Hyperosmotic urea activates basolateral NHE in proximal tubule from P-gp null and wild-type mice

Yukio Miyata, Yasushi Asano, and Shigeaki Muto

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


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Using the pH-sensitive fluorescent dye BCECF, we compared the effects of hyperosmotic urea on basolateral Na+/H+ exchange (NHE) with those of hyperosmotic mannitol in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type (WT) mice. All the experiments were performed in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH2O by adding mannitol or urea. NHE activity was assessed by the Na+-dependent acid extrusion rate (JH) after an acid load with NH4Cl prepulse. In WT mice, hyperosmotic mannitol had no effect on JH at over the entire range of intracellular pH (pHi) studied (6.20-6.90), whereas in KO mice it increased JH at a pHi range of 6.20-6.45. In contrast, in both WT and KO mice, hyperosmotic urea increased JH at a pHi range of 6.20-6.90. In KO mice, JH in a hyperosmotic urea solution were similar to those in a hyperosmotic mannitol solution at a pHi range of 6.20-6.40 but were greater than in a hyperosmotic mannitol solution at a pHi range of 6.45-6.90. In WT mice, hyperosmotic urea caused an increase in Vmax without changing Km for peritubular Na+. Staurosporine (the PKC inhibitor) inhibited hyperosmotic mannitol-induced NHE activation in KO mice, whereas it had no effect on hyperosmotic urea-induced NHE activation in WT or KO mice. Genistein (the tyrosine kinase inhibitor) inhibited hyperosmotic urea-induced NHE activation in WT and KO mice, whereas it caused no effect on hyperosmotic mannitol-induced NHE activation in KO mice. We conclude that hyperosmotic urea activates basolateral NHE via tyrosine kinase in tubules from both WT and KO mice, whereas hyperosmotic mannitol activates it via PKC only in tubules from KO mice.

mdr1a; mdr1b; isolated nonperfused tubule; intracellular pH measurement; PKC; tyrosine kinase


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P-GLYCOPROTEIN (P-gp) IS A member of the ATP-binding cassette superfamily of transporters and utilizes ATP to pump hydrophobic drugs out of the cells, decreasing their intracellular concentrations and hence their toxicity (reviewed in Ref. 15). Humans have one drug-transporting P-gp (MDR1), whereas mice have two genes encoding drug-transporting P-gps, mdr1a (also called mdr3) and mdr1b (also called mdr1) (15, 18, 32). The mdr1a and mdr1b genes in the mouse together fulfill the same function as MDR1 in humans, and similar levels of mdr1a and mdr1b expression have been observed in the kidney (11, 33). In the kidney, the apical membrane of the proximal tubule epithelium is particularly rich in P-gp (12, 38), placing this pump in the correct location to mediate the active excretion of xenobiotics. Consistent with a role for P-gp as an excretory transporter, our laboratory (40) previously reported in the isolated perfused mouse proximal tubule that P-gp-mediated drug efflux capacity indeed exists in the apical membrane of the proximal tubule S2 segment from wild-type (WT) mice but is lacking in that of mice in which both mdr1a and mdr1b genes were disrupted (KO mice).

In a variety of cell types, Na+/H+ exchange (NHE) is activated by shrinkage of a cell in a hyperosmotic solution, resulting in Na+ entry into the cell. Osmotic uptake of water due to this Na+ entry leads to cell swelling, a process termed regulatory volume increase (RVI) (reviewed in Refs. 19 and 23). In sharp contrast, when rabbit proximal tubule cells are suddenly exposed to hyperosmotic mannitol, NaCl, or raffinose, they rapidly shrink but remain reduced in size (13, 22). However, the underlying mechanisms for the lack of RVI have not fully been understood. Recently, our laboratory (24) used isolated nonperfused proximal tubule S2 segments from WT and KO mice to demonstrate that exposure of tubules from WT mice to hyperosmotic mannitol did not result in RVI, whereas exposure of tubules from KO mice to hyperosmotic mannitol elicited RVI. We also observed that in tubules from WT mice, peritubular addition of P-gp inhibitors (verapamil and cyclosporin A) resulted in RVI, whereas in tubules from KO mice it had no effect on RVI (24). Therefore, in the mouse proximal tubule, P-gp modulates RVI during exposure to hyperosmotic mannitol. We have also shown that P-gp-induced modulation of RVI occurs via PKC activation (27). Furthermore, we demonstrated that basolateral NHE partly contributes to P-gp-induced modulation of RVI (24). Recently, we reported in the mouse proximal tubule that in the absence of P-gp activity, hyperosmotic mannitol actually activates basolateral NHE via PKC, whereas in the presence of P-gp activity it does not (25). In that report, we observed that in KO mice exposure to hyperosmotic mannitol significantly increased Na+-dependent acid extrusion rates (JH) via NHE only at a pHi range of 6.20-6.45 and shifted JH vs. pHi by ~0.15 pH units in the alkaline direction at the low pHi (25). Here, the question arises of whether the stimulatory effect on NHE is specific to mannitol, because in a variety of cell types exposed to hyperosmotic stress, the alkaline shift in pHi sensitivity of NHE occurs along a wide range of pHi (17, 26, 34, 35).

Unlike the poorly permeating solutes (NaCl, mannitol, and raffinose), urea is relatively membrane permeant and has traditionally been considered to play a passive role in renal epithelial cell function (reviewed in Ref. 14). In fact, we previously observed that in the mouse proximal tubule, the effects of urea on the change in cell volume differ markedly from those of mannitol; when proximal tubules of both WT and KO mice were exposed to hyperosmotic urea, the reduction in cell volume was smaller compared with hyperosmotic mannitol and transient, and cells immediately returned to their control volume without RVI (24). Our laboratory also reported that in cultured rat inner medullary collecting duct cells, hyperosmolality induced by NaCl, mannitol, and raffinose stimulates both Na+-K+-ATPase alpha 1- and beta 1-subunit mRNA expression and Na+-K+-ATPase activity, whereas hyperosmolality induced by urea does not (29). However, the effect of hyperosmotic urea on NHE has received less attention. In addition, it is unknown whether the effect is dependent on P-gp. To solve the above problems, we used isolated nonperfused proximal tubule S2 segments from KO and WT mice to address the following issues: 1) whether hyperosmotic urea activates basolateral NHE in proximal tubules of WT and/or KO mice, and if so 2) how hyperosmotic urea modulates basolateral NHE. For this purpose, we compared the effects of hyperosmotic urea on basolateral NHE with those of hyperosmotic mannitol.


