tumor cells (reviewed in Ref. 15). P-gp is a member of the ATP-binding cassette superfamily of transporters (15) 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) (15, 19, 35). The mdr1a and mdr1b in the mouse together fulfill the same function as MDR1 in humans, and similar levels of mdr1a and mdr1b expression are observed in the kidney (5, 36). In the kidney, the apical membrane of the proximal tubule epithelium is particularly rich in P-gp (10, 39), 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, monolayers of LLC-PK1 cells (33), opossum kidney cells, and cultured mouse proximal tubule cells (9) exhibit net secretion of several MDR substrates, including verapamil and cyclosporin A. Also, Tsuruoka et al. (42) recently demonstrated in the isolated perfused mouse proximal tubule 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 the mice in which both the mdr1a and mdr1b genes were disrupted (KO mice).

Most animal cells regulate their volume in response to changes in the osmolality of the environment (reviewed in Refs. 20 and 25). After cell swelling in hyposmotic medium, a 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 (20, 25). Conversely, in most animal cells, after cell shrinkage in hyperosmotic medium, a regulatory volume increase (RVI) is generally achieved by the gain of ions and other osmolytes (20, 25). In sharp contrast, when rabbit proximal tubule cells are exposed suddenly to hyperosmotic mannitol, NaCl, or raffinose solutions, they rapidly shrink but remain reduced in size (12, 23). However, the mechanisms responsible for the lack of RVI have not been fully demonstrated.

In addition to the drug-transporting activity, in cultured cells overexpressing P-gp activity, P-gp has been suggested to be a regulator of the swelling-activated Cl- channel and RVD after the initial cell swelling (1, 13). However, Miyata et al. (27) very recently used isolated nonperfused proximal tubule S2 segments from WT and KO mice to demonstrate that in intact proximal tubule cells, P-gp is not involved in RVD under hyposmotic stress. Instead, they have shown that the exposure of the tubules from WT mice to the hyperosmotic mannitol solution lacked RVI, whereas the exposure of the tubules from KO mice to the hyperosmotic mannitol solution elicited RVI (27). They observed that in WT mice, the peritubular addition of the P-gp inhibitors (verapamil and cyclosporin A) resulted in RVI, whereas in KO mice it had no effect on RVI (27). Therefore, in mouse proximal tubule, P-gp modulates RVI during the exposure to the hyperosmotic mannitol solution. They also observed that the basolateral Na+/H+ exchange contributes to the P-gp-induced modulation of RVI (27). Presently, however, it is not known how P-gp modulates the hyperosmotic mannitol-induced RVI.

In Madin-Darby canine kidney (MDCK) cells, hyperosmolality induced by NaCl or raffinose has been shown to enhance inositol 1,4,5-triphosphate levels and thereby activate phorbol 12-myristate 13-acetate (PMA)-sensitive protein kinase C (PKC) (38). In Ehrlich mouse ascites tumor cells, PKC is involved in activation of the Na+-K+-2Cl- cotransporter induced by hyperosmolality (21). 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 (46). The PKC activation induces translocation of PKCalpha , PKCdelta , and PKCepsilon from the cytosol to the plasma membrane after the stimulation of NIH/3T3 cells with hyperosmotic NaCl (46). However, it is not known whether and how, in the mouse proximal tubule, PKC actually modulates P-gp-induced modulation of RVI after exposure to the hyperosmotic mannitol solution.

Cytoskeletal elements may interfere in several ways with volume regulatory mechanisms (reviewed in Ref. 20). RVD is inhibited in several tissues by cytochalasin B and D, which interfere with actin assembly (20, 22). Cell swelling increases microtubule stability and stimulates the expression of tubulin (22). The addition of the microtubule inhibitor vincristine to the rabbit proximal convoluted tubule impaired RVI after the initial cell swelling (22). Colchicine, which disrupts the microtubule network, inhibits RVD in peripheral neutrophils (7). However, it is not known whether the disruptors of the microtubule and microfilament affect the hyperosmotic cell volume regulation induced by P-gp.

Phosphatidylinositol 3-kinases (PI 3-kinases) are enzymes that phosphorylate position 3 of the head group of the membrane lipid phosphatidylinositol (41). In recent years, PI 3-kinase activity and its lipid products have been implicated in regulation of many cellular processes, including membrane ruffling, vesicular trafficking, and activation of membrane ion channels (3, 4, 31, 37). In human intestine 407 cells, PI 3-kinase has been shown to be involved in swelling-activated Cl- channels (40). In rat medullary thick ascending limb of Henle's loop, hyposmolality, but not hyperosmolality, induces an increase in PI 3-kinase activity, which, in turn, results in the stimulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (14). However, no information is available on the role of PI 3-kinase in the hyperosmotic cell volume regulation in the proximal tubule.

Therefore, we used isolated nonperfused proximal tubule S2 segments from KO and WT mice to examine the intracellular signaling mechanisms for the P-gp-induced modulation of RVI during exposure to a hyperosmotic mannitol solution.


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Animals. The KO mice used in this study were originally described by Schinkel et al. (35). Male KO and FVB (WT) mice, serving as 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 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).

Both proximal and distal ends of the tubule were drawn into and crimped by glass micropipettes as described by Dellasega and Grantham (6) and Miyata et al. (27). In this preparation, both ends of the tubule are occluded so that the lumen becomes and remains collapsed. A system of the flow-through bath 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 the control isosmotic bathing solution used in the present study was as follows (in mM): 110 NaCl, 5 KCl, 25 NaHCO3, 0.8 Na2HPO4, 0.2 NaH2PO4, 10 Na-acetate, 1.8 CaCl2, 1.0 MgCl2, 8.3 glucose, and 5 alanine. The control isosmotic solution was adjusted to an osmolality of 300 mosmol/kgH2O. The hyperosmotic bathing solution was made by the addition of 200 mM mannitol, to a final osmolality of 500 mosmol/kgH2O. The osmolality of all the solutions was measured by freezing-point depression before the experiments. All the solutions were continuously gassed with a mixture of 5% CO2-95% O2 and adjusted to pH 7.4 at 37°C.

