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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Inhibition of Rho-Kinase Reduces Renal Na-H Exchanger Activity and Causes Natriuresis in Rat

From Departments of Clinical Pharmacology (K.N., S.T., A.K., K.S., A.F.) and Pharmacology (M.S., M.I.), Jichi Medical School, Minamikawachi, Tochigi, Japan; and Department of Pediatrics (G.J.S.), University of Rochester School of Medicine and Dentistry, Rochester, New York

Received July 18, 2002; accepted October 22, 2002.

	Abstract

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Rho-kinase regulates the actin cytoskeleton and therefore modulates transport. The role of Rho-kinase in Na-H exchanger (NHE) activity of rat proximal convoluted tubules (PCTs) was investigated using (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), a specific inhibitor of Rho-kinase. In spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats, apical and basolateral NHE activities were determined by measuring cell pH recovery following luminal prepulse and basolateral sodium removal, respectively. Apical NHE activity was greater in 8 to 9 week old hypertensive SHR compared with WKY. Although Y-27632 suppressed pH_i recovery in both strains, sensitivity was 50-fold higher in adult SHR. Y-27632 suppressed basolateral NHE in both strains with similar sensitivity. Apical NHE activity was not greater in 5-week-old, not yet hypertensive, SHR rats compared with WKY. In clearance studies, Na excretion was less in SHR than in WKY rats. Y-27632 increased Na excretion and fractional excretion Na in both strains but more so in SHR. ²²Na uptake of the brush border membrane vesicle taken from Y-27632-treated rats decreased more than that from vehicle-treated animals in both adult SHR and WKY. We conclude that apical NHE activity is increased in SHR PCT compared with controls and that inhibition of Rho-kinase reduces PCT NHE activities and causes natriuresis.

The state of the cytoskeleton situated beneath the cell membrane can modulate activities of sodium transporters located on the membrane (Mills et al., 1994; Hamm-Alvarez and Sheetz, 1998). Shrinkage of the cell increases NHE activity in astrocytes by modulating myosin light chain kinase (Shrode et al., 1995). The state of actin organization modulates trafficking of NHE3 protein and thereby changes its activity in renal proximal tubular cells (Chalumeau et al., 2001). Furthermore, Kurashima et al. (1999) reported using CHO cells transfected with NHE3 and that the NHE3 requires an intact actin cytoskeleton to maintain its usual function.

Small GTP-binding proteins of Rho family (Rho, Rac, and Cdc40) are among the regulators of the actin cytoskeleton (Hall, 1998). Systemic inhibition of p160ROCK (a Rho-associated coil-forming protein kinase) (Hirata et al., 1992) by administration of a selective inhibitor, Y-27632, lowers blood pressure by the disruption of the cytoskeleton and inhibition of NHE1 activity in vascular smooth muscle cells, leading to the reduction in arterial resistance (Uehata et al., 1997). We have recently reported in neutrophils that Y-27632 inhibited superoxide anion production (Kawaguchi et al., 2000). Furthermore, myosin light chain phosphorylation by Rho-kinase is a prerequisite for normal NHE3 function in transfected CHO cells (Szaszi et al., 2000). Thus, it is possible that NHE may play a significant role in modulating blood pressure via Rho-kinase mediated mechanisms. It remains to be determined, however, whether the Rho-kinase actually affects intact NHE3 in native renal tubular cells and subsequent Na/fluid balance in the body.

The purpose of this study was as follows: 1) to examine the contribution of p160ROCK to maintaining NHE3 and NHE1 activities in rat isolated perfused proximal convoluted tubule cells in vitro by using a selective inhibitor Y-27632; 2) to compare this contribution in young (not yet hypertensive) (Yamori, 1974) and adult (hypertensive) rats; 3) to determine the effect of Y-27632 on urine volume and sodium excretion by an in vivo clearance study; and finally 4) to compare the roles of inhibiting p160ROCK in SHR versus control animals.