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Solutions. The composition of the solutions is given in Table 1. All solutions were nominally CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> free. Solutions 1-3 were adjusted to pH 7.4 at 37°C. The osmolality of all solutions was measured before the experiment and was verified to be within a range of 300 ± 5 mosmol/kgH2O. Hyperosmotic mannitol or urea solutions (500 mosmol/kgH2O) were made by adding mannitol or urea to solution 1 or 2, respectively. The nigericin calibrating solution was titrated to different pH values at 37°C with either HCl or N-methyl-D-glucamine (NMDG). For Na+ free solutions (solutions 2-4, Table 1), Na+ was replaced with NMDG titrated with the appropriate acid. BCECF-acetoxymethyl ester (AM) was prepared as a 10 mM stock solution and was diluted 1:1,000 to a final concentration of 10 µM. Nigericin was prepared as a 10 mM stock solution in ethanol and was diluted 1:1,000 into solution 4 (Table 1) to a final concentration of 10 µM. EIPA was prepared as a 100 mM stock solution in methanol and was diluted 1:1,000 to a final concentration of 100 µM. Staurosporine and genistein were dissolved in DMSO at 0.1% final concentrations. Equivalent concentrations of vehicle were added as a control for individual protocols.

                              
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Table 1.   Composition of solutions

Animals. KO mice used in this study were originally described by Schinkel et al. (32). Male KO and FVB (WT) mice, serving as a control (body wt 25-40 g), were purchased from Taconic Engineering (Germantown, NY). The animals were maintained under a controlled environment and had free access to a standard rodent chow and tap water ad libitum until the beginning of the experiments. Ages of the KO animals were matched with their WT controls.

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

Both proximal and distal ends of the tubule were drawn into and crimped by glass micropipettes as described by Miyata et al. (24, 25, 27). A flow-through bath system was utilized to permit rapid exchange of the bath fluid. The bathing solution flowed by gravity at 4.5-5.5 ml/min from the reservoirs through a water jacket to stabilize the bath temperature at 37°C. After tubules were tightly crimped between the two glass micropipettes, they were bathed in the isosmotic control solution (solution 1, Table 1) at 37°C for 10-15 min to allow them to equilibrate.

Optical measurement of pHi. The details of our techniques for measuring pHi in mouse proximal tubules have been published elsewhere (25). Briefly, both groups of tubules in the bath were exposed to the isosmotic control HEPES-buffered solution (solution 1, Table 1) containing BCECF-AM (10 µM). After a 15-min dye-loading period at 37°C, the dye was washed out. pHi was then measured microfluorometrically by alternately exciting the dye with a 7.5-µm diameter light at 440 and 490 nm while the emission was monitored at 530 nm (25, 26). The resulting fluorescence-to-excitation ratios were converted to pHi values as described (25, 26), using the high-K+/nigericin technique (39). We used the same intracellular dye calibration coefficients described for the proximal tubule cells from WT and KO mice (25).

Computation of JH. In the proximal tubule cells from the WT and KO mice, we computed JH as the product of the previously measured intrinsic buffering power (beta I) (25), which varies with pHi, and the rate of Na+-dependent pHi increase (dpHi/dt). To obtain the rate of Na+-dependent pHi increase, both groups of proximal tubule cells were incubated in the Na+-containing isosmotic solution (solution 1, Table 1) and then exposed to the Na+-free solution containing 20 mM NH4Cl for 2 min (solution 3, Table 1). During the pulse, pHi increased due to the entry of NH3 into the cell and then tended to decrease toward baseline due to a slower influx of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. In the continued presence of the Na+-free solution, NH4Cl was abruptly removed from the peritubular solution, causing pHi to decrease. Afterward, the readdition of Na+ to the peritubular solution gave rise to a rapid increase in pHi. The Na+-dependent pHi increase was divided into two or three sections, each of which was fitted to a third- or fourth-order polynomial. The derivatives of these equations (i.e., dpHi/dt values) were calculated at pHi intervals of 0.05. At each pHi, data from four or more experiments were averaged to produce a plot of mean JH vs. pHi (25, 26, 31).

Drugs and chemicals. All chemicals were obtained from Wako (Osaka, Japan) unless noted as follows: HEPES and BCECF-AM were from Dojindo (Kumamoto, Japan); and EIPA, staurosporine, genistein, and nigericin were from Sigma (St. Louis, MO).

Data analysis and statistics. The data are expressed as means ± SE. Comparisons were performed by Student's t-test or one-way ANOVA in combination with Fisher's protected least significant difference test where appropriate. P values <0.05 were considered significant.