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

Measurements of tubule cell volume. Measurements of tubule cell volume were carried out by the methods described by Miyata et al. (27). In brief, before, during, and after exposure of the tubules to the hyperosmotic solution, we took serial photographs, at a magnification of ×400, 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 in shape, the apparent cell volume could then be calculated according to the following equation
V<IT>=</IT>&pgr;(<IT>d</IT>/2)<SUP>2</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 the control, isosmotic tubule cell volume.

Measurement of PKC activity. We measured PKC activity in both groups of proximal tubules exposed to the hyperosmotic mannitol solution, using the previously described methods in our laboratory (8). Proximal straight tubules from both groups of mice were dissected at 4°C in the control isosmotic solution without enzymatic digestion of the tissue. On a given day, they were dissected over a 4-h period, and the pooled tubules were then divided into several groups. The groups of tubules were then incubated in the control isosmotic solution or hyperosmotic mannitol solution, in the absence and presence of PMA. The reaction was then stopped by the addition of 35 µl of extraction solution (20 mM Tris · HCl, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 µg/ml aprotinin, and leupeptin, pH 7.5). Cell extracts were centrifuged at 1,500 g for 5 min. The supernatant was then incubated with 25 µM of a synthetic peptide [4-14 amino acids of bovine myelin basic protein (MBP4-14)](45) and reaction mixture containing 20 mM Tris · HCl (pH 7.5), 5 mM Mg acetate, 0.1 mM CaCl2, 0.5 µg phosphatidyl serine, 50 ng diolein, and 50 µM [gamma -32P]ATP (specific activity; 10 Ci/mM) for 10 min at 30°C. The reaction products were placed on P-81 paper (Whatman International, Clifton, NJ) and were washed three times with 20 ml of ice-cold 10% phosphoric acid. The radioactivity was counted by a liquid scintillation counter (Aloka LSC-671, Tokyo, Japan). Specific radioactivity was obtained by subtracting the radioactivity of the synthetic peptide-free reaction from the synthetic peptide-directed radioactivity. PKC activity was represented as picomoles of ATP incorporated per milligram protein of cell extracts per minute. The protein content of the tubules was determined by the Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA), using BSA as the standard.

Chemicals. Verapamil, cyclosporin A, PMA, 4alpha -phorbol, staurosporine, colchicine, cytochalasin B, wortmannin, and MBP4-14 were purchased from Sigma (St. Louis, MO). LY-294002 was purchased from Calbiochem (La Jolla, CA). [gamma -32P]ATP was purchased from New England Nuclear (Boston, MA). Other high-grade chemicals were obtained from Wako (Osaka, Japan).

Verapamil, PMA, 4alpha -phorbol, staurosporine, calphostin C, cytochalasin B, and wortmannin were dissolved in dimethylsulfoxide at 0.1% final concentrations. Cyclosporin A, colchicine, and LY-294002 were dissolved in ethanol at 0.1% final concentrations. 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 ANOVA in combination with Fisher's protected least significant difference or Newman-Keuls multiple-range test where appropriate. P values <0.05 were considered significant.


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Effect of hyperosmotic mannitol solution on cell volume. At first, we examined the effect of hyperosmotic mannitol solution on cell volume in the proximal tubules from the WT and KO mice. For this purpose, the tubules were initially bathed in the control isosmotic solution (300 mosmol/kgH2O) and were then perfused with the hyperosmotic solution (500 mosmol/kgH2O) containing 200 mM mannitol for 10 min. Figures 1 and 2 are illustrations of the cell volume in the tubules from the WT and KO mice, respectively. When the tubules from the WT mice were rapidly exposed to the hyperosmotic mannitol solution, they abruptly shrank to 81.4 ± 1.4% (n = 5) of their control volume but remained in a shrunken state during the experimental period, indicating a lack of RVI (Fig. 1A). In sharp contrast, when the tubules from the KO mice were rapidly treated with the hyperosmotic mannitol solution for 10 min, they abruptly shrank to 80.0 ± 1.5% (n = 5) of their control volume and then gradually swelled to 88.1 ± 1.3% (n = 5) of their control volume, showing the presence of RVI (Fig. 2).


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Fig. 1.   Effects of hyperosmotic mannitol solution in the absence or presence of P-glycoprotein (P-gp) inhibitors (verapamil or cyclosporin A) on cell volume in isolated nonperfused proximal tubule S2 segments from wild-type (WT) mice. Data are expressed as a percentage relative to control, isosmotic tubule cell volume treated with control isosmotic solution. A: tubules were initially bathed in control isosmotic solution, and were then treated with hyperosmotic solution including 200 mM mannitol for 10 min. Data are means ± SE of 5 tubules from WT mice. B and C: tubules were initially bathed in control isosmotic solution, and were then treated with control isosmotic solution including verapamil (100 µM) or cyclosporin A (5 µM) for 2 min. Thereafter, they were exposed to hyperosmotic mannitol solution in the continued presence of verapamil or cyclosporin A for 10 min. Values are means ± SE of 6 or 5 tubules from WT mice in the presence of verapamil (B) or cyclosporin A (C), respectively. *P < 0.05 vs. maximum cell volume decrease.