	Materials and Methods

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Animals. Male Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR/Izm) were purchased from Funabashi No-en (Funabashi, Chiba, Japan). Systolic arterial blood pressure (BP) was measured by a tail-cuff method. For microperfusion, we selected five (young; developing phase of hypertension) (Yamori, 1974) and eight- to nine-week-old rats (adult; established phase of hypertension). The clearance study was performed using 8- to 9-week-old animals because of technical difficulties of cannulation to femoral veins in young rats. The number of animals was 10 and 20 in young and adult WKY rats, respectively, and 9 and 22 in young and adult SHR, respectively. All animals were allowed free access to standard rat chow (CE-2; Japan Clea Co. Ltd., Tokyo, Japan) and tap distilled water until the initiation of the experiments. This study was conducted in accordance with the Guideline for Animal Research at Jichi Medical School.

In Vitro Microperfusion. In vitro perfusion of isolated proximal convoluted (S1) tubule was performed in accordance with Burg et al. (1966), as modified in our laboratory (Tsuruoka et al., 2000, 2001). In brief, animals were anesthetized with intraperitoneal injection of pentobarbital sodium. The left kidney was removed and coronal slices were made. The slices were transferred to a chilled solution of the following composition: 14 mM KH₂PO₄, 44 mM K₂HPO₄, 15 mM KCl, 9 mM NaHCO₃, and 160 mM sucrose with pH 7.4. S1 segments with length ranging from 0.25 to 0.35 mm were dissected from the superficial labyrinth by fine forceps under stereomicroscope at 4°C. We dissected and used three to eight proximal tubules from each animal. Isolated tubules were transferred to a perfusion chamber mounted on an inverted microscope and perfused in vitro at 37°C. Tubules were hooked up to the holding pipette, and a single-barreled perfusion pipette was inserted into the tubular lumen. Triple-barreled polyethylene tubing was inserted into the perfusion pipette to allow rapid exchange of the perfusion fluid. The perfusion rate was controlled at 10 to 20 nl/min by adjusting the height of the fluid reservoir, which was connected to the back-end of the perfusion pipette. A flow-through bath system was used to allow rapid exchange of bathing fluid. The bath fluid was maintained at 37°C. The flow rate of bath fluid was 3 to 5 ml/min, which allowed the bath fluid to be exchanged within 2 s. The composition of the bicarbonate-free solution for perfusing and bathing solutions in this study was as follows: 135 mM NaCl, 5 mM KCl, 1.6 mM Na₂HPO₄, 0.4 mM NaH₂PO₄, 1.8 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 5.6 mM glucose, and 5 mM L-alanine at pH 7.4 and 293 ± 3 mOsm/kg. For the prepulse solution, 40 mM NaCl of the bicarbonate-free solution was replaced by NH₄Cl.

Measurement of Intracellular pH. Acetoxymethyl ester of 2',7'-bis(carboxyethyl) carboxyfluorescein (BCECF-AM) was used as an indicator of intracellular pH (pH_i). BCECF-AM was added to the bicarbonate-free solution from a stock solution (1 mM in dimethyl sulfoxide) to a final concentration of 1 µM. After the microperfused tubule was stabilized for 15 min, BCECF-AM was added to the bathing solution, and the tubules were incubated for about 20 min. Then, BCECF was removed from the bathing solution, and the experiments were started.

Measurement of intracellular pH was performed by a fluorescence ratio imaging technique using an Olympus OSP system (Olympus Optical, Tokyo, Japan), as described previously (Tsuruoka et al., 1993, 2001). Measurements were done at 400x magnification; the diameter of the beam of light focused on the tubule was 25 µm. BCECF fluorescence was measured at an emission wavelength of 530 nm in response to excitation wavelengths of 490 and 440 nm. Autofluorescence-corrected fluorescence ratio values (490/440 nm) were calculated by the apparatus every 1 s. We used only the tubules showing at least 20-fold greater intensity than that of background. To minimize photobleaching and cell damage, the duration of each experiment was made as short as possible. BCECF fluorescence was calibrated using the nigericin/high K method, and fluorescence ratios were converted to pH units, as described previously (Thomas et al., 1979).