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Na+-dependent pHi recovery from an acid load in proximal tubule cells from WT and KO mice under isosmotic and hyperosmotic conditions. First, we observed Na+-dependent pHi recovery from an acid load with NH4Cl in proximal tubule cells from WT and KO mice. Representative pHi recordings in WT and KO mice are shown in Figs. 1A and 2A, respectively. Both groups of cells were first bathed in the Na+-containing, HEPES-buffered solution (solution 1, Table 1) and then incubated in the Na+-free, HEPES-buffered solution containing 20 mM NH4Cl (solution 3, Table 1). At this time, pHi rapidly increased because of the rapid diffusion of NH3 into the cells. During the exposure to NH4Cl for 2 min, pHi tended to decrease toward baseline due to the slow inward diffusion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Afterward, removing NH4Cl from the Na+-free, HEPES-buffered solution rapidly decreased pHi, because accumulated internal NH<UP><SUB>4</SUB><SUP>+</SUP></UP> dissociates into H+ and NH3 (which exits from the cell). At this time, pHi values of proximal tubule cells from WT and KO mice were 6.14 ± 0.04 (n = 10) and 6.15 ± 0.03 (n = 8), respectively, and were not significantly different between the two groups. Subsequent addition of Na+ to the peritubular solution caused pHi to rapidly increase to values similar to those for initial steady-state pHi. In fact, final steady-state pHi values in cells from WT and KO mice were 6.95 ± 0.03 (n = 10) and 7.01 ± 0.02 (n = 8), respectively, and were not significantly different from those for the initial steady-state (WT mice: 6.99 ± 0.03, n = 10; KO mice: 7.00 ± 0.01, n = 8). In both groups of cells, pretreatment with EIPA (the specific NHE inhibitor; 100 µM) inhibited the Na+-dependent pHi recovery rate by ~80%. These findings confirmed our previous observations (25). To further characterize basolateral NHE in proximal tubules from WT and KO mice, dose-response experiments were performed with EIPA. As shown in Fig. 3, inhibition curves for tubules from both WT and KO mice indicated a single NHE activity, with IC50 of 11.3 and 10.8 µM, respectively. These data are consistent with the notion that basolateral NHE in both groups of proximal tubule cells is highly amiloride resistant (5, 7, 28).


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Fig. 1.   Na+-dependent intracellular pH (pHi) recovery from an acid load when the proximal tubule cells from wild-type (WT) mice were exposed to an isosmotic solution (A), hyperosmotic mannitol solution (B), and hyperosmotic urea solution (C). The cells were first bathed in a Na+-containing HEPES-buffered solution (solution 1, Table 1) and then incubated in a Na+-free HEPES-buffered solution containing 20 mM NH4Cl (solution 3, Table 1) for 2 min. During the pulse, pHi increased (due to the entry of NH3 into the cell) and then tended to decrease toward baseline due to a slower influx of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Thereafter, the abrupt removal of NH4Cl from the Na+-free solution caused a substantial and sustained decrease in pHi. Then, readdition of peritubular Na+ to the cells caused pHi to increase. When the cells were exposed to the hyperosmotic mannitol solution (500 mosmol/kgH2O), Na+-dependent pHi recovery was similar to that when the cells were exposed to the isosmotic solution. In cells exposed to the isosmotic solution and the hyperosmotic mannitol solution, final steady-state pHi values were similar to the initial steady-state pHi values. However, when cells was exposed to the hyperosmotic urea solution (500 mosmol/kgH2O), Na+-dependent pHi recovery was faster than when they were exposed to the isosmotic solution and the hyperosmotic mannitol solution. In cells treated with the hyperosmotic urea solution, the final steady-state pHi values were greater than the initial steady-state pHi values.



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Fig. 2.   Na+-dependent pHi recovery from an acid load when proximal tubule cells from KO mice were exposed to an isosmotic solution (A), hyperosmotic mannitol solution (B), and hyperosmotic urea solution (C). Cells were first bathed in the Na+-containing HEPES-buffered solution (solution 1, Table 1) and then incubated in the Na+-free HEPES-buffered solution containing 20 mM NH4Cl (solution 3, Table 1) for 2 min. During the pulse, pHi increased (due to the entry of NH3 into the cell) and then tended to decrease toward baseline due to a slower influx of NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. Thereafter, the abrupt removal of NH4Cl from the Na+-free solution caused a substantial and sustained decrease in pHi. Then, readdition of peritubular Na+ to the cells caused pHi to increase. When cells were exposed to the hyperosmotic mannitol or urea solutions (500 mosmol/kgH2O), Na+-dependent pHi recovery was faster than when they were exposed to the isosmotic solution. In cells exposed to the hyperosmotic mannitol solution, the final steady-state pHi values were similar to the initial steady-state pHi values, whereas in cells exposed to the hyperosmotic urea solution, the final steady-state pHi values were greater than the initial steady-state pHi values.



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Fig. 3.   Dose-response curves for inhibition of basolateral Na+/H+ exchange (NHE) activity of proximal tubule cells from WT and KO mice under isosmotic conditions by EIPA. NHE activity was assessed by Na+-dependent acid extrusion rates (JH) at a pHi of 6.30 on readdition of Na+ to the peritubular side after Na+ removal and an acid load with NH4Cl prepulse, in the presence of EIPA at various concentrations. Values are means ± SE of at least 3 tubules from WT and KO mice, expressed as the percentage of JH compared with those observed in the absence of EIPA (control).

Next, we examined whether the hyperosmotic mannitol solution affects Na+-dependent pHi recovery from an acid load in proximal tubule cells from WT (Fig. 1B) and KO (Fig. 2B) mice. Both groups of proximal tubule cells were first incubated in the Na+-containing HEPES-buffered solution (solution 1, Table 1) and then bathed in the Na+-free solution containing 20 mM NH4Cl (solution 3, Table 1) for 2 min. Afterward, NH4Cl was rapidly removed from the Na+-free HEPES-buffered solution, leading to a rapid decrease in pHi. Thereafter, both groups of cells were immediately exposed to the Na+-free HEPES-buffered solution (solution 2, Table 1) treated with 200 mM mannitol (500 mosmol/kgH2O). At this time, pHi values in both groups of cells were unchanged. Afterward, when external Na+ was readded in the presence of hyperosmotic mannitol, pHi recovery in cells from KO mice was substantially faster than in its absence, although pHi values for the final steady state (6.99 ± 0.03, n = 13) were not significantly different from those for the initial steady state (7.02 ± 0.02, n = 13) (Fig. 2B). At this time, Na+-dependent pHi recovery rates in the presence of hyperosmotic mannitol (132.5 ± 10.6 pH/s × 104, n = 13, P < 0.001) were significantly greater than in its absence (74.6 ± 5.0 pH/s × 104, n = 8). Furthermore, under hyperosmotic conditions, Na+-dependent pHi recovery was faster in KO mice than in WT mice (see Figs. 1B and 2B). At this time, Na+-dependent pHi recovery rates in KO mice (132.5 ± 10.6 pH/s × 104, n = 13, P < 0.001) were also significantly greater than those in WT mice (90.3 ± 7.7 pH/s × 104, n = 15). In sharp contrast to KO mice, in WT mice Na+-dependent pHi recovery and Na+-dependent pHi recovery rates under hyperosmotic conditions were similar to those under isosmotic conditions. Therefore, these findings were compatible with our previous report (25).