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Fig. 2.   Effects of hyperosmotic mannitol solution on cell volume in isolated nonperfused proximal tubule S2 segments from knockout (KO) mice. The tubules were initially bathed in control isosmotic solution, and were then treated with hyperosmotic solution including 200 mM mannitol for 10 min. Data are expressed as a percentage relative to control, isosmotic tubule cell volume treated with control isosmotic solution. Values are means ± SE of 5 tubules from the KO mice. *P < 0.05 vs. maximum cell volume decrease.

Next, we examined whether the hyperosmotic mannitol-induced cell volume changes in the tubules from the WT mice are influenced by the two P-gp inhibitors (verapamil and cyclosporin A). For this purpose, the tubules from the WT mice were initially incubated in the control isosmotic solution and were then treated with the control isosmotic solution involving verapamil (100 µM) or cyclosporin A (5 µM) for 2 min. Thereafter, they were exposed to the hyperosmotic mannitol solution in the continued presence of verapamil or cyclosporin A for 10 min. Results are shown in Fig. 1B (for the verapamil-treated tubules) and C (for the cyclosporin A-treated tubules). The peritubular addition of verapamil or cyclosporin A alone had no effect on cell volume under isosmotic conditions. 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 81.6 ± 2.3 (n = 6) or 80.7 ± 1.7% (n = 5) of their control volume and then gradually swelled to 90.0 ± 1.8 (n = 6) or 89.0 ± 1.3% (n = 5) of their control volume, respectively (Fig. 1, B and C). In the WT mice, the hyperosmotic mannitol-induced changes in cell volume in the presence of the two P-gp inhibitors were markedly similar to those of the KO mice. Similar to the 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 80.0 ± 1.3 (n = 4) or 79.7 ± 1.3% (n = 5) of their control volume and then gradually swelled to 88.5 ± 2.4 (n = 4) or 88.8 ± 1.2% (n = 5) of their control volume, respectively. In the KO mice, the hyperosmotic mannitol-induced changes in cell volume in the presence of the two P-gp inhibitors were not significantly different from those in its absence. Therefore, the exposure of the tubules from the KO mice to the hyperosmotic mannitol in the presence of the two P-gp inhibitors had no effect on RVI. Taken together, when the P-gp activity is acutely suppressed by the P-gp inhibitors or when both mdr1a and mdr1b genes are genetically disrupted, RVI in hyperosmotic medium occurs. The above findings confirm our previous report (27).

Role of PKC in the P-gp-induced modulation of RVI after initial cell shrinkage. Next, we examined whether PKC contributes to the P-gp-induced modulation of RVI in the proximal tubule from the WT mice. For this purpose, the tubules of the WT mice were initially exposed to the control isosmotic solution and were then bathed in the control isosmotic solution containing 100 nM PMA (the PKC activator) or 100 nM 4alpha -phorbol (the inactive phorbol) for 2 min. Thereafter, they were treated with the hyperosmotic mannitol solution in the continued presence of PMA or 4alpha -phorbol for 10 min. Results are shown in Fig. 3, A (for the PMA-treated tubules) and B (for the 4alpha -phorbol-treated tubules). The peritubular addition of PMA or 4alpha -phorbol alone had no effect on cell volume under isosmotic conditions. However, when the tubules of the WT mice were rapidly exposed to the hyperosmotic mannitol solution in the presence of PMA, they abruptly shrank to 81.4 ± 0.9% (n = 8) of their control volume and then gradually swelled to 88.8 ± 0.4% (n = 8) of their control volume, indicating a presence of RVI (Fig. 3A). The hyperosmotic mannitol-induced changes in cell volume in the presence of PMA were markedly similar to those seen in the presence of the two P-gp inhibitors. In sharp contrast, when the tubules were exposed to the hyperosmotic mannitol solution containing 4alpha -phorbol, they did not exhibit RVI after the initial cell shrinkage (Fig. 3B). Next, we examined the effects of the PKC inhibitors (staurosporine or calphostin C) on the hyperosmotic mannitol-induced cell volume regulation. For this purpose, the tubules of the WT mice were initially incubated in the control isosmotic solution, and were then bathed in the control isosmotic solution including staurosporine (100 nM) or calphostin C (500 nM) for 2 min. Thereafter, they were exposed to the hyperosmotic mannitol solution in the continued presence of the PKC inhibitors for 10 min. In the presence of either staurosporine or calphostin C, RVI after the initial cell shrinkage was not observed at all (data not shown). Next, we examined whether the PMA-induced RVI is inhibited by the PKC inhibitors (staurosporine or calphostin C). For this purpose, the tubules of the WT mice were initially incubated in the control isosmotic solution and were then treated with the control isosmotic solution involving PMA (100 nM) plus either of the two PKC inhibitors for 2 min. Thereafter, they were exposed to the hyperosmotic mannitol solution in the continued presence of PMA plus either of the two PKC inhibitors for 10 min. Results are shown in Fig. 3, C (for the PMA plus staurosporine-treated tubules) and D (for the PMA plus calphostin C-treated tubules). The peritubular addition of PMA plus staurosporine or calphostin C had no effect on cell volume under isosmotic conditions. However, when the tubules of the WT mice were rapidly incubated in the hyperosmotic mannitol solution including PMA plus either staurosporine or calphostin C, they abruptly shrank to 82.4 ± 1.0 (n = 6) or 81.8 ± 1.4% (n = 8) of their control volume, respectively, but remained reduced in size (Fig. 3, C and D). Therefore, the peritubular addition of PMA plus staurosporine or calphostin C completely suppressed RVI under hyperosmotic conditions. Collectively, in the tubules of the WT mice, PKC activation by PMA elicited RVI after the initial cell shrinkage.