Measurement of Apical and Basolateral NHE Activities. Apical NHE activity of the cell was assessed by the H-ATPase-independent pH_i recovery rate (dpH_i/dt) after intracellular acid loading in basal bicarbonate-free solution under the luminal presence of 5 nM bafilomycin A1, a specific inhibitor of the vacuolar H-ATPase (Dagher and Sauterey, 1992; Tsuruoka and Schwartz, 1997). The acid load was accomplished by the prepulse; after 5 min of exposure to 40 mM NH₄Cl, the ammonium was rapidly removed. The initial rate of recovery (dpH_i/dt) at around pH_i 7.0 was used for the evaluation (Fig. 1). To calculate the initial dpH_i/dt, a regression line was obtained using StatView software (SAS Institute, Inc., Cary NC). We monitored dpH_i/dt for more than 7 min after the pulse to determine the initial slope. From the increase in pH_i over 50 s, the initial slope was calculated. It took approximately 45 min from initiation of the perfusion to termination of the pH_i monitoring.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Representative tracings of cell pH (pHi) following ammonium prepulse (40 mM NH4Cl to lumen) and effect of Y-27632 (10–7 M) in rat proximal tubule cell. Apical NHE activity was estimated by slope of pHi change (dpHi/dt; dashed line) just after removal of ammonium from tubular lumen. A and B, adult WKY; C and D; adult SHR.

Preliminary studies confirmed that the bafilomycin A1-insensitive pH_i recovery was completely abolished by concomitant addition of amiloride (1 mM) to lumen, which was previously used for the evaluation of the activity (Hayashi et al., 1997). Thus, the bafilomycin A1-insensitive pH_i recovery was comparable to the amiloride-inhibitable pH_i recovery after the prepulse. Vehicle (water) or a concentration of Y-27632 was added with bafilomycin A1 10 min before the initiation of the prepulse.

Basolateral NHE activity was determined by the change of pH_i following the removal of Na from bathing solution (Geibel et al., 1989). The pH_i stabilized within 3 min after this maneuver. We have previously confirmed that this change of pH_i was completely abolished by concomitant addition of amiloride (10 µM) to the bath (Tsuruoka et al., 1993). During these experiments we perfused with a Na-free solution for the time that Na was removed from the basolateral solution (Hayashi et al., 1997).

In Vivo Clearance Study. The in vivo clearance study was performed as reported by Kawaguchi et al. (2001) from our laboratory. In brief, the animals were anesthetized using Inactin (BYK Gulden, Konstanz, Germany; 100 mg/kg, i.p.) and a tracheostomy performed. The femoral artery was cannulated with PE-10 tubing, which was connected to PE-50 tubing for blood sampling and blood pressure monitoring under anesthesia. The femoral vein was cannulated for infusion of medications and agents. Thereafter, bladder puncture was performed by insertion of PE-50 tubing after urethral ligation; animals with gross hematuria were discarded. Inulin (0.5% in saline) was continuously infused at 55 µl/min. After an equilibration period of 90 min, urine was collected during two successive 60-min periods (from 90 to 150 min and 210 to 270 min). The first period was considered the control period and the second period as the experimental one. This latter period began just after the first urinary collection (150 min) with a continuous infusion of Y-27632 (0.8 mg/kg/h; generous gift from Yoshitomi Pharmaceutical Co. Ltd., Osaka, Japan) or vehicle. The infusion continued until the end of the study (270 min). In preliminary studies, we found that this infusion rate of Y-27632 was the maximum dose that could be given without a reduction in systemic blood pressure. We carefully monitored mean arterial blood pressure during the study, and an experiment was discarded when the mean blood pressure decreased more than 5% of the basal level. Blood sampling (200 µl) was performed at the midpoint of each urine collection period; an equal volume of saline was injected to offset the loss of blood. Concentrations of inulin, Na, K, and creatinine were measured from both urine and serum specimens. Inulin concentration was determined by modified anthrone method and concentrations of Na and K were measured by an Auto-Analyzer. Data for urinary sodium excretion (milliequivalent per hour per 100 g b.wt.), fractional excretion of sodium and potassium (FE Na and FE K, in percentages), and GFR (milliliter per minute per 100 g b.wt.) were generated from standard calculations.