Next, we examined whether the hyperosmotic urea solution affects Na+-dependent pHi recovery from an acid load in proximal tubule cells from WT (Fig. 1C) and KO (Fig. 2C) mice. Both groups of cells were first incubated in the Na+-containing HEPES-buffered solution (solution 1, Table 1) and then bathed in the Na+-free solution containing 20 mM NH4Cl (solution 3, Table 1) for 2 min. Afterward, NH4Cl was rapidly removed from the Na+-free HEPES-buffered solution, leading to a rapid decrease in pHi. Thereafter, both groups of cells were immediately exposed to the Na+-free HEPES-buffered solution (solution 2, Table 1) treated with 200 mM urea (500 mosmol/kgH2O). At this time, pHi values in both groups of cells were unchanged. Afterward, when external Na+ was readded in the presence of hyperosmotic mannitol, pHi recovery in cells from WT mice was substantially faster than in its absence, and pHi values for the final steady state (7.13 ± 0.04, n = 8, P < 0.005) were also significantly greater than those for the initial steady state (6.97 ± 0.02, n = 8) (see Fig. 1C). At this time, Na+-dependent pHi recovery rates in the presence of hyperosmotic urea (125.2 ± 13.9 pH/s × 104, n = 8, P < 0.005) were also significantly greater than in its absence (81.8 ± 9.8 pH/s × 104, n = 10). Similarly, in cells from KO mice, Na+-dependent pHi recovery was substantially faster than in its absence, and pHi values for the final steady state (7.04 ± 0.02, P < 0.01, n = 7) were also significantly greater than those for the initial steady state (6.95 ± 0.02, n = 7) (see Fig. 2C). At this time, Na+-dependent pHi recovery rates in the presence of hyperosmotic urea (107.8 ± 12.2 pH/s × 104, n = 7, P < 0.05) were also significantly greater than in its absence (74.6 ± 5.0 pH/s × 104, n = 8). Furthermore, for the hyperosmotic urea solution, Na+-dependent pHi recovery rates in cells from KO mice (107.8 ± 12.2 pH/s × 104, n = 7) were not significantly different from those in cells from WT mice (125.2 ± 13.9 pH/s × 104, n = 8).

Next, we examined the effects on Na+-dependent pHi recovery of adding EIPA to both groups of cells treated with the hyperosmotic mannitol and urea solutions. Representative pHi recordings are shown in Fig. 4. When both groups of cells were exposed to EIPA (100 µM) alone, steady-state pHi values significantly decreased by ~0.05. In both groups of cells treated with the hyperosmotic mannitol or urea solutions, Na+-dependent pHi recovery from an acid load in the presence of EIPA was substantially slower than in its absence (see Figs. 1, B and C, and 2, B and C, and 3). When cells from WT mice were treated with the hyperosmotic mannitol or urea solutions, Na+-dependent pHi recovery rates in the presence of EIPA (mannitol: 33.0 ± 5.6 pH/s × 104, n = 6, P < 0.001; urea: 18.5 ± 2.4 pH/s × 104, n = 4, P < 0.001) were also significantly smaller than in its absence (mannitol: 90.3 ± 7.7 pH/s × 104, n = 15; urea: 125.2 ± 13.9 pH/s × 104, n = 8). Similar findings were observed in cells from KO mice that were exposed to the hyperosmotic mannitol or urea solutions. When cells from KO mice were exposed to the hyperosmotic mannitol or urea solutions, Na+-dependent pHi recovery rates in the presence of EIPA (mannitol: 25.2 ± 7.4 pH/s × 104, n = 7, P < 0.001; urea: 15.9 ± 0.4 pH/s × 104, n = 4, P < 0.001) were also significantly smaller than in its absence (mannitol: 132.5 ± 10.6 pH/s × 104, n = 13; urea: 107.8 ± 12.1 pH/s × 104, n = 7).


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Fig. 4.   Na+-dependent pHi recovery from an acid load when proximal tubule cells from WT (A and C) mice and KO (B and D) mice were treated with hyperosmotic mannitol or urea in the presence of peritubular EIPA. It should be noted that in both groups of cells treated with the hyperosmotic mannitol or urea solutions, EIPA (100 µM) alone decreased pHi, indicating that in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, basolateral NHE in proximal tubule cells from both WT and KO mice must be active in the normal steady-state pHi to balance a substantial rate of intracellular acid loading. In both groups of cells exposed to the hyperosmotic mannitol or urea solutions (500 mosmol/kgH2O), Na+-dependent pHi recovery in the presence of EIPA was substantially slower than in its absence.

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


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Fig. 5.   pHi dependence of JH when proximal tubule cells from WT (A) and KO (B) mice were exposed to an isosmotic solution, hyperosmotic mannitol solution, and hyperosmotic urea solution. The plots were computed from experiments such as those illustrated in Figs. 1 and 2. Values are means ± SE. Isosmo., isosmolality. *P < 0.05 compared with cells exposed to the isosmotic solution, at comparable pHi values. dagger P < 0.05 compared with cells from KO mice exposed to the hyperosmotic mannitol solution, at comparable pHi values.