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Fig. 3.   Effects of hyperosmotic mannitol solution in the presence of phorbol 12-myristate 13-acetate (PMA), 4alpha -phorbol, PMA plus staurospoine, or PMA plus calphostin C on cell volume in the tubules from WT mice. The tubules were initially bathed in control isosmotic solution, and were then treated with control isosmotic solution including PMA (100 nM), 4alpha -phorbol (5 µM), PMA (100 nM) plus staurospoine (100 nM), or PMA (100 nM) plus calphostin C (500 nM) for 2 min. Thereafter, they were exposed to hyperosmotic mannitol solution in the continued presence of PMA, 4alpha -phorbol, PMA plus staurospoine, or PMA plus calphostin C for 10 min. Data are means ± SE of 8, 4, 6, or 8 tubules from WT mice in the presence of PMA (A), 4alpha -phorbol (B), PMA plus staurospoine (C), or PMA plus calphostin C (D), respectively. Values are expressed as a percentage relative to control, isosmotic tubule cell volume treated with control isosmotic solution. *P < 0.05 vs. maximum cell volume decrease.

Next, we examined whether, in the proximal tubules from the WT mice, RVI induced by the P-gp inhibitors under hyperosmotic conditions is mediated by PKC, because the effects of the hyperosmotic mannitol solution in the presence of the two P-gp inhibitors on cell volume were markedly similar to those in the presence of PMA. For this purpose, the tubules of the WT mice were initially incubated in the control isosmotic solution and were then bathed in the control isosmotic solution including either of the two P-gp inhibitors (verapamil and cyclosporin A) plus either of the two PKC inhibitors (staurosporine and calphostin C) for 2 min. Thereafter, they were treated with the hyperosmotic mannitol solution in the continued presence of the drugs for 10 min. Results are shown in Fig. 4. The treatment with either of the two P-gp inhibitors plus either of the two PKC inhibitors caused no effect on cell volume under isosmotic conditions. In sharp contrast, when the tubules from the WT mice were rapidly treated with the hyperosmotic mannitol solution including verapamil plus either staurosporine or calphostin C, they abruptly shrank to 81.8 ± 1.2 (n = 6) or 82.7 ± 1.0% (n = 6) of their control volume, respectively, but remained reduced in size (Fig. 4, A and B). Similarly, when the tubules from the WT mice were rapidly exposed to the hyperosmotic mannitol solution including cyclosporin A plus either staurosporine or calphostin C, they immediately shrank to 80.8 ± 1.9 (n = 6) or 80.8 ± 1.0% (n = 7) of their control volume, respectively, but remained reduced in size (Figs. 4, C and D). Therefore, the addition of either of the two PKC inhibitors completely abolished RVI induced by the hyperosmotic mannitol solution involving either of the two P-gp inhibitors. Taken together, we conclude that in the proximal tubules from the WT mice, RVI induced by the P-gp inhibitors under hyperosmotic conditions is mediated by PKC.


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Fig. 4.   Effects of hyperosmotic mannitol solution in the presence of either of the two P-gp inhibitors plus either of the two protein kinase C (PKC) inhibitors on cell volume in the tubules from WT mice. Tubules were initially bathed in control isosmotic solution, and were then treated with control isosmotic solution including either of the P-gp inhibitors [verapamil (100 µM) or cyclosporin A (5 µM)] plus either of the PKC inhibitors [staurosporine (100 nM) or calphostin C (500 nM)] for 2 min. Thereafter, they were exposed to the hyperosmotic mannitol solution in the continued presence of either of the P-gp inhibitors plus either of the PKC inhibitors for 10 min. Data are means ± SE of 6, 6, 6, or 7 tubules from WT mice in the presence of verapamil plus staurosporine (A), verapamil plus calphostin C (B), cyclosporin A plus staurosporine (C), or cyclosporin A plus calphostin C (D), respectively. Values are expressed as a percentage relative to control, isosmotic tubule cell volume treated with control isosmotic solution.

Next, we examined whether, in the proximal tubule from the KO mice, PKC contributes to the RVI induced by the hyperosmotic mannitol solution. For this purpose, the tubules of the KO mice were initially bathed in the control isosmotic solution, and were then treated with the control isosmotic solution including PMA (100 nM), staurosporine (100 nM), or calphostin C (500 nM) for 2 min. Thereafter, they were exposed to the hyperosmotic mannitol solution in the continued presence of either of the drugs for 10 min. Results are shown in Fig. 5. The peritubular addition of PMA (100 nM) or either of the PKC inhibitors alone had no effect on cell volume under isosmotic conditions (Fig. 5). When the tubules of the KO mice were rapidly exposed to the hyperosmotic mannitol solution in the presence of PMA, they abruptly shrank to 80.8 ± 1.7% (n = 5) of their control volume and then gradually swelled to 90.9 ± 1.0% (n = 5) of their control volume. In sharp contrast, when the tubules of the KO mice were rapidly incubated in the hyperosmotic mannitol solution including staurosporine or calphostin C, they abruptly shrank to 80.7 ± 1.6 (n = 5) or 81.8 ± 1.3% (n = 5) of their control volume, respectively, but remained reduced in size (Fig. 5, B and C). Therefore, the peritubular addition of staurosporine or calphostin C completely inhibited RVI under hyperosmotic conditions. Taken together, in the tubules of the KO mice, RVI after the initial cell shrinkage is mediated through PKC.