²²Na Uptake Study with Brush-Border Membrane Vesicle. Brush-border membrane vesicle (BBMV) was prepared from the renal cortex of adult WKY and SHR. ²²Na uptake studies were performed according to Morduchowicz et al. (1989). In brief, purified BBMV were prepared by Mg²⁺ precipitation method from SHR and WKY treated with Y-27632 or vehicle in vivo. The way of treatment was similar with in vivo clearance study mentioned above. Transport study using ²²Na was performed at room temperature by a Millipore rapid-filtration procedure. ²²Na uptake was measured in the presence of an outward H⁺ gradient induced by preincubation of medium (273 mM mannitol, 10 mM MgSO₄, 9 mM Tris, 14 mM HEPES, and 30 mM MES, pH 6.0). Uptake was initiated by incubation of vesicles with medium (1 mM ²²NaCl, 286 mM mannitol, 2 mM MgSO₄, 13 mM Tris, 15 mM HEPES, 6 mM MES, pH 7.5) and was stopped on ice-cold isosmotic solution. The uptake was increased with time until 30 s. To evaluate initial uptake rate, we measured the uptake at 5 and 15 s after initiation of the incubation. Protein concentration was measured by Coomassie brilliant blue with bovine serum albumin as the reference protein. The initial ²²Na uptake rate was expressed in moles per milligram protein per 5 s (Morduchowicz et al., 1989).

Statistical Analysis. Data are expressed as the mean ± S.E. Statistical analysis was performed by two-way analysis of variance and repeated measures approach when necessary. Significance was asserted when P < 0.05.

	Results

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Mean Blood Pressure Is Higher in SHR than in WKY Rats. Systolic BP was 112 ± 4 and 110 ± 5 mm Hg in young (n = 10) and adult (n = 20), WKY rats, respectively, and 138 ± 10 and 158 ± 6 mm Hg in young (n = 9) and adult (n = 22) SHR, respectively. The systolic BP values in the SHR rats were significantly higher than those in the WKY rats at both age groups (P < 0.05). The BP in young SHR was significantly lower than adult SHR.

Apical NHE Activity Is Reduced by the Inhibition of Rho-Kinase Activity. Figure 1A shows a representative tracing of pH_i recovery before and after the prepulse, during which bafilomycin A1 was present in the perfusate, in proximal convoluted tubule cells of adult WKY rats. The pH_i under basal conditions was 7.33 ± 0.03 pH units (n = 82 cells). After the prepulse, the pH_i decreased to 7.0 and rapidly recovered. The dpH_i/dt was 60 ± 8 x 10^–2 pH units/s (n = 8; Fig. 2A, vehicle). Treatment with 10^–7 M Y-27632 significantly reduced dpH_i/dt recovery from the acid load (Fig. 1B), and this was evident in a concentration-dependent manner (Fig. 2A). The mean value at 1 x 10^–6 M was 10 ± 8 x 10^–2 pH units/s (n = 7; Fig. 2A). Figure 2B shows a 75% reduction of apical Na-H activity at 10^–7 M.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effect of Y-27632 on apical Na-H activity in SHR and WKY. A, change of dpHi/dt after apical NH4 prepulse in adult (established hypertension) SHR and WKY; B, change of dpHi/dt after apical NH4 prepulse in young (developing phase of hypertension) SHR and WKY; C, percent reduction of apical NHE activity compared with vehicle in adult and young SHR and WKY.

In proximal convoluted tubule cells from SHR, the pH_i recovery was significantly greater than that in the age-matched WKY (a representative tracing is shown in Fig. 1C). The mean value for dpH_i/dt was 95 ± 15 x 10^–2 pH units/s (n = 70). Y-27632 also significantly suppressed the dpH_i/dt (Fig. 1D) in a concentration-dependent manner in SHR (Fig. 2A), and the sensitivity to the drug as determined by the 50% reduction (percent reduction) was about 50-fold greater than that in WKY (Fig. 2B).

We evaluated pH_i recovery in proximal tubule cells of young SHR and WKY rats. Basal dpH_i/dt was 59 ± 10 x 10^–2 and 42 ± 10 x 10^–2 pH units/s in SHR and WKY, respectively, and there was no significant difference between the two groups (P = 0.08, n = 60; Fig. 2C). Treatment with Y-27638 significantly reduced pH_i recovery in a concentration-dependent manner in both groups (Fig. 2C). The sensitivity to the drug in young SHR was similar to that in young WKY rats (Fig. 2C).