Next, we examined whether, in cells from WT mice, changes in Km for peritubular Na+ concentrations and/or Vmax are involved in basolateral NHE activation induced by the hyperosmotic urea solution. For this purpose, the kinetics of basolateral NHE were determined by measuring JH at a pHi of 6.30 on readdition of varying concentrations of Na+ (0, 7, 14, 43, 100, and 142 mM) to the peritubular side of the tubules from the WT mice after peritubular Na+ removal and an acid load with NH4Cl prepulse in the absence and presence of hyperosmotic urea. Results are shown in Fig. 6A. As shown in Fig. 6B, a Lineweaver-Burk plot of the data obtained showed that under isosmotic conditions, apparent Km values for peritubular Na+ concentrations were 22.9 mM with a Vmax of 346.8 µM/s. Under hyperosmotic conditions, the apparent Km for peritubular Na+ was similar (21.6 mM), but Vmax increased to 480.8 µM/s.


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Fig. 6.   Kinetics of basolateral NHE when proximal tubule cells from WT mice were exposed to isosmotic solution and hyperosmotic urea (Urea) solution. A: after removal of peritubular Na+ and an acid load with NH4Cl prepulse, varying concentrations of Na+ (0, 7, 14, 43, 100, and 142 mM) were added to the peritubular side of the tubules from WT mice, and JH at a pHi of 6.30 were estimated. Each point represents the mean ± SE of at least 4 determinations. B: a Lineweaver-Burk analysis of the data indicates that exposure of cells from WT mice to the hyperosmotic urea solution had no effect on the apparent Km values for the peritubular Na+ (from 22.9 to 21.6 mM) but increased Vmax from 346.8 to 480.8 µM/s.

Next, we examined whether hyperosmotic urea dose dependently activates basolateral NHE in cells from WT mice. For this purpose, we added the Na+-free HEPES-buffered solution (solution 2, Table 1) containing 0, 100, 200, or 400 mM urea to cells from WT mice and then observed the Na+-dependent pHi recovery. JH values at a pHi of 6.30 when cells were treated with different concentrations of urea are summarized in Fig. 7. Urea at 100 mM (400 mosmol/kgH2O) had no effect on JH. On the other hand, urea at 200 (500 mosmol/kgH2O) and 400 mM (700 mosmol/kgH2O) significantly increased JH by a similar magnitude.


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Fig. 7.   JH at a pHi of 6.30 when proximal tubule cells from WT mice were exposed to different concentrations of urea. Values are means ± SE. The no. of tubules examined is in parentheses. NS, not significant.

Next, we examined whether the structural analogs of urea mimic the stimulatory effect of hyperosmotic urea on basolateral NHE in cells from WT mice. The urea analogs tested were methylurea, thiourea, and acetamide. For this purpose, we added the Na+-free HEPES-buffered solution (solution 2, Table 1) involving 200 mM methylurea, thiourea, and acetamide (500 mosmol/kgH2O) to cells from WT mice and then observed the Na+-dependent pHi recovery. JH values at a pHi of 6.30 when cells from WT mice were exposed to hyperosmotic urea, methylurea, thiourea, or acetamide are summarized in Fig. 8. In the cells treated with hyperosmotic methylurea, JH values were 563.0 ± 61.5 µM/s (n = 4), a value significantly greater than those in cells treated with the isosmotic solution (336.8 ± 30.2 µM/s, n = 8, P < 0.05) but not significantly different from those in cells treated with hyperosmotic urea (525.2 ± 42.0 µM/s, n = 7). On the contrary, thiourea and acetamide were without effect.


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Fig. 8.   JH at a pHi of 6.30 when proximal tubule cells from WT mice were exposed to an isosmotic solution (300 mosmol/kgH2O), hyperosmotic urea, methylurea, thiourea, acetamide, and glycerol (200 mM; 500 mosmol/kgH2O). Values are means ± SE. The no. of tubules examined is in parentheses.

Next, we examined whether another membrane-permeant solute, glycerol, mimics the stimulatory effect of hyperosmotic urea on basolateral NHE in cells from WT mice. For this purpose, we added the Na+-free HEPES-buffered solution (solution 2, Table 1), including 200 mM glycerol (500 mosmol/kgH2O), to cells from WT mice and then observed the Na+-dependent pHi recovery. JH values at a pHi of 6.30 when cells from WT mice were exposed to hyperosmotic glycerol are also summarized in Fig. 8. Hyperosmotic glycerol had no effect on JH.

Role of PKC in hyperosmotic mannitol- and urea-induced NHE activation. Previously, we have demonstrated that exposure to the hyperosmotic mannitol solution actually activated PKC in proximal tubules from KO mice but not in those from WT mice, whereas exposure of proximal tubules from WT mice to the hyperosmotic mannitol solution containing PMA (the PKC activator) activated PKC (27). Recently, we observed that in the cells from KO mice, PKC inhibitors (staurosporine and calphostin C) inhibited hyperosmotic mannitol-induced NHE activation, whereas PMA under isosmotic conditions mimicked the stimulatory effect of hyperosmotic mannitol on basolateral NHE (25). On the basis of this observation, we concluded that a hyperosmotic mannitol solution activates basolateral NHE via PKC in tubules from KO mice but not in those from WT mice (25). Therefore, we examined whether PKC is also involved in hyperosmotic urea-induced NHE activation. For this purpose, we added the hyperosmotic mannitol or urea solutions to both groups of the cells in the presence of staurosporine (100 or 500 nM) and then observed the Na+-dependent pHi recovery. JH values at a pHi of 6.30 when cells from WT and KO mice were exposed to the hyperosmotic mannitol or urea solutions (500 mosmol/kgH2O) in the absence and presence of staurosporine are summarized in Fig. 9. When cells from KO mice were treated with the hyperosmotic mannitol solution plus staurosporine (100 nM), the JH were 376.6 ± 44.1 µM/s (n = 10), which were significantly smaller than those in cells treated with the hyperosmotic mannitol solution alone (490.4 ± 25.1 µM/s, n = 12) but were not significantly different from those in cells treated with the isosmotic solution (336.8 ± 30.2 µM/s, n = 8). These findings were in good accord with our previous report (25). When the cells of the WT mice were exposed to the hyperosmotic urea solution plus staurosporine (100 or 500 nM), JH values at a pHi of 6.30 were not significantly changed. When the cells of the KO mice were exposed to the hyperosmotic urea solution plus staurosporine (500 nM), JH values at a pHi of 6.30 were not significantly influenced, either.