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Fig. 5.   Effects of hyperosmotic mannitol solution in the presence of PMA, staurosporine, or calphostin C on cell volume in tubules from KO mice. The tubules were initially bathed in control isosmotic solution, and were then treated with control isosmotic solution including PMA (100 nM), staurosporine (100 nM), or calphostin C (500 nM) for 2 min. Thereafter, they were exposed to hyperosmotic mannitol solution in the continued presence of PMA, staurosporine, or calphostin C for 10 min. Data are means ± SE of 5 tubules from KO mice in the presence of PMA (A), staurosporine (B), or calphostin C (C). Values are expressed as a percentage relative to control, isosmotic tubule cell volume treated with control isosmotic solution. *P < 0.05 vs. maximum cell volume decrease.

Next, we examined whether PKC is actually activated when the proximal tubules of the WT mice were exposed to the hyperosmotic mannitol solution involving PMA. For this purpose, the proximal straight tubules from the WT mice were exposed to the control isosmotic solution or hyperosmotic mannitol solution involving PMA (100 nM) during a 10-min period. The results are shown in Fig. 6A. Control isosmotic solution showed relatively constant levels of PKC activity throughout the 10-min period. On the other hand, treatment with the hyperosmotic mannitol solution including PMA time dependently increased PKC activity, with a maximum 2.4-fold increase at 3 min. This PKC activation was not sustained and was downregulated with time, so that PKC activity decreased to 1.6 times the basal levels at 5 min and returned to the basal levels after 10 min of continued exposure to the hyperosmotic mannitol solution including PMA. Figure 6B summarizes PKC activity when the proximal tubules of the WT mice were treated with the control isosmotic solution or hyperosmotic mannitol solution, in the absence and presence of PMA (100 nM) for 3 min. When the proximal tubules of the WT mice were exposed to the control isosmotic solution including PMA, PKC activity increased to 2.2 times the control isosmotic solution alone. Similarly, the exposure to the hyperosmotic mannitol solution including PMA caused a significant increase in PKC activity to 2.4 times the control isosmotic solution alone. In sharp contrast, the exposure to the hyperosmotic mannitol solution alone did not significantly influence PKC activity.


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Fig. 6.   Effects of hyperosmotic mannitol solution in the absence and presence of PMA on PKC activity in the proximal straight tubules from the WT and KO mice. A: tubules from WT mice were incubated in control isosmotic solution (Control) or hyperosmotic mannitol solution (Mannitol) containing PMA (100 nM) during 10 min. The PKC activity is expressed as a percentage relative to the activity measured at 0 min (basal activity). In control solution, the basal-specific radioactivity for PKC was 2,036 ± 116 cpm (n = 3). Each point is means ± SE of 3 separate experiments. *P < 0.01 compared with basal activity. B: tubules from WT mice were incubated in control isosmotic solution (Control) or hyperosmotic mannitol solution (Mannitol), in the absence and presence of PMA (100 nM) for 3 min. PKC activity is expressed as a percentage relative to the activity measured in control isosmotic solution. Values are means ± SE of 3 separate experiments. *P < 0.01 vs. Control. dagger P < 0.01 vs. Mannitol. C: tubules from the KO mice were incubated in control isosmotic solution (Control) or hyperosmotic mannitol solution (Mannitol), in the absence and presence of PMA (100 nM) for 3 min. PKC activity is expressed as a percentage relative to the activity measured in control isosmotic solution. Data are means ± SE of 3 separate experiments. *P < 0.01 vs. Control.

Next, we examined whether PKC is actually activated when the proximal tubules of the KO mice were exposed to the hyperosmotic mannitol solution. For this purpose, the proximal straight tubules from the KO mice were exposed to the control isosmotic solution or hyperosmotic mannitol solution, in the absence and presence of PMA (100 nM) for 3 min. The results are summarized in Fig. 6C. When the proximal tubules of the KO mice were exposed to the control isosmotic solution including PMA, PKC activity increased to 2.4 times the control isosmotic solution alone. In sharp contrast to the WT mice, exposure to the hyperosmotic mannitol solution alone caused a significant increase in PKC activity to 2.7 times the control isosmotic solution alone. The treatment with the hyperosmotic mannitol solution including PMA increased PKC activity 2.4-fold, a value that was not significantly different from that with the control isosmotic solution including PMA and that with the hyperosmotic mannitol solution alone. Taken together, these findings are consistent with the notion that the hyperosmotic mannitol solution indeed activates PKC in the proximal tubules from the KO mice, but not from the WT mice.

Role of cytoskeleton and PI 3-kinase in PKC-induced RVI after initial cell shrinkage. Next, we examined whether the cytoskeleton contributes to the PKC-induced RVI after the initial cell shrinkage. For this purpose, the tubules of the WT and KO mice were incubated in the control isosmotic solution involving the microtubule disruptor (colchicine; 10 µM) or the microfilament disruptor (cytochalasin B; 10 µM) for 2 min and were then exposed to the hyperosmotic mannitol solution in the continued presence of colchicine or cytochalasin B for 10 min. In the WT mice, the addition of colchicine or cytochalasin B had no effect on basal cell volume or the hyperosmotic mannitol-induced cell volume (data not shown). In the KO mice, the addition of either of the disruptors alone had no effect on cell volume under isosmotic conditions (Fig. 7, A and B). However, when the tubules from the KO mice were rapidly exposed to the hyperosmotic mannitol solution including colchicine or cytochalasin B, they abruptly shrank to 82.6 ± 1.3 (n = 7) or 81.2 ± 0.8% (n = 6) of their control volume, respectively, but remained in a shrunken state during the experimental period (Fig. 7, A and B). Therefore, in sharp contrast to WT mice, in KO mice, the addition of colchicine or cytochalasin B completely inhibited RVI induced by the hyperosmotic mannitol solution. Next, we observed the cell volume change, when the tubules of the WT mice were initially treated with the control isosmotic solution including PMA (100 nM) plus colchicine (10 µM) or cytochalasin B (10 µM) for 2 min, and were then exposed to the hyperosmotic mannitol solution in the continued presence of PMA plus colchicine or cytochalasin B for 10 min. The coadministration with PMA and either colchicine or cytochalasin B had no effect on cell volume under isosmotic conditions (Fig. 8, A and B). However, when the tubules from the WT mice were rapidly exposed to the hyperosmotic mannitol solution including PMA plus either colchicine or cytochalasin B, they abruptly shrank to 82.7 ± 1.0% (n = 6) or 83.0 ± 1.2% (n = 4) of their control volume, respectively, but remained reduced in size (Figs. 8, A and B). Therefore, the addition of colchicine or cytochalasin B to the tubules of the WT mice completely abolished RVI induced by the hyperosmotic mannitol solution including PMA. Collectively, both the microtubule and microfilament are involved in the PKC-induced RVI.