Basolateral NHE Activity Is Suppressed by the Inhibition of Rho-Kinase Activity. Removal of Na from bath-induced pH_i recovery to the same extent in proximal tubules from adult WKY rats (0.34 ± 0.05 pH units, n = 21) and SHR (0.36 ± 0.06 pH units, n = 22). Pretreatment with Y-27632 significantly suppressed the change of pH_i in a concentration-dependent manner (Fig. 3). The sensitivity of the two groups did not differ significantly. In young animals, the recovery of pH_i after Na removal and the sensitivity to Y-27632 were similar to those of adult rats (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Effect of Y-27632 on basolateral Na-H activity in adult and young SHR and WKY.

Y-27632 Increases Sodium Excretion and Urinary Volume at a Subdepressor Dose. In WKY rats, the systemic infusion of Y-27632 at a subdepressor dose significantly increased urinary volume, urinary Na excretion, and FE Na, whereas GFR and FE K were not influenced (Table 1). Vehicle alone did not change any parameters. In SHR, urine volume at basal condition was not different from that in WKY. Basal FE Na in SHR was significantly lower than that of WKY rat (0.26 ± 0.06 versus 0.40 ± 0.05%, P = 0.04), but total Na excretion during control period was not significantly lower than that in WKY rat (0.034 ± 0.009 versus 0.052 ± 0.007 mEq/h/100 g b.wt., P = 0.08). After the addition of Y-27632, urinary volume, Na excretion, and FE Na significantly increased in both groups of rats (Table 1). The changes of Na excretion and FE Na were greater in SHR, whereas the change of urinary volume of the two groups did not differ significantly. There were no changes in FE K induced by Y-27632. Mean arterial pressure during the experiments is listed in Table 1. Although mean arterial pressure in SHR was significantly higher than that in age-matched WKY rats, it was not changed by Y-27632 treatment in either group.

²²Na Uptake of BBMV Was Decreased by Treatment of Y-27632. ²²Na uptake of BBMV treated with vehicle was significantly higher in adult SHR than age-matched WKY (0.72 ± 0.24 and 1.37 ± 0.30 nmol/mg/5 s, WKY and SHR, respectively; n = 4 each, P < 0.05). In BBMV treated with Y-27632 in vivo, the uptake was significantly (P < 0.05) lower than that treated with vehicle (0.46 ± 0.19 and 0.59 ± 0.27 nmol/mg/5 s, WKY and SHR, respectively; n = 4 in each). In young animals, the uptake was not significantly different between SHR and WKY (0.60 ± 0.14 and 0.55 ± 0.18 nmol/mg/5 s, WKY and SHR, respectively; n = 4 in each). Y-27632 inhibited the uptake to a similar extent in both groups (0.43 ± 0.15 and 0.41 ± 0.12 nmol/mg/5 s, WKY and SHR, respectively; n = 4 each). These results further supported our data with in vitro microperfusion that apical NHE activity was reduced by inhibition of Rho-kinase.

	Discussion

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In this study, we found that in vitro pretreatment with Y-27632 reduced NHE activities in both apical (presumably NHE3) and basolateral (presumably NHE1) membranes of rat renal proximal convoluted tubule cells. These findings are compatible with previous results concerning NHE1 in smooth muscle cells and NHE3 in transfected CHO cells (Szaszi et al., 2000); however, we are not aware of any published studies concerning the effect of Y-27632 on these transporters in the intact kidney, one of the major organs that expresses NHE3 (Orlowski et al., 1992). Because Rho is one of the regulators of the actin cytoskeleton in cell membranes (Hall, 1998), the suppression of NHE activity by the drug observed in this study may be mediated by an alteration of the cytoskeletal network in the proximal tubule cells. Indeed, NHE3 activity can be reduced by the disruption of actin with colchicine and cytochalasin D in rat proximal tubule (Chalumeau et al., 2001).