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Fig. 9.   JH at a pHi of 6.30 when proximal tubule cells from WT and KO mice were exposed to an isosmotic solution, hyperosmotic mannitol solution, hyperosmotic mannitol solution plus staurosporine, hyperosmotic urea solution, and hyperosmotic urea solution plus staurosporine. Values are means ± SE. Stauro, staurosporine. The no. of tubules examined is in parentheses.

Role of tyrosine kinase in hyperosmotic mannitol- and urea-induced NHE activation. Cohen et al. (10) reported that in a murine inner medullary collecting duct cell line, hyperosmotic urea induced transcription of the zinc finger-containing transcription factor Egr-1 and urea-induced Egr-1 transcription was sensitive to genistein (the tyrosine kinase inhibitor). Their findings suggest the possibility that tyrosine kinase may be involved in hyperosmotic urea-induced NHE activation. To demonstrate this possibility, we added our hyperosmotic mannitol or urea solutions in the presence of genistein to both groups of cells and then observed the Na+-dependent pHi recovery. JH values at a pHi of 6.30 when cells from WT and KO mice were exposed to the hyperosmotic mannitol or urea solutions (500 mosmol/kgH2O) in the absence and presence of genistein (10 µM) are summarized in Fig. 10. When cells from WT mice were treated with the hyperosmotic urea solution plus genistein, JH values were 411.4 ± 10.7 µM/s (n = 9). The values were significantly smaller than those in cells treated with the hyperosmotic urea solution alone (527.2 ± 39.9 µM/s, n = 13) but were not significantly different from those in the cells treated with the isosmotic solution (357.5 ± 41.3 µM/s, n = 10). Similarly, when cells from KO mice were treated with the hyperosmotic urea solution plus genistein, JH values were 354.4 ± 28.5 µM/s (n = 7), values that were significantly smaller than those in cells treated with the hyperosmotic urea solution alone (511.4 ± 38.9 µM/s, n = 8) but were not significantly different from those in cells treated with the isosmotic solution (336.8 ± 30.2 µM/s, n = 8). In sharp contrast, when cells from KO mice were exposed to the hyperosmotic mannitol solution plus genistein, JH values at a pHi of 6.30 were not significantly changed at all.


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Fig. 10.   JH at a pHi of 6.30 when proximal tubule cells fromWT and KO mice were exposed to an isosmotic solution, hyperosmotic mannitol solution, hyperosmotic mannitol solution plus genistein, hyperosmotic urea solution, and hyperosmotic urea solution plus genistein. Values are means ± SE. The no. of tubules examined is in parentheses.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our laboratory has previously reported that exposure of isolated nonperfused proximal tubules from WT mice to hyperosmotic mannitol did not elicit RVI after initial cell shrinkage (24). On the other hand, RVI was observed with hyperosmotic mannitol, when P-gp activity is acutely suppressed by the P-gp inhibitors (verapamil and cyclosporin A) or when both mdr1a and mdr1b genes are genetically knocked out (24). The P-gp-induced modulation of RVI during exposure to hyperosmotic mannitol occurs via PKC activation (27). Basolateral NHE partly contributes to the P-gp-induced modulation of RVI (24). Furthermore, we demonstrated that in the absence of P-gp activity, hyperosmotic mannitol actually activates basolateral NHE via PKC, but in the presence of P-gp activity it does not (25). In the present study, we extend our previous study to compare the effects of hyperosmotic urea on basolateral NHE with those of hyperosmotic mannitol in mouse proximal tubule cells.

Basolateral NHE activity in both groups of cells treated with isosmotic, hyperosmotic mannitol, and hyperosmotic urea solutions. As shown in Fig. 5, in cells from WT and KO mice under both isosmotic and hyperosmotic conditions, JH decreased through a NHE process as pHi increased from the acidic to the normal range. Under isosmotic conditions, JH values in cells from KO mice were not significantly different from those in cells from WT mice over the entire range of pHi studied (6.20-6.90), consistent with the notion that under these conditions, basolateral membranes of proximal tubule cells from KO mice have a NHE activity similar to that in cells from WT mice. However, in KO mice, both JH values at a pHi range of 6.20-6.45 and maximal JH values were significantly greater in cells exposed to the hyperosmotic mannitol solution than in those exposed to the isosmotic solution. In KO mice, hyperosmotic mannitol shifted JH vs. pHi by ~0.15 pH units in the alkaline direction at the low pHi values. In sharp contrast to KO mice, when cells from WT mice were treated with the hyperosmotic mannitol solution and the isosmotic solution, JH values over the entire range of pHi examined (6.20-6.90) were not significantly different. In marked contrast to cells exposed to hyperosmotic mannitol, in WT and KO mice, both JH values over the entire range of pHi examined and maximal JH values were significantly greater in cells exposed to the hyperosmotic urea solution than in those exposed to the isosmotic solution. For the hyperosmotic urea solution, maximal JH values were not significantly different between the two groups. In both groups of cells, hyperosmotic urea shifted JH vs. pHi by 0.15-0.20 pH units in the alkaline direction for the wide range of pHi . In both groups of cells treated with the hyperosmotic mannitol or urea solutions, Na+-dependent pHi recovery was blocked by pretreatment with EIPA (see Fig. 4). Taken together, we conclude that in both groups of cells, hyperosmotic urea activates basolateral NHE by a similar magnitude, whereas only in cells from KO mice does hyperosmotic mannitol activate basolateral NHE. In other words, hyperosmotic urea-induced NHE activation is independent of P-gp, whereas hyperosmotic mannitol-induced NHE activation is dependent on P-gp. In contrast to the stimulatory effect of urea on NHE in the mouse proximal tubule, Leviel et al. (21) reported that in rat medullary thick ascending limb, hyperosmotic urea inhibits NHE activity.