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Fig. 7.   Effects of hyperosmotic mannitol solution in the presence of colchicine, cytochalasin B, wortmannin, or LY-294002 on cell volume in tubules from KO mice. Tubules were initially bathed in control isosmotic solution, and were then treated with control isosmotic solution including colchicine (10 µM), cytochalasin B (10 µM), wortmannin (100 nM), or LY-294002 (50 µM) for 2 min. Thereafter, they were exposed to hyperosmotic mannitol solution in the continued presence of colchicine, cytochalasin B, wortmannin, or LY-294002 for 10 min. Data are means ± SE of 7, 6, 5, or 5 tubules from KO mice in the presence of colchicine (A), cytochalasin B (B), wortmannin (C), or LY-294002 (D), respectively. Values are expressed as a percentage relative to control, isosmotic tubule cell volume treated with control isosmotic solution. *P < 0.05 vs. maximum cell volume decrease.



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Fig. 8.   Effects of hyperosmotic mannitol solution in the presence of PMA plus either of colchicine, cytochalasin B, wortmannin, or LY-294002 on cell volume in tubules from WT mice. Tubules were initially bathed in control isosmotic solution, and were then treated with control isosmotic solution including PMA (100 nM) plus either of colchicine (10 µM), cytochalasin B (10 µM), wortmannin (100 nM), or LY-294002 (50 µM) for 2 min. Thereafter, they were exposed to hyperosmotic mannitol solution in the continued presence of PMA plus either of the drugs for 10 min. Data are means ± SE of 6, 4, 6, or 6 tubules of WT mice in the presence of PMA plus colchicine (A), PMA plus cytochalasin B (B), PMA plus wortmannin (C), or PMA plus LY-294002 (D), respectively. Values are expressed as a percentage relative to control, isosmotic tubule cell volume treated with control isosmotic solution. *P < 0.05 vs. maximum cell volume decrease.

Next, we examined whether PI 3-kinase contributes to the PKC-induced RVI after the initial cell shrinkage. For this purpose, the tubules of the WT and KO mice were initially incubated in the control isosmotic solution involving either of the PI 3-kinase inhibitors [wortmannin, (100 nM), or LY-294002, (50 µM)] for 2 min and were then exposed to the hyperosmotic mannitol solution in the continued presence of either of the PI 3-kinase inhibitors for 10 min. In the WT mice, the addition of wortmannin or LY-294002 had no effect on basal cell volume or the hyperosmotic mannitol-induced cell volume (data not shown). In the KO mice, wortmannin or LY-294002 alone had no effect on cell volume under isosmotic conditions, either (Fig. 7, C and D). However, when the tubules of the KO mice were rapidly treated with the hyperosmotic mannitol solution containing wortmannin, they abruptly shrank to 82.1 ± 1.5% (n = 5) of their control volume but remained reduced in size (Fig. 7C). On the other hand, when the tubules of the KO mice were exposed to the hyperosmotic mannitol solution involving LY-294002, they abruptly shrank to 79.4 ± 1.1% (n = 5) of their control volume and then gradually swelled to 88.2 ± 1.4% (n = 5) of their control volume (Fig. 7D). Therefore, in the KO mice, the peritubular addition of another PI 3-kinase inhibitor, LY-294002, which is chemically unrelated to wortmannin and impairs the enzyme by a different mechanism (43), caused no effect on RVI induced by the hyperosmotic mannitol solution. Next, we observed the cell volume change, when the tubules of the WT mice were initially treated with the control isosmotic solution containing PMA (100 nM) plus either wortmannin or LY-294002 for 2 min and were then exposed to the hyperosmotic mannitol solution in the continued presence of PMA plus either wortmannin or LY-294002 for 10 min. The treatment with PMA plus either wortmannin or LY-294002 had no effect on cell volume under isosmotic conditions (Fig. 8, C and D). When the tubules from the WT mice were rapidly exposed to the hyperosmotic mannitol solution including PMA and wortmannin, they abruptly shrank to 82.5 ± 1.2% (n = 6) of their control volume but remained reduced in size (Fig. 8C). On the other hand, when the tubules from the WT mice were rapidly incubated in the hyperosmotic mannitol solution including PMA and LY-294002, they abruptly shrank to 80.6 ± 1.7% (n = 6) of their control volume and then gradually swelled to 88.3 ± 1.8% (n = 6) of their control volume (Fig. 8D). Therefore, in the WT mice, the addition of wortmannin, but not LY-294002, suppressed RVI induced by the hyperosmotic mannitol solution including PMA. Collectively, the wortmannin-sensitive, LY-294002-insensitive PI 3-kinase contributes to PKC-induced RVI.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We 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 (27). On the other hand, RVI was seen in the hyperosmotic mannitol solution when the P-gp activity was acutely suppressed by the P-gp inhibitors (verapamil and cyclosporin A) or when both mdr1a and mdr1b genes were genetically disrupted (27). In the present study, we extend our previous study to determine the intracellular signaling mechanisms for the P-gp-induced modulation of RVI after initial cell shrinkage.