We also compared the renal NHE activities in SHR with those in WKY rats. Compared with proximal tubules from WKY rats, there was increased NHE3 activity in tubules from SHR. These findings are compatible with previous results; Morduchowicz et al., 1989; Dagher and Sauterey, 1992; Hayashi et al., 1997). Furthermore, we showed that the sensitivity of the enhanced NHE3 activity in proximal tubules was about 50-fold higher in SHR compared with WKY rats. Because NHE3 mRNA expression is not different between SHR and WKY kidneys (Hayashi et al., 1997), the higher NHE3 activity in proximal tubules of SHR may be attributable to the special conditions of anchoring NHE3 to the apical membrane by the actin cytoskeleton of SHR. In aortic endothelium, stress fiber expression was more prominent in SHR than in WKY (White and Fujiwara, 1986). This may also be true in renal cells. Further studies, such as the evaluation of p160ROCK expression, are needed to address these differences.

It has also been reported that dpH_i/dt after prepulse is mediated largely by NHE activity, but also by vacuolar H-ATPase activity in the apical membrane of proximal convoluted tubules (Dagher and Sauterey, 1992; Aldred et al., 2000). For this reason, we preincubated proximal tubules with luminal bafilomycin A1, a specific inhibitor of the vacuolar H-ATPase (Tsuruoka and Schwartz, 1997), so that only sodium-dependent changes in pH_i were evaluated after acid loading the tubules.

Another important finding of this study is that administration of Y-27632 using a subdepressor dose enhanced Na excretion and total urine volume. These clearance results are compatible with the data obtained from our in vitro microperfusion study. Systemic infusion of this drug at a higher dose reduces arterial blood pressure by inhibiting NHE1 in vascular smooth muscle cell (Hirata et al., 1992; Uehata et al., 1997), which leads to a reduction of renal blood flow and subsequent increase in Na absorption and decrease in urine volume. To evaluate a more specific renal tubular response to this drug, we titrated its dose such that there was no change in blood pressure; under these conditions, we performed the renal clearance study. In general, a natriuresis, especially in patients with hypertension, reduces the blood pressure. Our findings suggest that the natriuresis induced by Y-27632 may contribute to the blood pressure lowering effect of this agent. Although the exact site of action of the drug in the kidney is uncertain, the proximal tubule may be one of the sites because the drug did not change urinary K excretion. We also found that urinary Na excretion induced by Y-27632 via the inhibition of Rho-kinase is more prominent in SHR than in WKY rats. This is in good agreement with our in vitro findings herein. Increases in FE Na and total Na excretion were higher in SHR, whereas changes in urine volume were similar in both strains. Basal FE Na was lower in SHR, which is compatible with previous results (Khraibi and Knox, 1989).

In conclusion, we have found that the inhibition of Rho-kinase (which may lead to the disruption of the actin cytoskeleton) reduced the activity of NHE in both apical and basolateral membranes of the native rat renal proximal convoluted tubule. The sensitivity in vitro was higher in adult SHR than in WKY, whereas such a difference was not observed in young animals. In vivo infusion of Y-27632 at a subdepressor dose enhanced urinary volume and Na excretion without affecting the GFR in both strains. The natriuresis was more prominent in SHR than in WKY rats. Thus, the normal actin network in renal proximal convoluted tubule cells is required to maintain NHE activity and urinary Na excretion. The network may be altered in SHR, thereby contributing to salt retention and hypertension. These findings may provide new clues toward better understanding the mechanisms of essential hypertension.

	Acknowledgements

 
We thank Mariko Hojo for technical assistance.

	Footnotes

 
DOI: 10.1124/jpet.102.041871.

ABBREVIATIONS: NHE, sodium-hydrogen exchanger; SHR, spontaneously hypertensive rat; Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-amino-ethyl)-cyclohexanecarboxamide; WKY, Wistar Kyoto; BP, blood pressure; BCECF-AM, acetoxymethyl ester of 2',7'-bis(carboxyethyl) carboxyfluorescein; FE, fractional excretion; GFR, glomerular filtration rate; BBMV, brush-border membrane vesicle; MES, 4-morpholineethanesulfonic acid; CHO, Chinese hamster ovary.

Address correspondence to: Dr. Shuichi Tsuruoka, Department of Clinical Pharmacology, Jichi Medical School, 3311 Yakushiji, Minamikawachi, Kawachi, Tochigi 329-0498, Japan. E-mail: tsuru{at}jichi.ac.jp

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