In both groups of cells treated with hyperosmotic urea, final steady-state pHi values after Na+-dependent pHi recovery were significantly greater than initial steady-state pHi values before the NH4Cl pulse. The exposure of both groups of cells to hyperosmotic urea caused an alkaline shift in pHi sensitivity of NHE at a wide pHi range. Similar to the effect of hyperosmotic urea, Miyata et al. (26) showed in cultured rat mesangial cells that hyperosmotic mannitol shifted JH vs. pHi by 0.15-0.3 pH units in the alkaline direction at a wide pHi range of 6.40-6.95. Similarly, Seo et al. (34) reported in perfused rat mandibular salivary gland that hyperosmotic sucrose shifted H+ flux via the NHE vs. pHi relationship in the alkaline direction at a pHi range of 7.05-7.45. Grinstein et al. (17) also studied the Na+-dependent component of pHi recovery from an acid load in thymic lymphocytes, finding that shrinkage shifts the flux vs. pHi relationship by 0.2-0.3 pH units in the alkaline direction over the entire range of pHi between ~6.2 and ~7.2. In addition, Shrode et al. (35) reported in C6 glioma cells treated with hyperosmotic mannitol that shrinkage caused an alkaline shift in pHi sensitivity of NHE at a wide range of pHi. Therefore, the stimulatory effect of hyperosmotic urea on NHE is common to many cell types. Because the pHi sensitivity of the exchange system appears to be largely determined by an allosteric modifier site located on the cytoplasmic surface of the membrane (2, 16), one of the mechanisms responsible for hyperosmotic urea-induced NHE activation in both groups of cells is a shift in pHi dependence of the antiport. In marked contrast to cells exposed to hyperosmotic urea, in cells from KO mice that were exposed to hyperosmotic mannitol, final steady-state pHi values after Na+-dependent pHi recovery were similar to initial steady-state pHi values before the NH4Cl pulse, and the alkaline shift in pHi sensitivity of NHE occurred only at the low pHi range. Therefore, this phenomenon is specific to mannitol only in proximal tubule cells from KO mice, although the underlying mechanisms remain unknown.

In many cell types, NHE, in addition to its important role in pHi homeostasis, contributes to RVI after a hyperosmotic stress such as mannitol (17, 19, 23, 26, 33, 35). In fact, we previously observed that only in proximal tubules from KO mice did hyperosmotic mannitol elicit RVI, whereas hyperosmotic mannitol-induced RVI was abolished by pretreatment with EIPA (24). On the other hand, we previously reported that in both groups of tubules exposed to hyperosmotic urea, the reduction in cell volume was smaller compared with hyperosmotic mannitol and transient, and washout of urea caused a large transient overshoot. This indicates that the volume change during the presence of urea is the result of simple diffusion but not RVI. Therefore, the effect of urea on basolateral NHE is not associated with RVI. We further examined whether another membrane-permeant solute, glycerol, activates basolateral NHE like urea, but we failed to demonstrate it. Of the structural analogs of urea, thiourea or acetamide had no effect on NHE, although methylurea activates NHE by a similar magnitude to urea. Accordingly, the stimulatory effect on basolateral NHE is relatively specific to urea. Similarly, Cohen and Gullans (9) observed that in Madin-Darby canine kidney (MDCK) cells, hyperosmotic urea increased [3H]thymidine incorporation, but hyperosmotic glycerol, acetamide, thiourea, or methylurea did not, and concluded that hyperosmotic urea selectively induced DNA synthesis in the cells.

Under isosmotic conditions, the apparent Km (22.9 mM) for peritubular Na+ of NHE and Vmax (346.8 µM/s) in basolateral membranes of proximal tubule cells from WT mice were similar to those reported in basolateral membranes of proximal tubule cells from KO mice (21.1 mM and 370.6 µM/s, respectively) (25). In the present study, we found that the exposure of cells from WT mice to hyperosmotic urea increased Vmax from 346.8 to 480.8 µM/s without changing the Km for peritubular Na+, indicating that in cells exposed to hyperosmotic urea, the affinity for peritubular Na+ remained the same, but the number of NHEs increased. This is also one of the mechanisms for hyperosmotic urea-induced NHE activation. Similar results were observed in cells from KO mice that were exposed to the hyperosmotic mannitol (25).

Functional and pharmacological properties of basolateral NHE. Amiloride and its analogs, including EIPA, have been widely used for pharmacological characterization of distinct NHE isoforms (7, 28). In the present experiments, IC50 values of basolateral NHE in proximal tubules from WT and KO mice suggested that basolateral NHE in both groups of proximal tubules is a highly amiloride-resistant type (NHE3 or NHE4) (7, 28). Furthermore, it should be noted that dose-response curves with EIPA are consistent with the presence of only one form of NHE. Although other NHE isoforms might be present at the basolateral membrane, the present results are compatible with the inhibition of a single population of NHE. These findings are unique because most epithelial cells, including proximal tubule cells, express NHE1 at the basolateral membrane (3, 28); however, our results clearly do not support the existence of a highly-sensitive NHE isoform at the basolateral membrane of both groups of proximal tubules.

Further studies concerning the effects of hyperosmolality on NHE activity were performed in an effort to discriminate between the drug-resistant isoforms, NHE3 and NHE4. Hyperosmolality-induced cell shrinkage has been shown to stimulate NHE4 activity (4), whereas it inhibits NHE3 activity (35). In our experiments, hyperosmotic mannitol activated basolateral NHE only in proximal tubules from KO mice, whereas hyperosmotic urea activated basolateral NHE in both groups of tubules. Previously, we reported that hyperosmotic mannitol and urea elicited cell shrinkage, although the response of cell volume to hyperosmotic urea was markedly different from that to hyperosmotic mannitol (24).