In the present study, we observed that in the proximal tubule from the WT mice, the peritubular addition of PMA elicited RVI after the initial cell shrinkage, but 4alpha -phorbol did not (Fig. 3). The treatment with the PKC inhibitors (staurosporine and calphostin C) inhibited the PMA-induced RVI (Fig. 3). PMA alone had no effect on cell volume under isosmotic conditions. Either of the PKC inhibitors had no effect on cell volume under is- or hyperosmotic conditions. These findings indicate that, in the tubules from the WT mice, PKC activation resulted in RVI under hyperosmotic conditions. This is supported by the fact that in the tubules from the WT mice, the hyperosmotic mannitol solution including PMA activated PKC, whereas the hyperosmotic mannitol solution alone did not (see Fig. 6B). We also observed that in the tubules of the KO mice, the two PKC inhibitors abolished RVI under hyperosmotic conditions, although they had no effect on cell volume under isosmotic conditions (Fig. 5). In the KO mice, PMA caused no effect on cell volume under is- or hyperosmotic conditions (Fig. 5). These findings indicate that in the tubules of the KO mice, PKC inhibition with the PKC inhibitors abolished RVI under hyperosmotic conditions. In the tubules of the KO mice, the hyperosmotic mannitol solution in the absence of PMA increased PKC activity with a similar magnitude to those in the presence of PMA (Fig. 6C). Therefore, the present results are compatible with the notion that the exposure of the proximal tubules from the KO mice to the hyperosmotic mannitol solution elicited RVI via PKC activation. In the WT mice, the hyperosmotic mannitol-induced cell volume changes in the presence of PMA were markedly similar to those seen in the presence of the P-gp inhibitors. The hyperosmotic mannitol-induced cell volume changes in the presence of PMA in the WT mice were markedly similar to those in the KO mice during exposure to hyperosmotic mannitol. In the WT mice, the P-gp inhibitors-induced RVI after the initial cell shrinkage was inhibited by the two PKC inhibitors (Fig. 4). On the basis of the above findings, we conclude that the exposure of the proximal tubule to the hyperosmotic mannitol in the absence of P-gp activity (when the P-gp activity is acutely suppressed by the P-gp inhibitors or when both mdr1a and mdr1b genes are genetically disrupted) induces PKC activation, which in turn results in RVI. On the other hand, exposure to the hyperosmotic mannitol in the presence of P-gp activity does not induce PKC activation and consequently abolishes RVI. In sharp contrast, in the presence or absence of P-gp activity, PKC is not involved in the cell volume regulation under isosmotic conditions. Collectively, PKC contributes to the P-gp-induced modulation of RVI after the initial cell shrinkage. This is the first demonstration of a link between PKC and the P-gp-induced modulation of RVI. Also, the present studies demonstrate one of the mechanisms responsible for the lack of RVI in the mammalian proximal tubule. In the isolated proximal tubule from frog kidney, both RVD and activation of Cl- channels during exposure to hyposmotic stress have also been reported to be mediated via PKC (32).

It should be noted that the exposure of the tubules from the WT mice to the hyperosmotic mannitol solution did not induce PKC activation (see Fig. 6B). In sharp contrast to the proximal tubules of the WT mice, hyperosmotic NaCl solution has been shown to activate PKC in MDCK cells (38) and NIH/3T3 cells (46). Similarly, the exposure of the tubules from the KO mice to the hyperosmotic mannitol solution resulted in PKC activation (see Fig. 6C). The mechanism for PKC activation by growth factors is well established in mammalian cells (24), and a partly common mechanism is also operating in hyperosmotic stress-induced PKC activation (38, 46). Stimulation of tyrosine kinase receptors activates phospholipase C and PI 3-kinase, leading to an increase in diacylglycerol and inositol 1,4,5-triphosphate levels, which, in turn, mediate activation of PKC (26, 28). In MDCK cells, hyperosmotic NaCl and raffinose solutions increased inositol 1,4,5-triphosphate levels and therby activated PKC (38). In NIH/3T3 cells, exposure to hyperosmotic NaCl solution triggers phospholipase C activation, then induces an increase in diacylglycerol levels, and as a consequence, PKC activation (46). However, we do not know at present how the hyperosmotic mannitol solution activates PKC in the proximal tubules of the KO mice but does not in those of the WT mice.

There are several mechanisms whereby PKC activation elicits RVI after the initial cell shrinkage. Pedersen et al. (29) have shown that, in Ehrlich ascites tumor cells, hyperosmotic shrinkage activates Na+/H+ exchanger via PKC. Recently, we demonstrated that in the mouse proximal tubule, the P-gp-induced modulation of RVI partly occurs through the basolateral Na+/H+ exchanger (27). Thus PKC-induced RVI could be partly mediated via the basolateral Na+/H+ exchange. Witters et al. (44) have reported that the purified human erythrocyte glucose transporter is phosphorylated on incubation with rat brain PKC. Mehrens et al. (26) demonstrated that in human embryonic epithelial cells (HEK-293 cells) transfected with the rat organic cation transporter 1 (rOCT1) gene, PKC phosphorylates rOCT1 protein and leads to a conformational change at the substrate binding site. Pewitt et al. (30) have also shown 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. Therefore, we propose that the PKC-induced RVI may be mediated via the direct phosphorylation of the volume-regulated transporters like the Na+/H+ exchanger and/or their regulatory proteins by PKC. In support of this concept, Sardet et al. (34) first described in hamster fibroblasts that Na+/H+ exchanger protein is rapidly phosphorylated in response to various mitogens, and that this phosphorylation of the Na+/H+ exchanger protein is temporally correlated with its activation. However, Grinstein et al. (17) observed in rat thymic lymphocytes that phosphorylation of the Na+/H+ exchanger protein is not affected by hyperosmotic cell shrinkage, although Na+/H+ exchanger is activated by phosphorylation. They concluded that activation of the Na+/H+ exchanger in lymphocyte RVI is due to phosphorylation by a protein kinase other than PKC. Instead, they have shown that direct PKC activation by PMA increases Na+/H+ exchanger activity by causing an alkaline shift in the cell pH dependence of the exchanger (16, 18). Therefore, it is also possible that Na+/H+ exchanger activation may occur via a PKC-dependent alkaline shift in the cell pH responsiveness of an allosteric modifier site on the cytoplasmic surface of the basolateral membrane (2, 16, 18). Further studies will be required to determine whether and how PKC activation by PMA actually activates the basolateral Na+/H+ exchanger in the mouse proximal tubule.

As shown in Fig. 7, in the tubules of the KO mice both colchicine and cytochalasin B inhibited RVI under hyperosmotic conditions, although they had no effect on cell volume under isosmotic conditions. Figure 8 shows that, in the tubules of the WT mice, both colchicine and cytochalasin B inhibited PMA-induced RVI under hyperosmotic conditions. On the other hand, exposure of the tubules from WT mice to colchicine or cytochalasin B had no effect on cell volume under is- or hyperosmotic conditions. These findings indicate that both colchicine and cytochalasin B inhibit PKC-induced RVI under hyperosmotic conditions. In other words, both microtubules and microfilaments are required for the PKC-induced RVI. These findings are also the first demonstration of the involvement of both microtubules and microfilaments in the PKC-induced RVI. Because neither colchicine or cytochalasin B affected cell volume under isosmotic conditions, both colchicine- and cytochalasin B-sensitive processes are activated under hyperosmotic conditions and consequently elicit RVI. Future studies will be required to clarify the mechanisms by which PKC activation under hyperosmotic conditions elicits RVI via the microtubule- and microfilament-dependent processes.

PI 3-kinases are a family of enzymes that can be distinguished by molecular characteristics, substrate specificity, and inhibitor sensitivities (3, 41, 43). Classic inhibitors of these enzymes are wortmannin, a fungal metabolite, which rapidly and irreversibly inhibits mammalian PI 3-kinase by alkylating the catalytic p110 subunit and is an inhibitor effective in the nanomolar concentration range, and LY-294002, a structurally unrelated compound, which interacts with the ATP-binding site of the enzyme and is an inhibitor effective in the micromolar concentration range (43). In the present study, we observed that in the tubules of the KO mice, wortmannin, but not LY-294002, inhibited RVI under hyperosmotic conditions, although neither of the inhibitors had an effect on cell volume under isosmotic conditions (see Fig. 7). We also show that in the tubules of the WT mice under hyperosmotic conditions, wortmannin abolished the PMA-induced RVI, but LY-294002 did not (see Fig. 8). On the other hand, the exposure of the tubules from the WT mice to either wortmannin or LY-294002 had no effect on cell volume under iso- or hyperosmotic conditions. Therefore, wortmannin, but not LY-294002, inhibits the PKC-induced RVI under hyperosmotic conditions, consistent with the notion that the wortmannin-sensitive, LY-294002-insensitive PI 3-kinase is required for the PKC-induced RVI after initial cell shrinkage. Because wortmannin caused no effect on cell volume under isosmotic conditions, the wortmannin-sensitive, LY-insensitive PI 3-kinase is activated under hyperosmotic conditions, and thereby elicits RVI, although the underlying mechanisms are unclear. This is also the first demonstration that PI 3-kinase contributes to the PKC-induced RVI after initial cell shrinkage. In human intestine 407 cells, PI 3-kinase has been shown to be involved in the activation of Cl- channels after hyposmotic stress (40). Feranchak et al. (11) also reported that, in HTC hepatoma cells, both RVD and activation of the Cl- channel under hyposmotic stress are mediated via PI 3-kinase. Good et al. (14) have very recently shown that in the medullary thick ascending limb of Henle's loop from rat kidney, PI 3-kinase is required for the hyposmolality-induced stimulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption but is not involved in the hyperosmolality-induced inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. Presently, however, the mechanisms whereby the wortmannin-sensitive, LY-insensitive PI 3-kinase contributes to the PKC-induced RVI under hyperosmotic conditions, remain unclear.

We concluded that in the mouse proximal tubule S2 segments, P-gp-induced modulation of RVI during hyperosmotic stress is mediated via PKC. On the basis of the use of pharmacological agents, we also found that the microtubule, microfilament, and wortmannin-sensitive, LY-294002-insensitive PI 3-kinase contribute to the PKC-induced RVI after initial cell shrinkage.


    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

A portion of this work was presented at the Annual Meeting of the American Society of Nephrology in Toronto, Ontario, Canada, and has been published in abstract form (J Am Soc Nephrol 11, 46A-46B, 2000).

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.

First published August 15, 2001; 10.1152/ajprenal. 0000036.2001

Received 6 February 2001; accepted in final form 7 August 2001.


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ABSTRACT
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METHODS
RESULTS
DISCUSSION
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