One might argue that in hyperosmotic medium, ubiquitously expressed NHE1 may become activated and contribute to basolateral NHE activity, because this isoform has also been shown to be activated by hyperosmolality (4, 35). However, under hyperosmotic conditions, addition of EIPA at a concentration that should completely inhibit NHE1 activity, resulted in a very slight inhibition of basolateral NHE activity. Considering the very low IC50 value (0.02 µM) (28) of NHE1 for EIPA, we conclude that NHE1 does not contribute to basolateral NHE activity under either isosmotic or hyperosmotic conditions. Also, if we compare EIPA data under hyperosmotic conditions to those in Fig. 3, the dose-response profile for inhibition of basolateral NHE activity by EIPA is exactly the same under isosmotic and hyperosmotic conditions. These functional properties strongly suggest that drug-resistant NHE4 is the isoform responsible for basolateral NHE activity in both groups of proximal tubules. This is also supported by the report of Chambrey et al. (6), in which the basolateral membrane of the rat proximal tubule was labeled with anti-NHE4 antibody.

Role of PKC and tyrosine kinase in hyperosmotic mannitol- and urea-induced NHE activation. In MDCK cells, hyperosmolality induced by NaCl or raffinose has been shown to enhance inositol 1,4,5-triphosphate levels and thereby activate PMA-sensitive PKC (37). In Ehrlich mouse ascites tumor cells, PKC is involved in activation of the Na+/K+/2Cl- cotransporter induced by hyperosmolality (20). Treatment of NIH/3T3 cells with hyperosmotic NaCl solution has also been reported to trigger phospholipase C activation, then induce an increase in diacylglycerol levels, and as a consequence, PKC activation (41). Previously, we reported that in KO mice, the effects of hyperosmotic mannitol on basolateral NHE (25) and RVI (27) indeed occur via PKC activation. In the present study, we confirmed that in KO mice, staurosporine (the PKC inhibitor) at 100 nM eliminated the hyperosmotic mannitol-induced NHE activation (see Fig. 9). Cohen et al. (8) reported that hyperosmotic urea upregulated expression at the mRNA level of two growth-associated immediate-early genes, Egr-1 and c-fos, in a renal epithelial cell-specific fashion. They also demonstrated that PKC is implicated in urea-induced Egr-1 transcription, because both the PKC inhibitors (staurosporine and calphostin C) and downregulation of PKC with chronic exposure to O-tetradecanoylphorbol 13-acetate inhibited the ability of urea to activate transcription of Egr-1 (10). Therefore, we expected that staurosporine might block hyperosmotic urea-induced NHE activation. However, staurosporine at 100 and 500 nM failed to do so in both WT and KO mice. Taken together with our previous and present findings, hyperosmotic mannitol-induced NHE activation is PKC dependent only in KO mice, whereas hyperosmotic urea-induced NHE activation is PKC independent in both WT and KO mice.

Finally, we examined the intracellular signaling mechanisms responsible for hyperosmotic urea-induced NHE activation. The result of the present study showed that tyrosine kinase is indeed involved in hyperosmotic urea-induced NHE activation in both groups of proximal tubule cells. Evidence supporting the conclusions of this study was obtained primarily from experiments examining the effects of the tyrosine kinase inhibitor genistein. Several findings support the view that genistein influenced basolateral NHE activity via its action on tyrosine kinase activity. At the concentration studied (10 µM), genistein is a selective tyrosine kinase inhibitor, with no significant activity against a variety of other protein kinases or phosphatases (1). The apparent specificity of this agent was supported in the present study by the observation that genistein, at the same concentration that completely abolished hyperosmotic urea-induced NHE activation in both groups of proximal tubule cells, had no effect on hyperosmotic mannitol-induced NHE activation in proximal tubule cells from KO mice. Also, these findings were not the results of a toxic or nonspecific metabolic effect on proximal tubule cells. Taken together, these results support the notion that genistein prevents hyperosmotic urea-induced NHE activation via its targeted action to inhibit tyrosine kinase activity.

Although our results suggest an important role for tyrosine phosphorylation in hyperosmotic urea-induced NHE activation, they do not establish the nature of the link between tyrosine kinase and hyperosmotic urea. An increase in tyrosine phosphorylation in response to hyperosmotic urea could result from stimulation of tyrosine kinase activity, inhibition of protein tyrosine phosphatase activity, or both. Pewitt et al. (30) have reported that exposure of duck red blood cells to hyperosmotic conditions stimulates phosphorylation of the bumetanide-sensitive Na+-K+-2Cl- cotransporter itself or a regulatory protein by activation of both cAMP-dependent and -independent kinases, thereby activating the cotransporter. Bianchini et al. (3) have shown in lymphocytes that okadaic acid (the phosphatase inhibitor) induces activation and phosphorylation of NHE protein. Future studies will be required to clarify whether hyperosmotic urea actually stimulates tyrosine kinase activity and how the tyrosine kinase induced by hyperosmotic urea increases basolateral NHE activity.

As shown in Figs. 9 and 10, in proximal tubule cells from KO mice, staurosporin suppressed hyperosmotic mannitol-induced NHE activation, whereas genistein did not. In marked contrast, in both groups of proximal tubule cells, genistein inhibited hyperosmotic urea-induced NHE activation, whereas staurosporin did not. Therefore, a genistein-sensitive tyrosine kinase does not appear to be involved in hypersomotic mannitol-induced NHE activation. On the basis of the use of pharmacological agents, we conclude that hyperosmotic mannitol-induced NHE activation occurs via a PKC-dependent pathway that does not involve tyrosine kinase, whereas hyperosmotic urea-induced NHE activation occurs via a tyrosine kinase-dependent pathway that functions independently of PKC. Therefore, hyperosmotic mannitol and urea differentially activate basolateral NHE in the mouse proximal tubule.


    ACKNOWLEDGEMENTS

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


    FOOTNOTES

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

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

10.1152/ajprenal.00025.2002

Received 18 January 2002; accepted in final form 17 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 283(4):F771-F783
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society




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